Methods for using pharmaceutical compositions comprising angiocrine factors in treating patients exposed to myeloablative insults

Abstract

To provide a pharmaceutical composition for use in a method for modulating an inflammatory stress response within a hematopoietic bone marrow microenvironment comprising bone marrow endothelial cells (BMECs), hematopoietic stem cells (HSCs), and bone marrow stromal cells following a myelosuppressive insult.SOLUTION: Wherein the pharmaceutical composition comprises pharmacological means for modulating the inflammatory stress response comprising administration of recombinant or synthetic Clec11 alpha stem cell factor (SCGF) and / or genetic means comprising suppression of endothelial NF - κ B signaling; The pharmaceutical composition protects the bone marrow (BM) hematopoietic compartment by inhibiting NF - κ B in endothelial cells, suppressing downstream NF - κ B signaling in the BM, down-regulating target NF - κ B genes in the BM, or both, and promotes recovery after myelosuppressive injury and resolves inflammation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 941,190, entitled "Endothelial MPAK Activation Disrupts Hematopoiesis by Inducing NF-kB-dependent Inflammatory Stress," filed November 27, 2019, and U.S. Provisional Application No. 62 / 980,108, entitled "Methods for Use of a Pharmaceutical Composition Comprising an Angiocrine Factor in Treating a Patient Exposed to a Myeloablative Insult," filed February 21, 2020, the contents of which are incorporated herein by reference in their entireties.

[0002] Government funding statement This invention was made with government support under contracts HL133021 and 1R01CA204308 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on November 25, 2020, is named 128533-02520_SL.txt and is 13,431 bytes in size.

[0004] FIELD OF THE INVENTION The described invention relates to hematopoietic recovery after myelosuppressive injury. [Background technology]

[0005] hematopoiesis Multipotent self-renewing hematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation. As used herein, the term "hematopoiesis" refers to the process by which the cellular components of blood are continuously replenished throughout an organism's lifespan by differentiation of hematopoietic stem cells (HSCs) into mature, functional cell types of the blood lineage. The hematopoietic lineage is divided into two major branches: the myeloid arm and the lymphoid arm. The common myeloid progenitor (CMP) gives rise to the myeloid arm, which can give rise to all myeloid cells. The common lymphoid progenitor (CLP) gives rise to the lymphoid arm, which can give rise to all lymphoid cells.

[0006] Hematopoietic stem cells (HSCs) reside in the bone marrow and are pluripotent stem cells capable of generating all cells of the blood and immune systems. They have the capacity to self-renew and differentiate into progeny of multiple lineages. Human HSC activity resides in the CD34Thy-1 population [Weiskopf, K. et al., "Myeloid cell origins, differentiation, and clinical implications," Microbiol.Spectr.(2016)4(5):10.1128 / microbiolspec.MCHD-0031-2016]. The CD90+CD45RA- population contains authentic human long-term HSCs, while the CD90-CD45RA- population represents intermediate downstream multipotent progenitor cells (MPPs) [ibid.]. The lin-CD34+CD38+ population of human bone marrow has limited self-renewal capacity and exhibits a high rate of myeloid-biased differentiation (ibid., citing Manz, MG, et al., "Prospective isolation of human clonogenic common myeloid progenitors," Proc. Natl Acad. Sci. USA (2002) 99(18):11872-77). This population was further subdivided by CD45RA and IL-3Rα expression, yielding three distinct subpopulations: IL-3RαloCD45RA-, IL-3RαloCD45RA+, and IL-3Rα-CD45RA- cells. In vitro, the IL-3RαloCD45RA- population gave rise to the full range of myeloid lineages, including mixed colonies, suggesting that this population represents a human common myeloid progenitor (CMP) [ibid., citing Manz, MG, et al., "Prospective isolation of human clonogenic common myeloid progenitors." Proc. Natl Acad. Sci. USA (2002) 99(18):11872-77].On the other hand, the IL-3RαloCD45RA+ population gave rise exclusively to cells of the granulocyte and macrophage lineages, whereas the IL-3Rα-CD45RA- population gave rise primarily to cells of the erythroid and megakaryocytic lineages, thereby indicating that these populations represent granulocyte / macrophage-lineage-restricted progenitors (GMPs) and megakaryocytic / erythroid-lineage-restricted progenitors (MEPs), respectively (ibid., citing Manz, MG, et al., "Prospective isolation of human clonogenic common myeloid progenitors." Proc. Natl. Acad. Sci. USA (2002) 99(18):11872-77).

[0007] Within the human MEP population, differentiation studies have identified unipotent human erythroid progenitors (EPs) as CD71 中間体(int) / + These cells helped define themselves as CD105+, and when sorted for purification, they gave rise to only erythroid cells in vitro, lacking megakaryocytic potential [ibid., citing Mori, Y. et al., "Prospective isolation of human erythroid lineage-committed progenitors," Proc. Natl. Acad. Sci. USA (2015) 112(31):9638-43]. Furthermore, erythroid-biased MEPs (E-MEPs) were identified as CD71+CD105-, intermediate between MEPs and EPs [ibid.]. Downstream stages of human erythropoiesis, including primitive erythroid progenitors (burst-forming unit-erythroid or BFU-E) and late colony-forming unit-erythroid (CFU-E), have also been isolated in pure form. These populations were primarily distinguished as IL-3R-CD34+CD36- and IL-3R-CD34-CD36+, respectively (ibid., citing Li, J. et al., "Isolation and transcriptome analyses of human erythroid progrenitors: BFU-E and CFU-E." Blood (2014) 124(24):3636-45).

[0008] Stem cell niche The effective function of the body's tissues and organs depends on innate regenerative processes to maintain adequate cell numbers (homeostasis) and replace damaged cells after injury (repair). In many, but not all, cases, regenerative capacity is determined by the presence and functionality of dedicated populations of stem and progenitor cells, which respond to exogenous cues to generate replacement cells when needed. (Wagers, A. J. The stem cell niche in regenerative medicine. Cell stem cell 10, 362–369, doi:10.1016 / j.stem.2012.02.018(2012)). These cells reside in a specialized environment called the “stem cell niche,” which provides sufficient spatial, temporal, and structural boundaries to protect these cells from damage or loss while maintaining communication with their surroundings to ensure appropriate responsiveness to physiological cues for cell replacement and repair. (Wagers, AJThe stem cell niche in regenerative medicine.Cell stem cell 10,362-369,doi:10.1016 / j.stem.2012.02.018(2012)).

[0009] Stem cell niches have been identified and characterized in many tissues, including the germ line, myeloid lineage, digestive and respiratory systems, skeletal muscle, skin, hair follicles, mammary glands, and the central and peripheral nervous systems (Wagers, AJ The stem cell niche in regenerative medicine. Cell stem cell 10, 362-369, doi:10.1016 / j.stem.2012.02.018(2012)).

[0010] The stem cell niche environment is composed of cellular and environmental components that are important for their function and maintenance. Cell-cell interactions provide structural support, regulate adhesive interactions, and generate soluble signals that control stem cell function. Environmental components include physical forces, such as pressure, structural and chemical signals, and temperature, as well as physiological parameters, such as interactions with the extracellular matrix (ECM). (Id.)

[0011] Heterogeneous cell-cell interactions in stem cell niches are tightly regulated and often exhibit complex bidirectional signaling that relies on cell-cell contact. Stem cell niches contain tissue-specific and general cell populations, each of which has specialized roles. (Lane, SW, Williams, DA & Watt, FM. Modulating the stem cell niche for tissue regeneration. Nature biotechnology 32, 795-803, doi:10.1038 / nbt.2978(2014)).

[0012] The hematopoietic microenvironment is localized in the bone marrow space of adult bones and contains a wide variety of cell types that clearly define the hematopoietic stem cell (HSC) niche, including osteoblasts, blood vessels, and neurons, megakaryocytes, macrophages, and immune cells. Secreted and membrane-bound factors, such as Wnt, SCF, Notch, and chemokines, directly bind to stem cell surface receptors to regulate cell fate, self-renewal, and polarity. (Lane, SW, Williams, DA, & Watt, FM. Modulating the stem cell niche for tissue regeneration. Nature biotechnology 32, 795-803, doi:10.1038 / nbt.2978(2014)).

[0013] The close association of many stem cell types with the vasculature and nervous system allows for the regulation of stem cell responses by metabolic cues and circadian rhythms, providing a conduit through which inflammatory and immune cells and humoral factors can be delivered to the niche. (Wagers, A. J. The stem cell niche in regenerative medicine. Cell stem cell 10, 362-369, doi:10.1016 / j.stem.2012.02.018(2012)) Immune cells dynamically regulate the niche during inflammation and tissue injury, which is tightly regulated by the existence of "immune privilege" (referring to the observation that tissue grafts placed in certain anatomical locations, including the brain and eye, can survive for long periods of time) and by escape from this privilege. (Lane,SW,Williams,DA& Watt,FM Modulating the stem cell niche for tissue regeneration.Nature biotechnology 32,795-803,doi:10.1038 / nbt.2978(2014)).

[0014] Extracellular matrix (ECM) proteins and stem cell interactions with the ECM provide retention cues and mechanical signals, based in part on the substrate's stiffness, that allow stem cells to respond to external physical forces. ECM proteins are important for maintaining the orientation and structure of the niche and provide instructive signals through ligand interactions with stem cell-expressed integrins. (Lane, SW, Williams, DA, & Watt, FM. Modulating the stem cell niche for tissue regeneration. Nature biotechnology 32, 795-803, doi:10.1038 / nbt.2978 (2014)). Additionally, the ECM may segregate or concentrate growth factors, chemokines, and other stem cell-regulating molecules by binding both locally and systemically produced factors within the niche. (Wagers, AJThe stem cell niche in regenerative medicine.Cell stem cell 10,362-369,doi:10.1016 / j.stem.2012.02.018(2012)).

[0015] Physical parameters such as topography, stiffness / elasticity, shear stress, temperature, oxygen tension, and blood flow guide stem cell maintenance and differentiation. Furthermore, many stem cell niches have varying environmental characteristics and require strict metabolic regulation to maintain long-term quiescence and self-renewal of stem cell populations. (Lane, SW, Williams, DA & Watt, FM. Modulating the stem cell niche for tissue regeneration. Nature biotechnology 32, 795-803, doi:10.1038 / nbt.2978(2014)).

[0016] Although the specific components that make up a particular stem cell niche may vary in various tissues under different physiological conditions, in all cases, signals provided by these cellular and acellular components appear to be internalized by stem cells to inform fate decisions, including choices of quiescence or proliferation, self-renewal or differentiation, migration or retention, and cell death or survival. (Wagers, A. J. The stem cell niche in regenerative medicine. Cell stem cell 10, 362-369, doi:10.1016 / j.stem.2012.02.018(2012)).

[0017] Hematopoietic stem cell niche The hematopoietic system supplies the human body with over 100 billion mature blood cells daily, which perform functions such as oxygen transport, immunity, and tissue remodeling. The hematopoietic system is composed of a diverse population of highly specialized cells with unique functions such as oxygen transport and immune defense. An adult human produces approximately 4-5 x 10 11 The hematopoietic stem cells (HSCs) are estimated to generate 100 hematopoietic cells. Continuous generation of many blood cell types requires a highly regulated and highly responsive system. Within mammalian hematopoietic tissues, rare hematopoietic stem cells (HSCs) occupy a high position in the hierarchy. (Pinho, S., Frenette, P.S. Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol 20, 303-320 (2019) doi:10.1038 / s41580-019-0103-9)

[0018] HSC niche development During development, HSCs migrate between niches to establish hematopoiesis. Primitive hematopoiesis occurs in the yolk sac at approximately embryonic day 7.0 (E7.0), when immature progenitor cells generate red blood cells that supply oxygen to the developing embryo. The first known definitive HSCs capable of fully reconstituting the hematopoietic system upon transplantation are found in the aorta-gonad-mesonephros of mice and humans. However, several studies suggest that E9.0–E10.0 yolk sac cells can mature into definitive HSCs when transplanted into newborns rather than adult mice. Furthermore, the placenta represents an important reservoir of developing HSCs. Once the vasculature develops, HSCs migrate to the fetal liver at or near E12.0, where they proliferate and differentiate. Fetal liver HSCs, in contrast to their bone marrow counterparts, actively circulate and may even outnumber adult bone marrow HSCs when transplanted into irradiated recipients. During HSC proliferation in the fetal liver, chondrocytes and osteoblasts are generated within mesenchymal condensations, resulting in the generation of cartilage and bone. Skeletal remodeling is associated with bone vascularization, enabling HSC homing and colonization of the fetal bone marrow by E17.5. This process is mediated by CXCL12 production by bone marrow stromal cells, which attracts specific adhesion molecules expressed on HSCs and bone marrow endothelium that express CXCR4. (Boulais, PE, & Frenette, PS (2015). Making sense of hematopoietic stem cell niches. Blood, 125(17), 2621-2629. doi:10.1182 / blood-2014-09-570192).

[0019] HSC niche and bone marrow microenvironment In adult bone, HSCs are essentially maintained in the G0 phase of the cell cycle, a stage of metabolic dormancy or quiescence, which maintains their function by limiting damage associated with cell replication. However, quiescent HSCs can rapidly respond to a wide range of niche or systemic signals by entering the cell cycle and proliferating. These instructive cues are therefore essential for orchestrating HSC differentiation and tailoring blood production to the needs of the organism. HSCs can also leave the BM niche and enter the bloodstream upon receiving recruitment signals to ensure immune surveillance of peripheral tissues and to engraft distant BM sites. Thus, by dynamically regulating the switch between quiescence / proliferation and anchorage / recruitment, HSCs crucially depend on short- and long-term instructive cues from the BM niche for many aspects of biology, including cell cycle and transport activity.

[0020] Resident Niche Cells. The HSC stem cell niche contains various cell types, each with distinct functions. For example, osteoblasts, vascular, and neural cells, megakaryocytes, macrophages, and immune cells each have important roles and can be considered to define distinct HSC niches. It also contains other specific niches, such as the osteoblastic and perivascular niches. Studies are conflicting as to whether these two niches have distinct, specific roles or whether there is coordinated regulation of HSCs, but they therefore overlap in function. For example, NG2+ periarteriolar cells regulate quiescence in long-term HSCs, and this quiescence appears to be essential for HSC function. Other cells, such as endosteal macrophages, maintain HSCs within the niche, and their loss triggers HSC recruitment from the supportive microenvironment. (Lane, SW, Williams, DA & Watt, FMModulating the stem cell niche for tissue regeneration. Nature biotechnology 32,795-803, doi:10.1038 / nbt.2978(2014)).

[0021] Direct cell-cell contact. Direct cell contact can be mediated by a variety of receptors, including cell-cell adhesion molecules and receptors with membrane-bound ligands. For example, in bone marrow, Notch ligands expressed by sinusoidal cells are essential for HSC self-renewal during recovery from myeloablative injury. (Lane, SW, Williams, DA & Watt, FM. Modulating the stem cell niche for tissue regeneration. Nature biotechnology 32, 795-803, doi:10.1038 / nbt.2978(2014)).

[0022] Secreted Factors. Indirect communication between stem cells and niche cells is mediated by secreted factors. For example, recruitment of HSCs from the niche using cytokines such as granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) is widely used to support the treatment of hematologic malignancies, bone marrow failure, and rare genetic disorders. These factors act in various ways, including promoting HSC proliferation and the release of HSC-niche adhesions. (Lane, SW, Williams, DA & Watt, FM. Modulating the stem cell niche for tissue regeneration. Nature biotechnology 32, 795-803, doi:10.1038 / nbt.2978(2014)). Specifically, secreted factors like stem cell factor (SCF), transforming growth factor beta 1 (TGF-b1), platelet factor 4 (PF4 or CXCL4), angiopoietin 1 (ANGPT1), and thrombopoietin (TPO) are all key executors of HSC quiescence. Adhesion molecules such as vascular cell adhesion protein 1 (VCAM-1), various selectins, and extracellular matrix (ECM) proteins like fibronectin or hyaluronan, along with the essential chemokine stromal-derived factor 1 (SDF1a or CXCL12) and its CXC chemokine receptor type 4 (CXCR4), are all essential regulators of HSC homing and anchoring in the niche.

[0023] The decision to remain quiescent or enter an actively proliferating state is controlled by numerous factors through both intrinsic and extrinsic mechanisms. HSCs can enter either quiescence or the cell cycle in response to exogenous soluble factors; inflammatory cytokines such as interferon (IFN)-α and IFN-γ; growth factors such as granulocyte colony-stimulating factor (GCSF), stem cell factor (SCF), and thrombopoietin (TPO); cytokines such as transforming growth factor (TGF)-β and tumor necrosis factor (TNF)-α; and chemokines such as stromal cell-derived factor (SDF)-1. Intrinsic factors that regulate HSC quiescence include cell cycle inhibitors such as p21 and p57, transcription factors (TFs) such as Gfi1, Egr1, FOXO, and PBX1, and ubiquitin ligases such as c-Cbl, Itch, Fbxw7, and A20. The proper maintenance of HSCs in the bone marrow niche requires the coordination of intrinsic and extrinsic factors (Nakagawa, MM, Chen, H., & Rathinam, CV (2018). Constitutive Activation of NF-κB Pathway in Hematopoietic Stem Cells Causes Loss of Quiescence and Deregulated Transcription Factor Networks. Frontiers in Cell and Developmental Biology, 6, 143).

[0024] Bone Microenvironment. Bone marrow can be subdivided into a hematopoietic cell compartment and a stroma composed primarily of fibroblasts, adipocytes, nerves, and the marrow vasculature. (Kopp, et al. "The Bone Marrow Vascular Niche: Home of HSC Differentiation and Mobilization." PHYSIOLOGY 20:349-356, 2005; 10.1152 / physiol.00025.2005).

[0025] Arterial blood vessels enter the bone marrow via the foramen alimentarii and then divide into several arterioles. These small arterioles and capillaries supply the sinusoids, which span the entire bone marrow and are interconnected by intersinusoidal capillaries. The sinusoids are distributed radially around the draining central sinus, which is approximately 100 mm in diameter. Bone marrow sinusoids are unique and should not be compared to ordinary veins. The sinusoidal walls are composed of a single layer of endothelial cells and lack supporting cells. The endothelial cells lack a connective tissue covering but rather are in direct contact with parenchymal cells. The surrounding hematopoietic bone marrow is the primary cellular component supporting the reconstruction and remodeling of the sinusoidal microcirculation.

[0026] Rapid induction of bone marrow hypocellularity by cytotoxic agents or radiation is followed by significant dilation and collapse of the sinusoids and central venous sinus. The absence of normal vascular walls in the sinusoids is reflected in high levels of permeability. The bone marrow microenvironment harbors HSCs and hematopoietic progenitor cells (HPCs), and the bone microanatomical environment, composed of adjacent stromal cells, supports and guides stem cells. It has been hypothesized that the stromal environment itself may determine the quality of hematopoiesis. (Kopp, et al., "The Bone-Marrow Vascular Niche: Home of HSC Differentiation and Mobilization." PHYSIOLOGY 20:349-356, 2005; 10.1152 / physiol.00025.2005)

[0027] HSCs and HPCs are not randomly distributed in bone marrow, but rather localize near the endosteum of bone and around blood vessels. Furthermore, embryonic bone marrow exhibits the first hematopoietic colonies adjacent to the endosteum. Researchers have identified a cellular developmental gradient in bone marrow, with undifferentiated cells located along the endosteum and differentiation and maturation associated with central movement into the highly vascularized bone marrow cavity. (Kopp, et al. "The Bone Marrow Vascular Niche: Home of HSC Differentiation and Mobilization." PHYSIOLOGY 20:349-356, 2005; 10.1152 / physiol.00025.2005)

[0028] The bone marrow microenvironment not only houses HSC niches but also osteoblastic or endosteal niches, and each vascular niche is defined by the role it plays in stem cell localization. The bone marrow osteoblastic niche provides signals to maintain repopulating cells in an undifferentiated state. These spatial differences in hematopoietic tissues do not reflect or translate into the properties of the stem cells themselves. Further studies have shown that stromal structures, such as bone marrow sinusoidal vessels, can serve as alternative cellular scaffolds where hematopoietic cells can reside and mature.

[0029] The term "vascular niche" is used to accurately outline the bone marrow sinusoidal and arteriolar network as a separate anatomical and functional entity derived from the endosteal zone. Ultrastructural studies have shown that differentiated, but not immature, hematopoietic cells are closely associated with the bone marrow microvasculature of the vascular niche. Furthermore, nearly all mature megakaryocytes were found adjacent to thin-walled sinusoids, demonstrating that megakaryocytes can migrate throughout intact endothelial cells. This observation is not limited to thrombopoiesis, but can also apply to erythroid and B lymphoid progenitors, as these lineages have also been reported to reside in defined niches within the bone marrow. These findings demonstrate progenitor-stromal cell interactions, which are crucial determinants of the maturation process and furthermore permissive for stem cell maintenance and differentiation, reinforcing the concept of the stem cell niche as an instructive microanatomical structure. Furthermore, bone marrow endothelial cells (BMECs) were found to have adhesive properties, interactions with angiogenic and chemokinetic factors, and contributions to supporting HSC self-renewal and differentiation, thereby demonstrating the interdependence of the bone marrow parenchyma and the vascular niche. (Crane, GM, et al., “Adult haematopoietic stem cell niches,” Nat.Rev.Immunol. (2017) 17(9):573-90; Ramalingam, P. et al., “Regulation of the hematopoietic stem cell llifecycle by the endothelial Yu,VW,and Scadden,DT,“Herterogeneity of the bone marrow niche,”Curr.Opin.Hematol.(2016)23(4):331-38;Kopp,et.al.“The Bone Marrow Vascular Niche:Home of HSC Differentiation and Mobilization.“PHYSIOLOGY 20:349-356,2005;10.1152 / physiol.00025.2005).

[0030] Angiocrine Factor There are numerous angiocrine growth factors that play multiple roles in bone tissue cell signaling in the bone microenvironment. Table 2 below describes such angiocrine factors and their crosstalk with bone tissue cells. (Sivan U, De Angelis J, Kusumbe AP. 2019 Role of angiocrine signals in bone development, homeostasis and disease. Open Biol. 9:190144. http: / / dx.doi.org / 10.1098 / rsob.190144) [Table 1-1] [Table 1-2]

[0031] The junction between hematopoietic cells and endothelial cells has been identified as the hemangioblast, a common precursor of endothelial and hematopoietic cells. There is a strong embryonic interdependence between HSC / HPC and endothelial cells that continues into adulthood (Kopp, et al., "The Bone Marrow Vascular Niche: Home of HSC Differentiation and Mobilization." PHYSIOLOGY 20:349-356, 2005; 10.1152 / physiol.00025.2005).

[0032] Bone marrow endothelial cells (BMECs) are key to mechanistically understanding the blood cell production capacity (i.e., hematopoiesis) of bone marrow. Studies on other endothelial cell types, such as human umbilical vein endothelial cells (HUVECs), have shown that transendothelial transport depends on the expression of surface receptors or adhesion molecules inducible by inflammatory cytokines. Therefore, it is likely that the release of mature blood cells, as well as the recruitment and homing of HSCs / HPCs, are regulated by similar mechanisms. BMECs have been shown to support the proliferation and differentiation of hematopoietic progenitor cells in vitro through the production of various cytokines and, in some cases, through physical contact. Coculture of megakaryocytes and BMECs prolonged the survival of BMECs, likely due to the secretion of the endothelial cell survival factor VEGF-A by megakaryocytes. (Kopp, et.al. “The Bone Marrow Vascular Niche:Home of HSC Differentiation and Mobilization.”PHYSIOLOGY 20:349-356,2005;10.1152 / physiol.00025.2005).

[0033] The adequacy of the vasculature of the bone marrow microenvironment is also key to hematopoiesis. Indeed, the function of the vascular niche interaction with BMECs is to provide a cellular platform conducive to HSC support, but the molecular mechanisms by which proper structural integrity of endothelial cells leads to this development remain unclear. (Kopp, et al. "The Bone Marrow Vascular Niche: Home of HSC Differentiation and Mobilization." PHYSIOLOGY 20:349-356, 2005; 10.1152 / physiol.00025.2005)

[0034] vascular integrity Vascular integrity is critical for vascular homeostasis. (Murakami, M., and Simons, M.J. Mol. Med. (Berl) (2009) 87(6):571-82). Maintenance of the vascular system is an active biological process requiring continuous basal cell signaling. Disruption of this system can have serious consequences, including hemorrhage, edema, inflammation, and tissue ischemia.

[0035] As currently understood, the steps in new blood vessel formation include endothelial cell proliferation and migration, followed by assembly into new vascular structures, lumen formation, and finally, maturation of the newly formed endothelial tube. Ibid. The latter stage, tube stabilization and restoration of barrier function, is crucial for the maturation of newly formed vessels. Ibid. This stabilization stage requires the activation of distinct cell signaling pathways that are distinct from those that initiate vascular cell proliferation and migration. Furthermore, not only newly formed vessels but also existing vessels must be actively maintained to maintain their integrity and tissue homeostasis. Ibid. Experience with various angiogenic therapeutic approaches has demonstrated that simply inducing new vessel growth is not sufficient to achieve functionally meaningful improvements in blood flow; preventing vascular regression and promoting vascular maturation are equally important. (Ibid., citing Simons, M., Circulation (2005) 111:1556-66)

[0036] Studies in various animal models, as well as genetic studies in mice and humans, have identified many factors that play important roles in actively maintaining vascular integrity during embryonic vascular development or in the adult vasculature. These factors act coordinately in an organized manner across many steps of vascular stabilization and maintenance. During the angiogenesis process, after new blood vessels are established, endothelial cells develop cell-cell junctions to establish an effective barrier, a process in which the Ang1-Tie2 and FGF systems play crucial roles (ibid., citing Fiedler, U, Augustin, HG, Trends Immunol. (2006) 27:552-58; Murakami, M., Simons, M.; Curr. Opin. Hematol. (2008) 15:215-220). Concomitantly, mesenchymal progenitor cells differentiate into pericytes or smooth muscle cells under the action of TGF-β, while PDGF-BB derived from endothelial tip cells promotes pericyte recruitment and proliferation (ibid., citing Pepper, MS, Cytokine Growth Factor Rev. (1997) 8:21-43; Betsholtz, C. Cytokine Growth Factor Rev. (2004) 15:215-228; Andrrae, J. et al., Genes Dev. (2008) 22:1276-1312). Throughout this process, integrins mediate extracellular matrix (ECM) cell signaling, which in turn directs vascular stabilization (ibid., citing Hynes, RO, J. Thromb. Haemost. (2007) 5(Suppl. 1):32-40).

[0037] Vascular integrity is tightly regulated by many factors that ensure the proper function of various components of the vascular wall. One of the early hallmarks of impaired vascular integrity is increased permeability, which is primarily controlled by the stability of endothelial junctions. Selective regulation of vascular permeability is achieved by regulating the size and state of the paracellular gap and by controlling transcellular transport. Normal vasculature exhibits a certain level of basal permeability that varies from bed to bed. Early studies revealed constitutively open junctions in a subset of vascular beds (ibid., citing Simionescu, N. et al., J. Cell Biol. (1978) 79:27-44). Under normal conditions, approximately 30% of the endothelial cell-cell junctions in postcapillary venules, where active permeability regulation occurs, are open and permeable to molecules approximately 60 Å in size (ibid.). Upon stimulation with either histamine or 5-HT (5-hydroxytryptamine), the cell junctions of postcapillary venules selectively open, allowing the passage of larger molecules, but outflow from the venular junctions is restricted and confined to the perivascular space (ibid., citing Simionescu, N. et al., J. Cell Biol. (1978) 79:27-44 (suggesting the presence of an external barrier in the perivascular tissue)) (ibid.).

[0038] Increased endothelial permeability induced by physiological and pathological stimuli is usually reversible and does not permanently impair vascular integrity. However, interference with endothelial junctional components can lead to severe impairment of vascular integrity. In this scenario, junction disruption is usually accompanied by eventual endothelial detachment from the vessel wall and subsequent thrombus formation. While the sequence of events in this process is not fully understood, it is possible that the duration of the permeability-inducing stimulus may influence the outcome. (Ibid.) Unlike transient increases in vascular permeability, in which endothelial cells can rapidly restore barrier function by reestablishing VE-cadherin-based junctions, prolonged stimulation can lead to more serious effects, such as the accumulation of reactive oxygen species (ROS). Excessive amounts of ROS, known to have many adverse effects on endothelial function, may mediate such a scenario. Indeed, ROS can irreversibly inactivate protein tyrosine phosphatases (PTPs) by oxidizing active site Cys residues, thereby affecting tyrosine phosphorylation-dependent signaling events. (Ibid., citing Tonks, N.K., Nat. Rev. Mol. C Vell. Biol. (2006) 7:833-846)

[0039] Endothelial Junctions—In endothelial cells, there are three types of intercellular junctions: adherens, adherens, and gap junctions. Of these, adherens and tight junctions contribute to the structural integrity of the endothelium. (Ibid., citing Dejana, E., Nat. Rev. Mol. Cell Biol. (2004) 5:261-270) Although it is difficult to precisely outline the functional differences between these two types of junctions, it has been shown that the assembly of tight junctions depends on the prior formation of adherens junctions. It is generally believed that adherens junctions are primarily important in controlling endothelial permeability, while tight junctions are involved in blocking the movement of lipids and integral membrane proteins between the apical and basolateral surfaces of the cells. (Molecular fence) (Ibid., citing Dejana, E., Nature Rev. Mol. Cell Biol. (2004) 5:261-270; Taddei, A., et al., Nat. Cell Biol. (2008) 10:923-34).

[0040] Each type of junction has a different set of proteins. Cadherins are a family of transmembrane proteins that constitute adherens junctions and mediate cell-cell contact in a calcium-dependent manner through trans-homophilic interactions. In endothelial cells, VE-cadherin is localized at sites of cell contact, where it regulates the formation of adherens junctions and links the junctions to the actin cytoskeleton.

[0041] The stability of VE-cadherin at adherens junctions, regulated by binding to catenins, particularly p120-catenin, is crucial for maintaining endothelial permeability and integrity. Src family kinases are known to play a key role in VEGF-induced increased endothelial permeability, and Src-mediated VE-cadherin phosphorylation leads to the disruption of cell-cell contacts and the internalization of VE-cadherin (ibid., citing Weis, SM, Chesh, DA, Nature (2005) 437:497-504). This process is thought to be important for endothelial motility and the establishment of the angiogenic phenotype of "activated" endothelial cells. Thus, endothelial junctions are dynamic structures that actively assemble and disassemble even in quiescent monolayers, suggesting that the balance of forces controlling net VE-cadherin dynamics determines endothelial behavior.

[0042] Inflammation and the niche Inflammatory signals play a key role in a variety of processes, including embryonic specification of hematopoietic stem cells (HSCs) during development, emergency granulopoiesis during infection, and hematopoietic regeneration after transplantation. 4~8

[0043] Although all stem cell niches are dynamic and exhibit cell turnover, it is useful to distinguish between niche cells that are "permanent residents" (e.g., endothelial cells, neurons, and connective tissue fibroblasts) and cells that temporarily occupy the niche (e.g., immune cells and cells that respond to tissue injury, e.g., to protect against pathogens or promote healing). In contrast to resident niche cells, many cells of the innate and adaptive immune systems move in and out of tissues. Immune cell function can be modulated to promote stem cell function. (Lane, SW, Williams, DA, & Watt, FM. Modulating the stem cell niche for tissue regeneration. Nature biotechnology 32, 795-803, doi:10.1038 / nbt.2978 (2014)).

[0044] Proinflammatory mediators that regulate HSC development include Toll-like receptors (TLRs), cytokines, and eicosanoids, each of which activates the immune system to combat injury. Tissue destruction by injury or pathogens leads to the release of proinflammatory cytokines, which trigger typical inflammation. In summary, myeloid cells (e.g., macrophages and neutrophils) possess Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which induce the release of proinflammatory cytokines and eicosanoids upon recognition of damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). TLRs promote the induction of gene expression and intracellular accumulation of the key proinflammatory cytokines interleukin (IL)-1β and IL-18 through the master inflammatory / immune transcription factor nuclear factor kappa B (NF-κB). Subsequently, recognition of PAMPs / DAMPs in the cytoplasmic compartment by NLRs promotes caspase-1-mediated proteolytic cleavage and release of proinflammatory cytokines and cytosolic phospholipase A2-mediated eicosanoid biosynthesis. These cytokines and eicosanoids then activate immune cells to eliminate the cause of infection and restore healthy tissue. (Espin-Palazon, R., Weijts, B., Mulero, V. & Traver, D. Proinflammatory Signals as Fuel for the Fire of Hematopoietic Stem Cell Emergence. Trends in cell biology 28, 58-66, doi:10.1016 / j.tcb.2017.08.003(2018)).

[0045] HSCs are thought to sense immune or tissue injury through both intrinsic and extrinsic mechanisms. HSCs dynamically respond to cytokines produced locally (in the niche / microenvironment) and peripherally (injury or infection), including proinflammatory cytokines, chemokines, and PAMPs. HSCs and hematopoietic stem and progenitor cells (HSPCs) sense immune or tissue injury indirectly (through proinflammatory cytokines or DAMPs) or directly (through PAMPs). Normally, HSCs respond to proinflammatory signals by biasing normal hematopoiesis toward myelopoiesis, often at the expense of lymphopoiesis and erythropoiesis. This is thought to occur to replenish bone marrow cell numbers as existing cells are recruited to the site of infection. (Espin-Palazon, R., Weijts, B., Mulero, V. & Traver, D. Proinflammatory Signals as Fuel for the Fire of Hematopoietic Stem Cell Emergence. Trends in cell biology 28,58-66, doi:10.1016 / j.tcb.2017.08.003(2018).

[0046] Like differentiated immune cells, HSCs recognize injury through the expression of TLRs. Ligation of TLR signals in HSCs leads to proliferation and differentiation. The cell-extrinsic mode of recognition of tissue or immune injury by HSCs involves signaling via receptors for proinflammatory cytokines. (Nakagawa, MM, Chen, H., & Rathinam, CV (2018). Constitutive Activation of NF-κB Pathway in Hematopoietic Stem Cells Causes Loss of Quiescence and Deregulated Transcription Factor Networks. Frontiers in Cell and Developmental Biology, 6, 143)

[0047] Various proinflammatory cytokines and chemokines, including IL1, IL6, IL8, TNF, CC-chemokine ligand 2 (CCL2), IFN-α, and IFN-γ, have been shown to affect HSCs. Indeed, in vitro stimulation of TLR2, TLR7, and TLR8 with agonists has been shown to induce cytokine production, such as IL-1b, IL-6, IL-8, TNF-α, and GM-CSF, and myeloid lineage differentiation. Exposure of human CD34+ HSPCs to IFN-γ has been shown to result in dramatic transcriptional changes in genes involved in proapoptotic processes, immune responses, and myeloproliferation, leading to an increase in viable cell numbers. While some transcriptional changes are specific to HSPCs, others, such as cell growth and signaling, generally occur in stromal cells incubated with IFN-γ. In contrast, studies have shown that in vitro stimulation with IFN-γ and TNF significantly impairs the ability of HSPCs to undergo multilineage reconstitution in xenografted mice. (Kovtonyuk, LV, Fritsch, K., Feng, X., Manz, MG & Takizawa, H. Inflamm-Aging of Hematopoiesis, Hematopoietic Stem Cells, and the Bone Marrow Microenvironment. Frontiers in immunology 7, 502, doi:10.3389 / fimmu.2016.00502(2016)). Membrane-anchored TNF-α has been shown to enhance engraftment of purified HSCs in allogeneic and syngeneic recipients. (Espin-Palazon, R., Weijts, B., Mulero, V. & Traver, D. Proinflammatory Signals as Fuel for the Fire of Hematopoietic Stem Cell Emergence.Trends in cell biology 28,58-66,doi:10.1016 / j.tcb.2017.08.003(2018)).

[0048] Prolonged exposure of HSCs to proinflammatory cytokines leads to a decrease in self-renewal and quiescence. In this regard, NF-κB can be considered the gatekeeper of HSC inflammation control, as the proinflammatory cytokines produced and secreted by HSCs depend on NF-κB function. In summary, NF-κB proteins function as transcription factors and are considered master regulators of innate and adaptive immunity. In mammals, there are five major members of this family: Rel A (p65), Rel B, c-Rel, p50 / p105 (also known as NF-κB1), and p100 / 52 (also known as NF-κB2). NF-κB signaling is activated in response to various upstream stimuli. In the absence of any activating signal, inhibitors of NF-κB (IκB) proteins form complexes with inactive NF-κB proteins and remain in the cytoplasm. Following activation signals, the IκB kinase (IKK) complex, composed of two kinases, IKK1 (IKKα), IKK2 (IKKβ), and the regulatory subunit NEMO (IKKγ), phosphorylates IκB, which leads to ubiquitination and subsequent degradation of IκB. This releases NF-κB complexes from the cytoplasm to the nucleus, where they activate target gene expression. Therefore, the IKK complex plays a key role in the entire NF-κB signaling cascade, as deregulated activation of the IKK complex can lead to deleterious downstream consequences. The IKK complex phosphorylates IκB proteins on two amino (N)-terminal regulatory serine residues. In most canonical NF-κB signaling pathways, IKK2 is required and sufficient to phosphorylate IκB and activate NF-κB. (Nakagawa, MM, Chen, H., & Rathinam, CV (2018). Constitutive Activation of NF-κB Pathway in Hematopoietic Stem Cells Causes Loss of Quiescence and Deregulated Transcription Factor Networks. Frontiers in cell and developmental biology, 6, 143).

[0049] In the context of NF-κB in the HSC niche, NF-κB is a major downstream effector of signals transduced by both TLRs and proinflammatory cytokines. Uncontrolled NF-κB activity leads to increased expression of proinflammatory cytokines, including TNF, IL-1, IL-6, and IFN-γ. Defects in the negative regulatory circuitry of NF-κB signaling cause loss of quiescence and premature exhaustion of HSCs. Although these studies highlight the importance of the NF-κB pathway in HSC quiescence, the downstream effects of NF-κB signaling and the precise mechanisms by which NF-κB regulates HSCs remain largely unknown. Constitutive activation of NF-κB has been described in various types of human diseases, including myeloid neoplasms. For example, NF-κB has been shown to be constitutively active in leukemia stem cells (LSCs). (Nakagawa, MM, Chen, H., & Rathinam, CV (2018). Constitutive Activation of NF-κB Pathway in Hematopoietic Stem Cells Causes Loss of Quiescence and Deregulated Transcription Factor Networks. Frontiers in cell and developmental biology, 6, 143).

[0050] Activation of quiescent ECs to generate a proinflammatory response is normally driven by the transcription factor nuclear factor kappa B (NF-κB), which not only activates the transcription of proinflammatory genes including TNF-α, interleukin-1 (IL-1), E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1), but also renders ECs more susceptible to apoptosis (Jin, Z., et al., Int. J. Mol. Sci. (2019) 20(1):172; Pober, J. S., Sessa, W. C., Nat. Rev. Immunol. (2007) 7:803-815; Aoki, M. et al., Hypertension (2001) 38:48-55; Kempe, S. et al., Nucleic Acids Res. (2005) 33:5308-5319).

[0051] The role of proinflammatory immunoregulators is not limited to adult HSC function. Studies have shown that proinflammatory pathways, such as the archetypal proinflammatory transcription factor NF-κB, are associated with the formation of the hematopoietic system during embryogenesis in both vertebrates and invertebrates. Other immunomodulators also influence HSC specification (identity), emergence, and maintenance during hematopoietic system formation. For example, IL-3, a cytokine that regulates immune cell function, proliferation, and differentiation, has been shown to promote HSC survival in the mouse aorta-gonad-mesonephros (AGM) region, the site of HSC specification, by acting downstream of Runx1, an essential transcription factor for HSC specification. In another example, IL-1, a regulator of inflammation, plays an active role in HSC development by promoting HSC proliferation. Prostaglandin E2 (PGE2), a key regulator of inflammation, has also been shown to be a potent inducer of HSC emergence or proliferation by autonomously regulating HSC Wnt at the level of beta-catenin degradation via cAMP / PKA-mediated stabilizing phosphorylation events. (Espin-Palazon, R., Weijts, B., Mulero, V. & Traver, D. Proinflammatory Signals as Fuel for the Fire of Hematopoietic Stem Cell Emergence.Trends in cell biology 28,58-66,doi:10.1016 / j.tcb.2017.08.003(2018)).

[0052] HSC fate determination is linked to the proinflammatory cytokines TNFα, IFNγ, and IL-1β; these cytokines, along with TLR4 signaling, are each critical determinants of HSC specification. TNFα acts through TNF receptor 2 (TNFR2) to specify HSCs from hemogenic endothelial cells (HEs), a specialized subset of developing vascular endothelial cells that can acquire hematopoietic potential and give rise to multilineage hematopoietic stem and progenitor cells during a narrow developmental window, e.g., in the extraembryonic yolk sac and embryonic aorta-gonad-mesonephros (EGV). (Griz, E. “Specification and function of hemogenic endothelium during embryogenesis,” Cell Mol. Life Sci. (2016) 73:1547-67). TNFR2 action is required for the expression of jag1a, a Notch ligand essential for HSC specification in the dorsal aorta. Expression of Jag1 signals to the Notch1a receptor on adjacent hemogenic endothelial cells (i.e., specialized endothelial cells from which HSPCs originate) to help establish HSC fate. The proinflammatory transcription factor NF-κB has been found to be active in nascent HSCs. TNFR2, NF-κB member p65, and TLR4 are all upregulated in HSCs. Furthermore, TLR4, IL-1β, and TNFα are required for HSC specification by acting upstream of NF-κB and Notch. It has been demonstrated that HSCs, but not endothelial cells, respond rapidly to IFN. IFN-α4 and IFN-γ are also required for HSC specification throughout vertebrates via IFNαR1 and IFNγR1, respectively. Unlike TNF-α and TLR4 signaling, IFN-γ acts downstream of Notch signaling and blood flow by activating Stat3. IFN-γ signaling acts autonomously in HE.(Espin-Palazon,R.,Weijts,B.,Mulero,V.& Traver,D.Proinflammatory Signals as Fuel for the Fire of Hematopoietic Stem Cell Emergence.Trends in cell biology 28,58-66,doi:10.1016 / j.tcb.2017.08.003(2018))。

[0053] The cellular source of proinflammatory cytokines during hematopoietic lineage formation is unknown. (Espin-Palazon, R., Weijts, B., Mulero, V. & Traver, D. Proinflammatory Signals as Fuel for the Fire of Hematopoietic Stem Cell Emergence. Trends in Cell Biology 28, 58-66, doi:10.1016 / j.tcb.2017.08.003(2018)). Such cytokines are known to influence HSC differentiation. At steady state, platelet-biased HSCs are at the top of the hematopoietic hierarchy and can generate myeloid-biased and lymphoid-biased HSCs. Myeloid-biased HSCs can generate both balanced-biased and lymphoid-biased HSCs, but lymphoid-biased HSCs do not generate their myeloid-biased counterparts. Platelet-biased HSCs have the potential to repopulate the platelet population more quickly than other HSC subsets. Myeloid-biased HSCs preferentially give rise to myeloid lineage cells via myeloid-committed progenitors. Balanced HSCs contribute equally to both myeloid and lymphoid lineages. Lymphoid-biased HSCs primarily generate lymphocytes over myeloid lineage cells via lymphoid-committed progenitors. Inflammation, especially chronic inflammation, enhances the production of myeloid lineages, including myeloid progenitors and mature myeloid cells, resulting in a myeloid bias in hematopoiesis. (Kovtonyuk, L.V., Fritsch, K., Feng, X., Manz, M.G., & Takizawa, H. Inflamm-Aging of Hematopoiesis, Hematopoietic Stem Cells, and the Bone-Marrow Microenvironment. Frontiers in Immunology 7, 502, doi:10.3389 / fimmu.2016.00502(2016)).

[0054] Therefore, inflammation is a major driver of hematopoiesis, and persistent inflammation has been proposed as a major driver of age-related hematopoietic disorders, including loss of HSC self-renewal capacity, myeloid-biased differentiation, and leukemia predisposition (Kovtonyuk, LV, et al. Inflamm-Aging of Hematopoiesis, Hematopoietic Stem Cells, and the Bone Marrow Microenvironment. Frontiers in immunology (2016) 7, 502, doi:10.3389 / fimmu.2016.0050); Pietras, EM et al. Chronic interleukin-1 exposure drives hematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal. Nature cell biology (2016) 18, 607-618, doi:10.1038 / ncb3346; Lussana, F. & Rambaldi, A. Inflammation and myeloproliferative neoplasms.Journal of autoimmunity(2017)85,58-63,doi:10.1016 / j.jaut.2017.06.010;Pietras,EMInflammation:a key regulator of hematopoietic stem cell fate in health and disease.Blood(2017)130,1693-1698,doi:10.1182 / blood-2017-06-780882).

[0055] Growing evidence indicates that crosstalk between hematopoietic cells and niches initiates and maintains chronic inflammation within the bone marrow (BM), but their precise contribution in this process remains unclear. (Kovtonyuk,LV,et al.Inflamm-Aging of Hematopoiesis,Hematopoietic Stem Cells,and the Bone Marrow Microenvironment.Frontiers in immunology(2016)7,502,doi:10.3389 / fimmu.2016.0050);Pietras,EMet al.Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal.Nature cell biology(2016)18,607-618,doi:10.1038 / ncb3346;Lussana, F. & Rambaldi, A.Inflammation and myeloproliferative neoplasms.Journal of autoimmunity(2017)85,58-63,doi:10.1016 / j.jaut.2017.06.010;Pietras,EMInflammation: a key regulator of hematopoietic stem cell fate in health and disease.Blood(2017)130,1693-1698,doi:10.1182 / blood-2017-06-780882).

[0056] Within the BM microenvironment, endothelial cells (ECs) are established as essential components of the perivascular niche that support HSCs, as indicated by their expression of a diverse array of HSC-regulating paracrine factors (Hooper, A. T. et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell stem cell (2009) 4, 263-274, doi:10.1016 / j.stem.2009.01.006; Butler, J. Met al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell stem cell (2010) 6, 251-264, doi:10.1016 / j.stem.2010.02.001; Kobayashi, H. et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells.Nature cell biology(2010)12,1046-1056,doi:10.1038 / ncb2108;Winkler,IGet al.Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance.Nature medicine(2012)18,1651-1657,doi:10.1038 / nm.2969;Ding,L.,et al.,Endothelial and perivascular cells maintain haematopoietic stem cells.Nature(2012)481,457-462,doi:10.1038 / nature10783;Poulos,M.G.et al.Endothelial jagged-1 is necessary for homeostatic and regenerative 947 hematopoiesis.Cell reports(2013)4,1022-1034,doi:10.1016 / j.celrep.2013.07.048;Greenbaum,A.et al.CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance.Nature(2013)495,227-230,doi:10.1038 / nature11926;Doan,P.L.et al.Epidermal growth factor regulates hematopoietic regeneration after radiation injury.Nature medicine(2013)19,295-304,doi:10.1038 / nm.3070;Poulos,M.G.et al.Endothelial-specific inhibition of NF-kappaB enhances functional haematopoiesis.Nat Commun(2016)7,13829,doi:10.1038 / ncomms13829;Kusumbe,A.P.et al.Age-dependent modulation of vascular niches for haematopoietic stem cells.Nature(2016)532,380-384,doi:10.1038 / nature17638;Morrison,S.J.& Scadden,D.T.The bone marrow niche for haematopoietic stem cells.Nature(2014)505,327-334,doi:10.1038 / nature12984;Rafii,S.,Butler,J.M.& Ding,B.S.Angiocrine functions of organ-specific endothelial cells.Nature(2016)529,316-325,doi:10.1038 / nature17040). Modulation of signaling pathways within the endothelium has also been shown to directly influence niche activity, thereby regulating HSC self-renewal and lineage commitment decisions (Kobayashi, H. et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of hematopoietic stem cells. Nature cell biology (2010) 12, 1046-1056, doi:10.1038 / ncb2108 (2010); Poulos, M. Get al. Endothelial-specific inhibition of NF-kappaB enhances functional hematopoietic stem cell differentiation. Nat Commun (2016) 7, 13829, doi:10.1038 / ncomms13829; Kusumbe, AP et al. Age-dependent modulation of vascular niches for hematopoietic stem cells cells. Nature (2016) 532, 380-384, doi:10.1038 / nature17638). In addition to functioning as tissue-specific niche cells, the endothelium is a key determinant of chronic inflammation (Rafii, S., Butler, JM & Ding, BS Angiocrine functions of organ-specific endothelial cells. Nature (2016) 529, 316-325, doi:10.1038 / nature17040; Pober, JS & Sessa, WC Evolving functions of endothelial cells in inflammation. Nature reviews. Immunology (2007) 7, 803-815, doi:10.1038 / nri2171) and appears as an important source of niche-derived inflammatory signals within the BM, including IL-1 and G-CSF, which promote myelopoiesis in response to acute demand (Pietras, EM et al.Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal.Nature cell biology(2016)18,607-618,doi:10.1038 / ncb3346;Boettcher,S.et al.Endothelial cells translate pathogen signals into G-CSF-driven emergency granulopoiesis.Blood(2014)124,1393-1403,doi:10.1182 / blood-2014-04-570762). Persistent endothelial inflammation has been implicated in the initiation of myeloproliferative disorders through the expression of G-CSF and TNFα (Wang, L. et al. Notch-dependent repression of miR-155 in the bone marrow niche regulates hematopoiesis in an NF-kappaB-dependent manner. Cell stem cell (2014) 15, 51-65, doi:10.1016 / j.stem.2014.04.021). However, the signaling pathways mediating chronic endothelial inflammation within the BM microenvironment that affect niche activity and HSC function remain poorly understood.

[0057] NF-κB and MAPK are major signaling pathways regulating chronic inflammatory responses in endothelial cells (Pober, J.S. & Sessa, W.C. Evolving functions of endothelial cells in inflammation. Nature reviews. Immunology (2007) 7, 803-815, doi:10.1038 / nri2171). However, their role in regulating inflammation within the BM endothelial niche and the accompanying effects on HSC function remains unexplored. Previous studies have shown that suppression of NF-κB signaling within the endothelium promotes steady-state hematopoiesis and regeneration after myelosuppression, in part by reducing proinflammatory cytokines (Poulos, M.G. et al. Endothelial-specific inhibition of NF-kappaB enhances functional hematopoiesis. Nat Commun (2016) 7, 13829, doi:10.1038 / ncomms13829). Recent reports suggest that endothelial MAPKs play an important role in inflammatory processes, including LPS-induced granulopoiesis and chronic vascular inflammation associated with atherosclerosis (Sanchez, A. et al. Map3k8 controls granulocyte colony-stimulating factor production and neutrophil precursor proliferation in lipopolysaccharide-induced emergency granulopoiesis. Sci Rep (2017) 7, 5010, doi:10.1038 / s41598-017-04538-3; Roth Flach, RJ et al. Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis. Nat Commun (2015) 6, 8995, doi:10.1038 / ncomms9995).Using an ex vivo niche model system, it has been demonstrated that endothelial MAPK activation leads to myeloid-biased differentiation of co-cultured HSCs at the expense of self-renewal, a feature suggestive of inflammatory stress (Kobayashi, H. et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of hematopoietic stem cells. Nature cell biology (2010) 12, 1046-1056, doi:10.1038 / ncb2108).

[0058] Myeloablative therapy In patients requiring hematopoietic reconstitution, a preparative or conditioning regimen is administered as part of the procedure to achieve two goals: providing sufficient immunoablation to prevent host rejection and providing tumor cytoreduction / disease eradication. Conditioning regimens have many variations, as intensity can vary based on disease-related factors such as diagnosis and remission status, as well as patient-related factors such as age, donor availability, and the presence of comorbid conditions. Conditioning regimens have been classified as high-dose (myeloablative), reduced-intensity, and non-myeloablative. (Gyurkocza, Boglarka, and Brenda M. Sandmaier. "Conditioning regimens for hematopoietic cell transplantation: one size does not fit all." Blood (2014) vol. 124, 3:344-53). Myeloablative therapy (MBT) refers to the treatment of patients with high-dose chemotherapy (HDC) or total-body irradiation (TBI) to eradicate the immune and hematopoietic systems and all malignant cells in the body. Typically, patients who undergo MBT do so as preparation for bone marrow transplant, stem cell transplant, or hematopoietic cell transplant (referred to herein as "stem cell rescue" or "SCR"); however, as shown in Table 3 below, MBT can also be used as a type of treatment for various types of malignancies for which SCR has not been shown to be beneficial. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79). [Table 2-1] [Table 2-2]

[0059] Generally, MBT regimens consist of HDC with alkylating agents (single or multiple agents) and are administered regardless of whether or not TBI has occurred. Such regimens are expected to ablate bone marrow hematopoiesis and therefore not allow for autologous hematologic recovery. (Gyurkocza, Boglarka, and Brenda M. Sandmaier. “Conditioning regimens for hematopoietic cell transplantation: one size does not fit all.” Blood (2014) vol. 124, 3:344-53) Examples of specific MBT regimens are shown in Table 4 below and can be reproduced in part in: Atilla, E., Ataca Atilla, P., & Demirer, T. A. Review of Myeloablative vs. Reduced-Intensity / Non-Myeloablative Regimens in Allogeneic Hematopoietic Stem Cell Transplantations. Balkan Medical Journal, (2017) 34(1), 1-9. [Table 3-1]

[0060] Total-body irradiation (TBI). TBI and high-dose TBI are widely used as part of conditioning regimens due to their immunosuppressive properties, efficacy against most leukemias and lymphomas, and ability to penetrate protected areas. The majority of regimens combine 12–16 Gy of TBI, usually fractionated (meaning the total dose of radiation is divided into several smaller doses over several days), with other chemotherapeutic agents, most commonly cyclophosphamide, based on their antineoplastic and immunomodulatory properties. Generally, high-dose TBI reduces the risk of recurrence but results in increased, often fatal, gastrointestinal, hepatic, and pulmonary toxicity, secondary malignancies, and impaired growth and development in children. In addition to the radiation dose, other factors such as dose rate, fractionation, fraction interval, and radiation source (e.g., cobalt-60 vs. linear accelerator) can also affect both the antitumor and toxic effects of TBI. Fractionation resulted in reduced organ toxicity due to a higher proportion of intact repair mechanisms retained in normal tissues, as opposed to leukemia cells, while maintaining sustained antitumor effects. Hyperfractionation (multiple fractions per day) with lung shielding reduced the incidence of interstitial pneumonia by 4%, down from the 50% observed with single TBI without lung shielding. Most currently used TBI schedules are either fractionated or hyperfractionated. In addition to cyclophosphamide, various agents, such as cytarabine (AraC), etoposide, melphalan, and busulfan, have been combined with high-dose TBI as conditioning regimens. (Gyurkocza, Boglarka, and Brenda M. Sandmaier. “Conditioning regimens for hematopoietic cell transplantation: one size does not fit all.” Blood (2014) vol. 124, 3:344-53).

[0061] The administration of high-dose TBI is associated with immediate and delayed toxicities, although it is not always possible to distinguish which components of the conditioning regimen are responsible for any given toxicity. Nausea, vomiting, transient acute parotitis, xerostomia, mucositis, and diarrhea are commonly observed acute complications. Interstitial pneumonia, idiopathic pulmonary fibrosis, and decreased pulmonary function may also be associated with high-dose TBI. Furthermore, renal damage may occur after high-dose TBI and may be delayed (i.e., up to approximately 2 years). The development of sinusoidal obstruction syndrome (SOS; formerly known as hepatic veno-occlusive disease) is more common with chemotherapy-based regimens, as described below. Long-term side effects of high-dose TBI include infertility, cataract formation, hyperthyroidism and thyroiditis, and secondary malignancies. (Gyurkocza, Boglarka, and Brenda M Sandmaier. “Conditioning regimens for hematopoietic cell transplantation: one size does not fit all.” Blood (2014) vol. 124, 3: 344-53).

[0062] High-Dose Chemotherapy (HDC). A key component of HDC is the delivery of alkylating agents due to their favorable toxicity profile (myelotoxicity as the dose-limiting toxicity) and their effectiveness against non-dividing tumors or malignant cells. Other agents that can be used include anthracyclines and taxanes. To avoid the short- and long-term toxicities associated with high-dose TBI, particularly in patients who have previously received radiation therapy, high-dose chemotherapy-based regimens have been developed in both autologous and allogeneic settings, where TBI is replaced by additional chemotherapy. Alkalyzing agents are often delivered with immunosuppressants; treatments may include busulfan, cyclophosphamide, or fludarabine, melphalan, thiotepa, etoposide, and treosulfan, as well as combinations of such therapies. (Gyurkocza, Boglarka, and Brenda M Sandmaier. “Conditioning regimens for hematopoietic cell transplantation: one size does not fit all.” Blood (2014) vol. 124, 3: 344-53).

[0063] Morphological Effects of MBT. The morphological characteristics of the bone marrow in patients undergoing MBT are determined by the overlapping processes of cell death and hematopoietic reconstitution. Aggressive chemotherapy, alone or in combination with TBI, results in the elimination of nearly all hematopoietic and immune cells within a few days. At the end of this period, the bone marrow is highly hypocellular, and the intact stroma contains a homogeneous periodic acid-Schiff (PAS)-positive proteinaceous transudate resembling fibrinoid necrosis. Minimal residual plasma cells and macrophages are usually present, and vascular congestion, areas of nonspecific hemorrhage, small noncaseating granulomas, interstitial edema, eosinophilia, mild reticulin fibrosis, sinus dilation, osteonecrosis, and other abnormalities may be seen. With or without stem cell rescue, recovery of normal levels of red blood cells, platelets, and granulocytes after MBT requires a period of several weeks, but complete functional reconstitution of the hematopoietic system occurs over several years. Furthermore, despite relatively rapid recovery of peripheral blood cell counts after myeloablative chemotherapy or bone marrow transplantation, severe cellular and humoral immunodeficiency persists for months to years (Gyurkocza, Boglarka, and Brenda M Sandmaier. “Conditioning regimens for hematopoietic cell transplantation: one size does not fit all.” Blood vol. 124, 3 (2014): 344-53).

[0064] Myeloablative and conditioning regimens that purge the bone marrow of malignant progenitor cells may also remove or damage nonmalignant hematopoietic and stromal progenitor cells, resulting in a reduced capacity for transplantation and natural stem cell regeneration. This defect is not always evident from examination of posttransplant peripheral blood smears or bone marrow biopsies. Although peripheral blood cell counts and bone marrow cellularity can reach pretransplant levels, severe, long-lasting defects in erythroid and megakaryocytic bone marrow progenitors can persist for years after bone marrow transplantation. Colony-forming units-fibroblasts (CFU-f), the progenitor stromal compartment of cells of the osteogenic lineage, are critical for hematopoietic cell survival, proliferation, and differentiation. Reconstitution of CFU-f can take as long as 12 years to reach pretransplant numbers and is solely of host origin.

[0065] Stem Cell Rescue (SCR) Therapy Stem cell rescue (or rescue transplant) is a method of replacing hematopoietic stem cells destroyed by treatment with high doses of anticancer drugs or radiation therapy. This is usually done using the patient's own stem cells that were stored before treatment. The stem cells help the bone marrow recover and produce healthy blood cells. Stem cell rescue may also allow more chemotherapy or radiation therapy to be given so that more cancer cells are killed.

[0066] Typically, after MBT, patients will receive an infusion of hematopoietic stem cells isolated from either bone marrow or peripheral blood with the goal of curing systemic malignancies, inherited metabolic disorders, or potentially fatal diseases of the hematopoietic or immune systems. The underlying reason for bone marrow transplantation in patients with bone marrow failure, malignancies, and congenital hematopoietic and immunodeficiency conditions is to provide normal stem cells for bone marrow repopulation after removal of diseased bone marrow. Regeneration of new bone marrow ("bone marrow reconstitution") occurs during recovery from myeloablative therapy from stem cell progenitors or, less frequently, from residual host progenitor cells. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79)

[0067] Morphological Features of Stem Cell Rescue. Immediately after chemotherapy or in the post-transplant period, marked bone marrow aplasia (meaning incomplete, delayed, or defective development) usually follows for 1–2 weeks. Adipocyte regeneration provides the first morphological evidence of bone marrow regeneration, followed by the appearance of minute clusters of immature monotypic hematopoietic cells that gradually mature and expand on days 6–14. These colonies are composed of cells of a single hematopoietic lineage ("monolineage"), usually myeloid or erythroid, and likely arise from committed stem cells in bone marrow transplant patients. Regenerating colonies tend to be paratrabecular in patients receiving bone marrow transplant alone and interstitial after stem cell transplant. Very early hematopoiesis after transplantation is usually polyclonal but may be monoclonal. Early erythropoietic islands are usually affected by large basophilic normoblasts, which may exhibit features of erythroid dysplasia. As hematopoietic reconstitution continues, the distribution of hematopoietic cells in the bone marrow is often atypical, with clusters of myeloid precursors aberrantly localized in the intertrabecular regions and erythroid precursors occurring near the endosteum. Megakaryocytes are usually the last to engraft. They are usually localized in the central portion of the intertrabecular regions and may appear in clusters rather than the usual scattered distribution. Macrophages, pseudo-Gaucher cells, and sheets of regenerating promyelocytes may also appear. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone-Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79)

[0068] Gradual recovery to normal bone marrow cellularity is accompanied by resolution of edema, reticulin fibrosis (representing increased reticulin staining), and fibrinoid necrosis (representing a type of necrosis occurring in the walls of small arteries or arteries that is pink and resembles fibrin (hence fibrinoid); it represents actual death or necrosis of the cell wall). The bone marrow should be approximately 50% normal by 3 weeks post-transplant and normal by 8–12 weeks. The time course of progression from early hematopoiesis to normal bone marrow cellularity is highly variable; some patients may achieve normal cellularity in as little as 14 days, whereas others require several months. However, 28 days is typical. Engraftment kinetics depend on the source of donor cells (e.g., peripheral blood stem cells, umbilical cord blood, bone marrow), the dose of CD34+ cells infused, the type and dose of exogenous hematopoietic growth factors (i.e., G-CSF, rhGM-CSF, erythropoietin), and HLA cross-matching. Bone marrow recovery rates are affected by the homing efficiency and clonogenic potential of the transplanted cells, as well as whether the infused cells are expanded in vitro prior to infusion (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79).

[0069] In peripheral blood, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce increases in total white blood cell counts and absolute numbers of neutrophils, monocytes, and eosinophils. Their effects on bone marrow include eosinophil hyperplasia and increases in cellularity and myeloid:erythroid (M:E) ratios. Furthermore, prominently granulated and / or vacuolated neutrophils and neutrophil precursors appear in both peripheral blood and bone marrow. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79)

[0070] Hematological effects of MBT and SCR Stem cell rescue via bone marrow transplantation is often compared to solid organ transplantation, but it is unique in several respects. Because bone marrow is a liquid "organ," compatible organ size, bile duct and ureteral obstruction, and other surgical problems are not encountered. Because bone marrow is rapidly replenished by healthy individuals, cadaveric organs are not required, and living donors do not sustain permanent organ dysfunction. Patients can donate their own bone marrow for subsequent transfusion (autologous SCR). However, transplant recipients who receive bone marrow donations from another individual (allogeneic SCR) face the problem of graft rejection, also known as graft-versus-host disease (GVHD). Furthermore, bone marrow transplant recipients are highly immunosuppressed until bone marrow reconstitution occurs, making them highly susceptible to opportunistic infections and other disorders during this period. Therefore, SCR via bone marrow transplantation is very costly and carries significant morbidity and mortality. Therefore, indications for this procedure are limited, and potential recipients undergo a thorough and potentially lengthy screening process. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79). The following table provides a brief summary of adverse hematologic outcomes of MBT and / or SCR. [Table 4]

[0071] Graft failure, acute graft rejection, and delayed engraftment Many factors can cause poor outcomes after SCR with bone marrow transplantation ("graft failure") or loss of recently engrafted bone marrow tissue ("graft rejection"). Initial engraftment failure (primary graft failure) is generally due to genetic differences between the donor and recipient, damaged or insufficient stem cells, inadequate immunosuppression or pretransplant conditioning, prior alloimmunization with multiple blood transfusions, excessive T-cell depletion of the graft material, an abnormal microenvironment in the host bone marrow, abnormal donor bone marrow, drug toxicity, or viral infection. Post-engraftment graft failure (secondary graft failure) results from drug toxicity, infection, fibrosis, or cell-mediated immune responses. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79)

[0072] Immunologically mediated acute bone marrow graft rejection is particularly common in three situations: 1) patients with aplastic anemia who have been multiply transfused, 2) patients who have received bone marrow from major histocompatibility-mismatched donors, and 3) patients who have received T-cell-depleted bone marrow. The incidence of acute rejection is only about 1% in patients who receive non-immunologically engineered HLA-matched transplants from siblings, but increases to 8-15% in patients who receive T-cell-depleted phenotypically matched transplants.

[0073] Graft rejection is primarily caused by host T lymphocytes that survive pretransplant conditioning regimens, proliferate in the allografted bone marrow, and then suppress the growth of donor cells and initiate cell-mediated responses against donor targets. Subtly different mechanisms of rejection may be involved within different patient populations. This is because suppressor T lymphocytes (CD3+CD8+CD57+) predominate in HLA-matched siblings undergoing allograft rejection, whereas cytotoxic T lymphocytes (CD3+CD8+CD57-) are evident in rejecting bone marrow transplant patients receiving HLA-matched allografts from unrelated donors. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79)

[0074] Severe thrombocytopenia after primary platelet transplant recovery ("secondary failed platelet recovery" or "SFPR") is associated with serious complications, poor clinical outcomes, or even death. Thrombocytopenia occurs in as many as 20% of patients receiving allogeneic transplants, but the incidence is much lower (8%) in autologous transplants. Cytomegalovirus infection has been implicated by several investigators as a significant risk factor for the development of SFPR. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79)

[0075] Morphologically, bone marrow aspirate smears from patients with failed or delayed engraftment are markedly hypocellular, with a predominance of stromal cells, whereas core biopsies and clot sections often show a diffuse proliferation of histiocytes, stationary phagocytes present in connective tissue (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone-Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79).

[0076] Clinically, early graft failure (>50 days after transplantation) is manifested by host T lymphocytosis (CD3+, CD8+, DR+), whereas late graft failure (>50 days after transplantation) is associated with a syndrome of delayed granulocytic regeneration, fever of unknown origin, and abdominal complaints. Graft rejection often occurs after progressive lymphocytosis (increased lymphocyte count) and a sudden decline in absolute neutrophil count. The prognosis for persistently successful engraftment is poor if lymphocytosis occurs, but donor lymphocyte infusion has been successful in a small number of patients. Prophylactic therapy for high-risk patients is directed toward preconditioning regimens that include total body irradiation, total lymph node irradiation, or increased immunosuppressive agents. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79)

[0077] Minimal residual disease (MRD) MRD is the persistence of leukemic cells in the bone marrow after remission induction therapy (meaning initial treatment with anticancer drugs) below the limit of detection by conventional morphological assessment. These residual leukemic cells are thought to be a potential cause of disease relapse in many patients who achieve "complete" morphological remission from various forms of leukemia, resulting in residual or recurrent bone marrow disease. The sensitivity of detecting MRD at clinically relevant levels has not been established, nor has additional therapy that eradicates very few residual cells been shown to improve survival in patients in clinical and morphological remission. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone-Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79)

[0078] Graft-versus-host disease (GVHD) GVHD is a major cause of morbidity and mortality after allogeneic bone marrow transplantation. It occurs in approximately 50% of cases of histocompatible bone marrow transfusion and in nearly all cases of bone marrow transplantation using HLA-mismatched bone marrow. In moderate to severe cases, GVHD has a significant mortality rate (40-80%). GVHD is a complex immunological phenomenon that is poorly understood, but it is usually a T-cell-mediated process that occurs in the setting of an imbalance in lymphocyte subsets, alloantigen presentation, and aberrant production or increased responsiveness to cytokines. Both acute and chronic forms of GVHD are recognized.

[0079] Acute GVHD (aGVHD) tracks lymphocyte reactivity to mismatched "minor" histocompatibility antigens in the skin, gastrointestinal tract, and liver. The increased likelihood of GVHD is associated with donor-host HLA disparity, increasing donor and host age, donor allosensitization, donor-recipient gender mismatch, increased intensity of the preparative regimen, and donor T-cell dose. Clinical manifestations range from mild skin rash, gastrointestinal (GI) disorders (nausea, vomiting, diarrhea), and liver function test abnormalities to life-threatening disease with skin breakdown, liver failure, bloody diarrhea, and severe immunosuppression. Approximately 5–10% of bone marrow transfusion patients die from GVHD. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47–79)

[0080] Chronic GVHD (cGVHD) can follow an acute process or develop de novo. It occurs in 25–65% of bone marrow transplant recipients. Platelet count is a predictor of survival; a platelet count below 100,000 / mL is associated with an overall mortality rate of over 50%. cGVHD is thought to represent an immune dysregulation state characterized by autoimmune phenomena, and the clinical picture resembles autoimmune diseases involving the skin, GI tract, and liver. Circulating autoantibodies are present, and complement and immunoglobulin deposits have been identified at the dermal-epidermal junction. Risk factors for cGVHD include previous aGVHD, older donor or recipient age, HLA mismatch, use of unrelated donors, viral infection, splenectomy, donor lymphocyte infusion (DLI), and the use of peripheral blood stem cells to treat cGVHD. (Riley, et al., “Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation.”Journal of Clinical Laboratory Analysis (2005) 19:47-79).

[0081] Myelofibrosis While mild, transient reticulin fibrosis is not uncommon after chemotherapy, severe collagen fibrosis is characteristic of chronic myeloid leukemia. Myelofibrosis is characterized by increased reticulin fiber density and, in severe cases, an increased number of CD61+ megakaryocytopoietic cells, an increased number of CD68+ macrophages, a decreased number of erythroid precursors, and an increased platelet count. While there is usually an initial regression of myelofibrosis after transplantation, it often recurs in areas of hematopoietic regeneration and is associated with the presence of atypical dwarf megakaryocytes, severe acute GVHD, and a significant delay in achieving transfusion independence. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone-Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79)

[0082] Treatment-related acute leukemia Therapy-related acute myeloid leukemia (t-AML) is a form of secondary leukemia resulting from cytotoxic chemotherapy and / or radiation therapy. The incidence of t-AML after high-dose chemotherapy for a previous malignancy has gradually increased, and t-AML is one of the most common secondary malignancies in both pediatric and adult populations. Polymorphisms or homozygous genetic defects in glutathione S-transferases P1, M1, and T1 may contribute to the increased incidence of t-AML due to insufficient detoxification of chemotherapy drugs. Patients treated for Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), myeloma, polycythemia vera, breast cancer, ovarian cancer, testicular cancer, or de novo acute lymphoblastic leukemia (ALL) are at highest risk for developing t-AML, with more than 50% of secondary AML patients also having breast cancer, NHL, and Hodgkin lymphoma. As shown, in contrast to t-AML, the occurrence of therapy-related ALL is rare, and the indications for the use of previous drugs such as those used in MBT are limited (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79).

[0083] Post-transplant lymphoproliferative disorder (PTLD) PTLD is a lymphoid neoplasm that develops as a result of immunosuppressive therapy in patients who have received bone marrow or solid organ transplants. Posttransplant lymphoproliferative disorders range from benign to malignant monoclonal or polyclonal lymphoproliferation. They occur in approximately 2% of solid organ transplant recipients, approximately 1% of autologous bone marrow transplant recipients, and up to 20% of patients with multiple risk factors, including HLA-mismatched allogeneic bone marrow transplants and immunosuppressive therapy for GVHD, such as anti-CD3 monoclonal OKT3, cyclosporine A, and FK506. Epstein-Barr virus, either primary or reactivated, is strongly associated with the development of PTLD. Impaired immune surveillance, chronic antigen stimulation from the allograft, and the oncogenic effects of immunosuppressive therapy are additional factors that contribute to PTLD. In contrast to the typical extranodal disease in solid organ transplant recipients with PTLD, bone marrow allograft recipients with PTLD often have widespread disease, involving both nodal and extranodal sites. (Riley, et al., “Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation.”Journal of Clinical Laboratory Analysis (2005) 19:47-79).

[0084] Toxic myelopathy Toxic myelopathy is a rare bone marrow lesion caused by toxic damage to the stromal and mesenchymal components of the bone marrow. Persistent cytopenias are the clinical hallmark of toxic myelopathy; the bone marrow is hypocellular with prominent interstitial damage, including edema, perivascular plasmacytosis, neutrophilic granulocytic necrosis, and cellular debris. Toxic myelopathy occurs in less than 1% of patients receiving chemotherapy or radiation therapy. (Riley, et al., "Hematologic Aspects of Myeloablative Therapy and Bone Marrow Transplantation." Journal of Clinical Laboratory Analysis (2005) 19:47-79).

[0085] Many, if not all, of these conditions result from incomplete removal of disease / malignant cells via inadequate MBT therapy, inadequate stem cell rescue, the use of immunosuppressants typically required after MBT, or damage to the bone marrow environment. The prevention of successful SCR and / or related conditions may be associated with the hematopoietic system in patients undergoing MBT. It is hypothesized that chronic inflammation within the tissue-specific microenvironment impairs the ability of supportive niche cells to properly nurture their cognate stem cells, thereby preventing SCR and hematopoietic reconstitution. (Wagers,AJThe stem cell niche in regenerative medicine.Cell stem cell(2012)10,362-369,doi:10.1016 / j.stem.2012.02.018;Lane,SW,Williams,DA& Watt,FMModulating the stem cell niche for tissue regeneration.Nature biotechnology(2014)32,795-803,doi:10.1038 / nbt.2978;Schepers, K., Campbell, TB & Passegue, E.Normal and leukemic stem cell niches:insights and therapeutic opportunities.Cell stem cell(2015)16,254-267,doi:10.1016 / j.stem.2015.02.014 911).

[0086] Recovery from bone marrow suppression. Immune reconstitution follows a general pattern of progression from immature to mature immune function. (Carson K. et al.,Chapter 35-Reimmunization after stem cell transplantation,”in Hematopoietic Stem Cell Transplantation in Clinical Practice(2009);(Butler,JMet al.Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells.Cell stem cell(2010)6,251-264,doi:10.1016 / j.stem.2010.02.001;Kobayashi,H.et al.Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells.Nature cell biology(2010)12,1046-1056,doi:10.1038 / ncb2108;Winkler,IGet al.Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance.Nature medicine(2012)18,1651-1657,doi:10.1038 / nm.2969;Ding,L.,et al.,Endothelial and perivascular cells maintain haematopoietic stem cells.Nature(2012)481,457-462,doi:10.1038 / nature10783;Poulos,MGet al.Endothelial jagged-1 is necessary for homeostatic and regenerative 947 hematopoiesis.Cell reports(2013)4,1022-1034,doi:10.1016 / j.celrep.2013.07.048;Greenbaum,A.et al.CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance.Nature(2013)495,227-230,doi:10.1038 / nature11926;Doan,PLet al.Epidermal growth factor regulates hematopoietic regeneration after radiation injury.Nature medicine(2013)19,295-304,doi:10.1038 / nm.3070)Immune reactivity during the first month post graft is extremely low.Id.Innate immunity is the first to regain function.Ogonek,J.et al.,“Immune reconstitution after allogeneic hematopoietic stem cell Hematopoietic lineage regeneration follows a reproducible sequence, with monocyte-like cells appearing first in peripheral blood, followed by granulocytes and then NK cells. NK cell recovery is significantly prioritized over T and B cells, both in terms of cell number and functional maturation (Grzywacz, B.et al, Natural Killer Cell differentiation by myeloid progenitors, Blood (2011) 117(13):3548-58). Cytotoxicity and phagocytic functions are restored by day 100, but more specialized functions of T and B lymphocytes may remain impaired for more than a year. After a period of time, the various components of the immune system of most healthy bone marrow recipients begin to function in a synchronized fashion, but the immune system of patients with chronic graft-versus-host disease (GvHD) remains suppressed. Delayed and incomplete immune reconstitution predisposes patients to infections, which are associated with high morbidity and mortality after allogeneic HCT.

[0087] It has long been known that hematopoietic regeneration and vascular regeneration in the bone marrow cavity after radiation exposure are temporally related, and that hematopoietic regeneration is not possible without bone marrow vascularization. It is now recognized that hematopoietic regeneration after myelosuppression by cytotoxic drugs or total body irradiation is interdependent on the bone marrow sinusoidal network and hematopoietic cell and megakaryocyte maturation. (Kopp, et al., "The Bone-Marrow Vascular Niche: Home of HSC Differentiation and Mobilization." PHYSIOLOGY 20:349-356, 2005; 10.1152 / physiol.00025.2005)

[0088] Bone marrow suppression leads not only to apoptosis of circulating hematopoietic cells but also to destruction of the bone marrow vasculature. The complex network of sinusoids, lacking regular vascular walls, is particularly susceptible to ionizing radiation and exhibits obvious ultrastructural signs of destruction, including necrosis, marked dilation, and leakage of plasma and blood cells. Bone marrow sinusoids appear to be supported by the adjacent hematopoietic cells themselves. Loss of this support means a loss of stability, which can lead to bleeding within the bone marrow cavity after radiation therapy or myelosuppressive chemotherapy. During hematopoietic regeneration, the sinusoids are remodeled. Therefore, the processes of hematopoiesis and angiogenesis are closely related. (Kopp, et al., "The Bone Marrow Vascular Niche: Home of HSC Differentiation and Mobilization." PHYSIOLOGY 20:349-356, 2005; 10.1152 / physiol.00025.2005)

[0089] The vasculature provides a protective niche for HSCs after chemotherapy, promoting bone and hematopoietic regeneration. Long-term quiescent HSCs are associated with both sinusoids and arteries. The vascular niche is essential for regenerating HSC populations after irradiation. Transplantation of bone marrow ECs after irradiation promotes hematopoiesis and protects radiosensitive tissues. Irradiated mice transplanted with bone marrow EC-conditioned medium showed increased survival, indicating that angiocrine factors can enhance survival but do not compensate for complete HSC loss. Endothelial-specific deletion of the Notch ligand JAG-1 leads to impaired HSC regeneration and increased mortality after irradiation. In addition to Notch signaling, ECs upregulate Fgf-2, Bmp4, Igfbp2, and angiopoietin-1 to promote hematopoietic stem and progenitor cell (HSPC) proliferation, indicating that these angiocrine factors may be useful for protecting HSCs after irradiation. (Sivan U, De Angelis J, Kusumbe AP.2019 Role of angiocrine signals in bone development, homeostasis and disease.Open Biol.9:190144.http: / / dx.doi.org / 10.1098 / rsob.190144).

[0090] The described invention provides a method of treating a subject exposed to a myeloablative insult, comprising administering to the subject a pharmaceutical composition containing a therapeutic amount of an angiocrine factor, which method is effective in maintaining vascular integrity, increasing bone marrow cellularity, increasing the likelihood of long-term engraftment, achieving multilineage reconstitution, suppressing vascular inflammation, and maintaining HSC function. Summary of the Invention

[0091] According to one aspect, the described invention provides a method for reducing vascular inflammation in a hematopoietic bone marrow microenvironment comprising bone marrow endothelial cells (BMECs), hematopoietic stem cells (HSCs), and bone marrow stromal cells following myelosuppressive insult, wherein reduced BMEC activity results in impaired steady-state hematopoiesis and HSC function, the method comprising administering to a subject a pharmaceutical composition comprising a recombinant or synthetic angiocrine factor and a pharmaceutically acceptable carrier; and enhancing hematopoietic recovery in the hematopoietic bone marrow microenvironment following myelosuppressive insult by one or more of: reducing inflammation in the hematopoietic microenvironment of the bone marrow; preserving vascular integrity in the hematopoietic microenvironment of the bone marrow; increasing the frequency and number of cell types in the hematopoietic compartment, including one or more of hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), multipotent progenitor cells (MPPs), and hematopoietic progenitor cell subsets, to effect multilineage reconstitution, wherein the vascular inflammation comprises one or more of increased vasodilation, decreased vascular integrity, including increased bone marrow vascular leakage, and increased levels of inflammatory mediators.

[0092] According to one embodiment of the method, the angiocrine factor is one or more recombinant or synthetic proteins selected from the group consisting of Clec11a, Hapln1, Hspd1, Igfbp1, Bgn, Wnt7a, Sparc, RP53, Bmpr1a, Ighm, Thbs4, Camk2d, Sirt2, Camk2b, Slitrk5, Dctpp1, Hnrnpa2b, and Erap1. According to another embodiment, the angiocrine factor is recombinant or synthetic Clec11α (stem cell growth factor). According to another embodiment, inflammation in the hematopoietic microenvironment of the bone marrow includes vascular inflammation, inflammation of BM stromal cells, and inflammation of hematopoietic cells. According to another embodiment, defective HSC function includes impaired HSC quiescence and increased HSC apoptosis. According to another embodiment, reducing vascular inflammation includes suppressing downstream NFkB signaling in BMECs within the bone marrow, downregulating target NFkB genes in bone marrow endothelial cells, or both. According to another embodiment, the myelosuppressive insult comprises exposure to radiation, chemotherapy, or both. According to another embodiment, the radiation is sublethal radiation, total body irradiation, or total lymph node irradiation. According to another embodiment, the myelosuppressive insult comprises chemotherapy. According to another embodiment, the myelosuppressive insult is myeloablative. According to another embodiment, the bone marrow (BM) microenvironment comprises BMECs, BM stromal cells, BM Lepr+ cells, and BM osteoblasts. According to another embodiment, the BMECs are sinusoidal and arteriolar BMECs. According to another embodiment, the immunophenotype of the BMECs is CD45-Ter119-CD31+VE-cadherin+. According to another embodiment, the immunophenotype of the BM stromal cells is CD45-Ter119-CD31-VE-cadherin-. According to another embodiment, the immunophenotype of the BM Lepr+ cells within the BM stromal population is CD45-Ter119-CD31-Lepr+. According to another embodiment, the immunophenotype of the mouse HSCs comprises lin-Ter119-CD11b-GR1-B220-CD3-CD41-ckit+SCA1+CD48-CD150+. According to another embodiment, the immunophenotype of the human HSCs comprises CD45RA-CD38-CD34+CD90+.According to another embodiment, reduced BMEC activity following myeloablative insult results in defects in steady-state hematopoiesis and HSC function.

[0093] According to another aspect, the described invention provides a method for improving hematopoietic homing, engraftment, reconstitution, and regeneration of bone marrow after myelosuppressive insult in a subject in need thereof, the method comprising: administering to the subject a pharmaceutical composition comprising a recombinant or synthetic angiocrine factor and a pharmaceutically acceptable carrier; administering a stem cell combination therapy comprising transplantation of a therapeutic amount of multipotent self-renewing hematopoietic stem cells (HSCs) effective to regenerate the blood system and promote hematopoietic reconstitution of the bone marrow; administering a vascular endothelial combination therapy comprising transplantation of a therapeutic amount of bone marrow endothelial cells (BMECs) effective to regenerate the blood system and promote hematopoietic reconstitution of the bone marrow; and promoting hematopoietic reconstitution of bone marrow within the hematopoietic bone marrow microenvironment comprising bone marrow endothelial cells (BMECs), hematopoietic stem cells (HSCs), and bone marrow stromal cells after myelosuppressive insult. and enhancing hematopoietic recovery in the hematopoietic bone marrow microenvironment after myelosuppressive insult by one or more of: reducing inflammation in the hematopoietic microenvironment of the bone marrow, maintaining vascular integrity in the hematopoietic microenvironment of the bone marrow, and increasing the frequency and number of cell types in the hematopoietic compartment, including one or more of hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), multipotent progenitor cells (MPPs), and hematopoietic progenitor cell subsets, to effect multilineage reconstitution, wherein the vascular inflammation comprises one or more of increased vasodilation, decreased vascular integrity, including increased bone marrow vascular leakage, and increased levels of inflammatory mediators.

[0094] According to one embodiment of the method, the angiocrine factor is one or more recombinant or synthetic proteins selected from the group consisting of Clec11a, Hapln1, Hspd1, Igfbp1, Bgn, Wnt7a, Sparc, RP53, Bmpr1a, Ighm, Thbs4, Camk2d, Sirt2, Camk2b, Slitrk5, Dctpp1, Hnrnpa2b, and Erap1. According to another embodiment, the angiocrine factor is recombinant or synthetic Clec11α (stem cell growth factor). According to another embodiment, the defective HSC function comprises impaired HSC quiescence and increased HSC apoptosis.

[0095] According to another embodiment, the stem cell combination therapy comprises isolating hematopoietic stem cells from a population of mononuclear cells isolated from a tissue source, enriching the isolated population of mononuclear cells for hematopoietic stem cells by positive or negative selection, and administering the enriched isolated population of hematopoietic stem cells to a subject.

[0096] According to another embodiment, the vascular endothelial cell combination therapy comprises isolating endothelial cells from human umbilical cord, enriching the isolated population for vascular endothelial cells by positive or negative selection, and administering the enriched isolated population of vascular endothelial cells to a subject.

[0097] According to another embodiment, the tissue source is autologous. According to another embodiment, the tissue source is allogeneic. According to another embodiment, reducing vascular inflammation comprises suppressing downstream NFkB signaling in BMECs within the bone marrow, downregulating target NFkB genes in bone marrow endothelial cells, or both. According to another embodiment, the myelosuppressive insult comprises exposure to radiation, chemotherapy, or both. According to another embodiment, the radiation is sublethal radiation, total body irradiation, or total lymph node irradiation. According to another embodiment, the myelosuppressive insult is chemotherapy. According to another embodiment, the myelosuppressive insult is myeloablative. According to another embodiment, the bone marrow (BM) microenvironment comprises BMECs, BM stromal cells, BM Lepr+ cells, and BM osteoblasts. According to another embodiment, the BMECs are sinusoidal and arteriolar BMECs. According to another embodiment, the BMECs are immunophenotyped as CD45-Ter119-CD31+VE-cadherin+. According to another embodiment, the immunophenotype of the BM stromal cells is CD45-Ter119-CD31-VE-cadherin-. According to another embodiment, the immunophenotype of the BM Lepr+ cells within the BM stromal population is CD45-Ter119-CD31-Lepr+. According to another embodiment, the immunophenotype of the mouse HSCs comprises lin-Ter119-CD11b-GR1-B220-CD3-CD41-ckit+SCA1+CD48-CD150+. According to another embodiment, the immunophenotype of the human HSCs comprises CD45RA-CD38-CD34+CD90+. According to another embodiment, the method enhances stable long-term engraftment of bone marrow, reduces myeloid bias in peripheral blood, or both. According to another embodiment, the pharmaceutical composition is administered before, after, or simultaneously with administration of stem cell combination therapy. According to another embodiment, inflammation in the hematopoietic microenvironment of the bone marrow comprises vascular inflammation, inflammation of BM stromal cells, and inflammation of hematopoietic cells.

[0098] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0099] [Figure 1A] Figure 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Total cell number per femur (n=5 mice / cohort). [Figure 1B] Figure 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Frequency of phenotypic HSCs per 10 femoral cells assessed by flow cytometry (n=5 mice / cohort). [Figure 1C] 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Representative contour plots showing the gating strategy for quantification of BM HSC and HSPC frequencies by flow cytometry. [Figure 1D] Figure 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Frequency of phenotypic HSPCs per 10 femoral cells assessed by flow cytometry (n=5 mice / cohort). [Figure 1E] Figure 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Methylcellulose-based progenitor cell assay. Bar graph shows the number of CFU per 105 whole bone marrow (WBM) (n=3 mice / cohort). [Figure 1F] Figure 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Competitive repopulation assays assessing total CD45.2+ cell engraftment and CD45.2+ lineage distribution over 4 months are shown (n = 10 recipients / cohort; n = 5 donors / cohort). To assess competitive repopulation, 5 × 105 donor WBM cells (CD45.2) were transplanted into preconditioned CD45.1 recipient mice along with 5 × 105 competitor WBM cells (CD45.1). [Figure 1G]Figure 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Competitive repopulation assays assessing total CD45.2+ cell engraftment and CD45.2+ lineage distribution over 4 months are shown (n = 10 recipients / cohort; n = 5 donors / cohort). To assess competitive repopulation, 5 × 105 donor WBM cells (CD45.2) were transplanted into preconditioned CD45.1 recipient mice along with 5 × 105 competitor WBM cells (CD45.1). [Figure 1H] 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Table 1 shows the number of recipients who were positive for long-term multiple lineage reconstitution (LTMR) after whole bone marrow limiting dilution transplantation assay (n=10 recipients / cohort; n=5 donors / cohort per dose of cells). [Figure 1I]

[0023] Figure 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Figure 2 shows line graphs displaying estimates of HSC frequency for genotypes indicated with dashed lines representing 95% confidence intervals. Extreme Limiting Dilution Analysis (ELDA) was used to determine stem cell frequency and significance. [Figure 1J] 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Cell cycle analysis of HSCs of the indicated genotypes by flow cytometry is shown (n=5 mice / cohort). [Figure 1K] Figure 1 shows that CDH5-MAPK mice exhibit impaired HSC and hematopoiesis. Quantification of HSC apoptosis in the indicated genotypes by flow cytometry is shown (n=5 mice / cohort). Error bars represent sample mean ± SEM. Statistical significance was determined using a two-tailed, unpaired Student's t-test (*P<0.05; **P<0.01; and ***P<0.001). [Figure 2A] CDH5-MAPK mice exhibit systemic and BM-localized inflammation. Representative immunofluorescence images of femurs labeled in vivo with vascular-specific CD144 / VE-cadherin antibody (red) show vasodilation in CDH5-MAPK mice. [Figure 2B]Figure 1 shows that CDH5-MAPK mice exhibit systemic and BM-localized inflammation. Quantification of Evan's Blue Dye (EBD) extravasation is shown (n=5 mice / cohort). [Figure 2C] 1 shows that CDH5-MAPK mice exhibit systemic and BM-localized inflammation. Representative images of femurs isolated from mice injected with EBD are shown. [Figure 2D] Figure 1 shows that CDH5-MAPK mice exhibit systemic and BM-localized inflammation. Microtiter plates showing the extracted EBD intensity for each sample are shown. Uninjected controls were used to determine the baseline. [Figure 2E] Figure 1 shows that CDH5-MAPK mice exhibit systemic and BM-localized inflammation. Figure 2 shows a heat map of 242 proteins differentially expressed in the plasma of CDH5-MAPK mice (n=7 control and n=8 CDH5-MAPK mice) identified by proteomic analysis. [Figure 2F] 1 shows that CDH5-MAPK mice exhibit systemic and BM-localized inflammation. 2 shows Ingenuity Pathway Analysis of differentially expressed proteins showing that inflammatory responses are overexpressed in CDH5-MAPK mice. [Figure 2G] Figure 1 shows that CDH5-MAPK mice exhibit systemic and BM-localized inflammation. Immunoblot analysis and quantification of bone marrow endothelial cells (BMECs) isolated from CDH5-MAPK mice (n=3 biological replicates per genotype) indicate that MEK1DD expression in BMECs leads to increased ERK1 / 2 and p65 phosphorylation, respectively. [Figure 2H] Figure 1 shows that CDH5-MAPK mice exhibit systemic and BM-localized inflammation. Immunoblot analysis and quantification of bone marrow endothelial cells (BMECs) isolated from CDH5-MAPK mice (n=3 biological replicates per genotype) indicate that MEK1DD expression in BMECs leads to increased ERK1 / 2 and p65 phosphorylation, respectively. [Figure 2I]Figure 1 shows that CDH5-MAPK mice exhibit systemic and BM-localized inflammation. Representative immunofluorescence images and quantification are shown demonstrating increased levels of nuclear p65 in BMECs derived from CDH5-MAPK mice compared to controls. Control BMECs treated with TNFα (10 ng / mL for 15 minutes) were used as a positive control for the assay. Each dot in the bar graph represents the nuclear p65 staining intensity per individual cell. Error bars represent the sample mean ± SEM. Statistical significance was determined using a two-tailed, unpaired Student's t-test (*P<0.05; **P<0.01; ***P<0.001). [Figure 2J] Figure 1 shows that CDH5-MAPK mice exhibit systemic and BM-localized inflammation. Representative immunofluorescence images and quantification are shown demonstrating increased levels of nuclear p65 in BMECs derived from CDH5-MAPK mice compared to controls. Control BMECs treated with TNFα (10 ng / mL for 15 minutes) were used as a positive control for the assay. Each dot in the bar graph represents the nuclear p65 staining intensity per individual cell. Error bars represent the sample mean ± SEM. Statistical significance was determined using a two-tailed, unpaired Student's t-test (*P<0.05; **P<0.01; ***P<0.001). [Figure 3A] Figure 1 shows that endothelial NF-κB inhibition resolves endothelial inflammation and restores vascular integrity in CDH5-MAPK mice. Immunoblot analysis and quantification are shown, respectively, demonstrating that expression of IkB-SS in BMECs isolated from CDH5-MAPK mice does not affect ERK1 / 2 or p65 phosphorylation. Black arrowheads represent endogenous IκBα, while red arrowheads represent the IkB-SS transgene. [Figure 3B] Figure 1 shows that endothelial NF-κB inhibition resolves endothelial inflammation and restores vascular integrity in CDH5-MAPK mice. Immunoblot analysis and quantification are shown, respectively, demonstrating that expression of IkB-SS in BMECs isolated from CDH5-MAPK mice does not affect ERK1 / 2 or p65 phosphorylation. Black arrowheads represent endogenous IκBα, while red arrowheads represent the IkB-SS transgene. [Figure 3C] Figure 1 shows that endothelial NF-κB inhibition resolves endothelial inflammation and restores vascular integrity in CDH5-MAPK mice. Representative immunofluorescence images and quantification are shown, respectively, demonstrating increased levels of nuclear p65 in BMECs derived from CDH5-MAPK mice compared to controls. It should be noted that expression of IkB-SS in BMECs isolated from CDH5-MAPK mice (CDH5-MAPK::IkB) reduces nuclear p65 levels. Each dot in the bar graph represents the nuclear p65 staining intensity per individual cell. [Figure 3D] Figure 1 shows that endothelial NF-κB inhibition resolves endothelial inflammation and restores vascular integrity in CDH5-MAPK mice. Representative immunofluorescence images and quantification are shown, respectively, demonstrating increased levels of nuclear p65 in BMECs derived from CDH5-MAPK mice compared to controls. It should be noted that expression of IkB-SS in BMECs isolated from CDH5-MAPK mice (CDH5-MAPK::IkB) reduces nuclear p65 levels. Each dot in the bar graph represents the nuclear p65 staining intensity per individual cell. [Figure 3E] Figure 3 shows that endothelial NF-κB inhibition resolves endothelial inflammation and restores vascular integrity in CDH5-MAPK mice. Figure 3E shows a heat map and bar graph (Figure 3F) demonstrating increased expression of NF-κB-dependent inflammatory genes in BMECs from CDH5-MAPK mice. Crossing CDH5-MAPK with Tie2.IkB-SS mice (CDH5-MAPK::IkB) resulted in decreased expression of NF-κB-dependent target genes. Expression of the housekeeping gene Actb was used for normalization. Dendrograms represent unsupervised hierarchical clustering of the entire dataset (n = 3 mice / cohort). [Figure 3F]Figure 3 shows that endothelial NF-κB inhibition resolves endothelial inflammation and restores vascular integrity in CDH5-MAPK mice. Figure 3E shows a heat map and bar graph (Figure 3F) demonstrating increased expression of NF-κB-dependent inflammatory genes in BMECs from CDH5-MAPK mice. Crossing CDH5-MAPK with Tie2.IkB-SS mice (CDH5-MAPK::IkB) resulted in decreased expression of NF-κB-dependent target genes. Expression of the housekeeping gene Actb was used for normalization. Dendrograms represent unsupervised hierarchical clustering of the entire dataset (n = 3 mice / cohort). [Figure 3G] Endothelial NF-κB inhibition resolves endothelial inflammation and restores vascular integrity in CDH5-MAPK mice. Representative immunofluorescence images of femurs labeled in vivo with a vascular-specific CD144 / VE-cadherin antibody (red) are shown, demonstrating that suppression of NF-κB signaling in endothelial cells of CDH5-MAPK mice resolves vasodilation. Error bars represent sample mean ± SEM. One-way ANOVA for multiple comparisons and Tukey's correction was performed to determine significance (*P<0.05; **P<0.01; ***P<0.001). [Figure 4A] Endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. Total cell numbers per femur are shown (n = 7-10 mice / cohort). [Figure 4B] Endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. Phenotypic HSC frequencies per 10 femoral cells assessed by flow cytometry are shown (n = 7-10 mice / cohort). [Figure 4C] Figure 1 shows that endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. Results of a methylcellulose-based progenitor assay are shown. Bar graphs show the number of CFU per 10 WBM (n = 4 mice / cohort). [Figure 4D]Endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. Results of a competitive repopulation assay evaluating total CD45.2+ cell engraftment and CD45.2+ lineage distribution 4 months after transplantation are shown. To assess competitive repopulation, 5 × 105 donor WBM cells (CD45.2) were transplanted together with 5 × 105 competitor WBM cells (CD45.1) into preconditioned CD45.1 recipient mice (n = 9–10 recipients / cohort; n = 5 donors per cohort). [Figure 4E] Endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. Results of a competitive repopulation assay evaluating total CD45.2+ cell engraftment and CD45.2+ lineage distribution 4 months after transplantation are shown. To assess competitive repopulation, 5 × 105 donor WBM cells (CD45.2) were transplanted together with 5 × 105 competitor WBM cells (CD45.1) into preconditioned CD45.1 recipient mice (n = 9–10 recipients / cohort; n = 5 donors per cohort). [Figure 4F] Endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. Table depicts the number of recipients who were positive for long-term multilineage reconstitution (LTMR) after whole bone marrow limiting dilution transplantation assays (n = 10 recipients / cohort per cell dose; n = 5 donors per cohort). [Figure 4G] Figure 1 shows that endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. Log fraction plot of limiting dilution analysis showing that CDH5-MAPK::IkB mice exhibit increased frequencies of LTMR-capable HSCs compared to CDH5-MAPK mice. Dashed lines indicate 95% confidence intervals. Extreme Limiting Dilution Analysis (ELDA) was used to determine stem cell frequencies and significance. [Figure 4H]Endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. Representative whole-mount immunofluorescence images of femurs and quantification of HSC distance from blood vessels in mice in vivo labeled with a vascular-specific CD144 / VE-cadherin antibody (red) are shown, demonstrating that endothelial MAPK activation disrupts HSC interaction with the vascular niche (white), which is restored in CDH5-MAPK::IkB mice. CD48+ and Lineage+ cells (blue channel) are not shown to better visualize HSC interaction with the vasculature. The yellow arrowhead indicates a representative HSC (defined as Lineagenegative, CD48negative, CD150bright) located in close proximity to a sinusoidal vessel. The yellow asterisk indicates a megakaryocyte. Each dot in the bar graph represents the distance of an individual HSC from the nearest blood vessel (n = 3 mice / cohort). [Figure 4I] Endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. Representative whole-mount immunofluorescence images of femurs and quantification of HSC distance from blood vessels in mice in vivo labeled with a vascular-specific CD144 / VE-cadherin antibody (red) are shown, demonstrating that endothelial MAPK activation disrupts HSC interaction with the vascular niche (white), which is restored in CDH5-MAPK::IkB mice. CD48+ and Lineage+ cells (blue channel) are not shown to better visualize HSC interaction with the vasculature. The yellow arrowhead indicates a representative HSC (defined as Lineagenegative, CD48negative, CD150bright) located in close proximity to a sinusoidal vessel. The yellow asterisk indicates a megakaryocyte. Each dot in the bar graph represents the distance of an individual HSC from the nearest blood vessel (n = 3 mice / cohort). [Figure 4J]Endothelial NF-κB inhibition restores HSC activity in CDH5-MAPK mice. The time course of peripheral blood recovery after irradiation (650 rad) is shown (n = 6–10 mice per cohort). Significant asterisks indicate a comparison with CDH5-MAPK mice of the indicated genotype. Results demonstrate a myeloprotective effect in CDH5-MAPK::IkB mice, indistinguishable from Tie2.IkB-SS mice. Error bars represent sample mean ± SEM. One-way ANOVA with Tukey's correction for multiple comparisons was performed to determine significance. *P<0.05; **P<0.01; ***P<0.001. [Figure 5A] Endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. Total cell numbers per femur of the indicated hematopoietic progenitor cells, estimated by flow cytometry, are shown (n = 4–5 mice / cohort). [Figure 5B] Endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. Total cell numbers per femur of the indicated hematopoietic progenitor cells, estimated by flow cytometry, are shown (n = 4–5 mice / cohort). [Figure 5C] Endothelial NF-κB inhibition restores hematopoietic progenitor activity in CDH5-MAPK mice. Lineage composition of CD45+ cells in the BM is shown (n=4-5 mice / cohort). [Figure 5D] Endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. Steady-state peripheral blood counts are shown (n = 4–6 mice / cohort). [Figure 5E] Endothelial NF-κB inhibition restores hematopoietic progenitor activity in CDH5-MAPK mice. The lineage composition of CD45+ cells in peripheral blood is shown (n=5 mice / cohort) (Fig. 5F). [Figure 5F] Figure 1 shows that endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. HSPC frequencies in peripheral blood are shown (n=5 mice / cohort). [Figure 5G]Figure 1 shows that endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. Whole images of spleens of the indicated genotypes are shown. [Figure 5H] Figure 1 shows that endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. Splenic cellularity is shown for the indicated genotypes (n = 4-5 mice / cohort). [Figure 5I] Figure 1 shows that endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. The total number of hematopoietic progenitor cells per spleen is shown (n = 4–5 mice / cohort). [Figure 5J] Figure 1 shows that endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. The total number of hematopoietic progenitor cells per spleen is shown (n = 4–5 mice / cohort). [Figure 5K] Figure 1 shows that endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. The total number of hematopoietic progenitor cells per spleen is shown (n = 4–5 mice / cohort). [Figure 5L] Endothelial NF-κB inhibition restores hematopoietic progenitor cell activity in CDH5-MAPK mice. The lineage composition of CD45+ cells in the spleen is shown (n = 4–5 mice per cohort). Note that crossing CDH5-MAPK with Tie2.IkB-SS mice (CDH5-MAPK::IkB) restores hematopoietic and HSPC attributes of CDH5-MAPK mice to control levels. Error bars represent sample mean ± SEM. Significance was determined by one-way ANOVA with Tukey's correction for multiple comparisons (*P<0.05; **P<0.01; ***P<0.001). [Figure 6A] Figure 1 shows that CDH5-MAPK and Tie2.IkB-SS mice exhibit endothelial-specific expression of the transgene.Figure 2 is a schematic diagram illustrating the breeding strategy for generating CDH5-MAPK mice. [Figure 6B]Figure 1 shows that CDH5-MAPK and Tie2.IkB-SS mice exhibit endothelial-specific expression of the transgene. Figure 2 shows a schematic diagram illustrating the tamoxifen regimen prior to experimental analysis. Figure 3 shows representative flow cytometry contour plots showing GFP expression in total bone marrow cells from CDH5-MAPK mice. Numbers indicate the mean frequency of cells within the indicated quadrant as a percentage of total BM cells ± SEM (n = 4-5 mice / cohort). Note that GFP expression was detected exclusively in cells within the CD45- fraction of total bone marrow cells, indicating surface expression of an endothelial marker. [Figure 6C] Figure 1 shows that CDH5-MAPK and Tie2.IkB-SS mice exhibit endothelial-specific expression of the transgene. Analysis of phospho-ERK1 / 2 expression by flow cytometry confirms in vivo activation of the MAPK pathway in BMECs from CDH5-MAPK mice after tamoxifen administration (n=3 mice / cohort). [Figure 6D] Figure 1 shows that CDH5-MAPK and Tie2.IkB-SS mice exhibit endothelial-specific expression of the transgene. GFP+ BMECs compared with GFP- BMECs in CDH5-MAPK mice exhibit increased phospho-ERK1 / 2 expression by flow cytometry, confirming the fidelity of the GFP reporter for tracking cre-mediated recombination in vivo. (n=3 mice / cohort). [Figure 6E] Figure 1 shows that CDH5-MAPK and Tie2.IkB-SS mice exhibit endothelial-specific expression of the transgene. Endothelial cells in the BM of CDH5-MAPK mice (defined as CD45-Ter119-CD31+VE-cadherin+) but not stromal cells (defined as CD45-Ter119-CD31-VE-cadherin-) exhibit cre-mediated recombination (n=4 mice / cohort). [Figure 6F] 1 shows that CDH5-MAPK and Tie2.IkB-SS mice exhibit endothelial-specific expression of the transgene. 2 is a schematic diagram illustrating the Tie2.IkB-SS mouse model. [Figure 6G]CDH5-MAPK and Tie2.IkB-SS mice show endothelial-specific expression of the transgenes. Agarose gel electrophoresis images of RT-PCR amplicons for the indicated genes are shown using RNA isolated from FACS-sorted endothelial cells and CD45+ hematopoietic cells of the indicated genotypes (n = 3 mice / cohort). Note that the IkB-SS transgene is expressed in endothelial cells, with no detectable expression in hematopoietic cells. Also note that there is expression of the cre transgene in endothelial cells of CDH5-MAPK mice, but no detectable expression in hematopoietic cells. NTC indicates "no template control." Sorting purity was confirmed using expression of Cdh5 (for endothelial cells) and Ptprc (for hematopoietic cells). [Figure 7A] Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury of HSPCs and BM niche cells. Estimation of the oxygenation status of BM HSPCs based on quantification of Hypoxyprobe by flow cytometry (n=5 mice / cohort). [Figure 7B] Endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. Quantification of ROS levels in HSPCs by flow cytometry-based quantification of CellROX Orange is shown (n=5 mice / cohort). Note that CDH5-MAPK HSPCs exhibit increased hypoxia and ROS levels, which are resolved by crossing with Tie2.IkB-SS mice. [Figure 7C] Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury of HSPCs and BM niche cells. Cell cycle analysis of HSPCs based on Ki67-Hoechst staining by flow cytometry (n=5 mice / cohort). [Figure 7D]Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. Quantification of HSPC apoptosis by flow cytometry quantifying the percentage of cells in sub-G0 / G1 phase is shown (n=5 mice / cohort). HSPCs from CDH5-MAPK mice exhibit loss of quiescence and increased apoptosis, which is reversed upon suppression of endothelial NF-κB signaling. [Figure 7E] Endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. Quantification of hypoxia, ROS levels, quiescence, and apoptosis in the indicated BM niche cells by flow cytometry is shown (n=4-5 mice / cohort). [Figure 7F] Endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. Quantification of hypoxia, ROS levels, quiescence, and apoptosis in the indicated BM niche cells by flow cytometry is shown (n=4-5 mice / cohort). [Figure 7G] Endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. Quantification of hypoxia, ROS levels, quiescence, and apoptosis in the indicated BM niche cells by flow cytometry is shown (n=4-5 mice / cohort). [Figure 7H] Endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. Quantification of hypoxia, ROS levels, quiescence, and apoptosis in the indicated BM niche cells by flow cytometry is shown (n=4-5 mice / cohort). [Figure 7I] Endothelial NF-κB inhibition rescues hypoxic injury of HSPCs and BM niche cells. Flow cytometric estimation of BMECs and stromal cells per femur (n=4-5 mice / cohort). [Figure 7J]Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. Expression of pro-HSC paracrine factors in FACS-sorted BM niche cells by RT-qPCR (n=3 mice / cohort). Actb was used for normalization. [Figure 7K] Endothelial NF-κB inhibition rescues hypoxic injury of HSPCs and BM niche cells. Flow cytometric estimation of BMECs and stromal cells per femur (n=4-5 mice / cohort). [Figure 7L] Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. Expression of pro-HSC paracrine factors in FACS-sorted BM niche cells by RT-qPCR (n=3 mice / cohort). Actb was used for normalization. [Figure 7M] Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. Figure 2 shows the identification of commonly upregulated NF-κB target genes from qPCR array data in BM stromal cells, hematopoietic cells, and ECs from CDH5-MAPK mice using Venny. [Figure 7N] Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. RT-qPCR confirmation of Il1b and Csf1 expression in the indicated cell types is shown (n=3 mice / cohort). Actb was used for normalization. Note that inhibition of endothelial NF-κB signaling significantly suppressed Il1b and Csf1 expression in the whole bone marrow of CDH5-MAPK mice. Error bars represent sample mean ± SEM. One-way ANOVA for multiple comparisons and Tukey's correction was performed to determine significance. *P<0.05; **P<0.01; ***P<0.001. [Figure 7O]Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. RT-qPCR confirmation of Il1b and Csf1 expression in the indicated cell types is shown (n=3 mice / cohort). Actb was used for normalization. Note that inhibition of endothelial NF-κB signaling significantly suppressed Il1b and Csf1 expression in the whole bone marrow of CDH5-MAPK mice. Error bars represent sample mean ± SEM. One-way ANOVA for multiple comparisons and Tukey's correction was performed to determine significance. *P<0.05; **P<0.01; ***P<0.001. [Figure 7P] Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. RT-qPCR confirmation of Il1b and Csf1 expression in the indicated cell types is shown (n=3 mice / cohort). Actb was used for normalization. Note that inhibition of endothelial NF-κB signaling significantly suppressed Il1b and Csf1 expression in the whole bone marrow of CDH5-MAPK mice. Error bars represent sample mean ± SEM. One-way ANOVA for multiple comparisons and Tukey's correction was performed to determine significance. *P<0.05; **P<0.01; ***P<0.001. [Figure 7Q] Figure 1 shows that endothelial NF-κB inhibition rescues hypoxic injury in HSPCs and BM niche cells. RT-qPCR confirmation of Il1b and Csf1 expression in the indicated cell types is shown (n=3 mice / cohort). Actb was used for normalization. Note that inhibition of endothelial NF-κB signaling significantly suppressed Il1b and Csf1 expression in the whole bone marrow of CDH5-MAPK mice. Error bars represent sample mean ± SEM. One-way ANOVA for multiple comparisons and Tukey's correction was performed to determine significance. *P<0.05; **P<0.01; ***P<0.001. [Figure 8A] Figure 1 shows that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice. The frequency of phenotypic HSCs per 10 WBM is shown (n = 9-10 mice / cohort). [Figure 8B]Figure 1 shows that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice.Representative contour plots are shown showing that SCGF infusion rescues the phenotypic HSC defects observed in CDH5-MAPK mice. [Figure 8C] 1 shows that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice. The results of a methylcellulose-based progenitor cell assay are shown (n=5 mice / cohort). [Figure 8D] We demonstrate that SCGF infusion resolves hematopoietic and vascular defects in CDH5-MAPK mice. Results of a competitive repopulation assay assessing total CD45.2+ cell engraftment and CD45.2+ lineage distribution 4 months after transplantation are shown (n = 5 recipients / cohort; n = 5 donors / cohort). For the competitive repopulation assay, 5 x 10 donor WBM cells (CD45.2) were transplanted together with 5 x 10 competitor WBM cells (CD45.1) into preconditioned CD45.1 recipient mice. It should be noted that donor cells derived from CDH5-MAPK mice treated with SCGF demonstrated significantly increased engraftment efficiency, accompanied by abrogation of myeloid bias and an increase in lymphoid products. It should also be noted that SCGF did not significantly affect hematopoietic function or phenotype in control mice. [Figure 8E] We demonstrate that SCGF infusion resolves hematopoietic and vascular defects in CDH5-MAPK mice. Results of a competitive repopulation assay assessing total CD45.2+ cell engraftment and CD45.2+ lineage distribution 4 months after transplantation are shown (n = 5 recipients / cohort; n = 5 donors / cohort). For the competitive repopulation assay, 5 x 10 donor WBM cells (CD45.2) were transplanted together with 5 x 10 competitor WBM cells (CD45.1) into preconditioned CD45.1 recipient mice. It should be noted that donor cells derived from CDH5-MAPK mice treated with SCGF demonstrated significantly increased engraftment efficiency, accompanied by abrogation of myeloid bias and an increase in lymphoid products. It should also be noted that SCGF did not significantly affect hematopoietic function or phenotype in control mice. [Figure 8F]We show that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice. Secondary transplantation assays show that WBM cells from long-term engrafted primary recipients were isolated and transplanted into preconditioned CD45.1 recipient mice (2 x 10 donor WBM cells per recipient) (n = 5 recipients / cohort; n = 5 donors / cohort). [Figure 8G] We show that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice. Secondary transplantation assays show that WBM cells from long-term engrafted primary recipients were isolated and transplanted into preconditioned CD45.1 recipient mice (2 x 10 donor WBM cells per recipient) (n = 5 recipients / cohort; n = 5 donors / cohort). [Figure 8H] We show that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice. Analysis of BM vascular leakage by Evan's Blue Dye (EBD) extravasation reveals that SCGF infusion significantly reduces vascular leakage in CDH5-MAPK mice (n = 3-5 mice / cohort). [Figure 8I] Figure 1 shows that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice. Representative immunofluorescence images of femurs labeled in vivo with a vascular-specific VECAD antibody (red) showing the reversal of vasodilation in CDH5-MAPK mice treated with SCGF. [Figure 8J] Figure 1 shows that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice. Normalized gene expression of NF-κB target genes within the BM microenvironment of the indicated genotypes compared to PBS-treated control mice is shown. B2m was used for normalization (n=3 mice / cohort). [Figure 8K]Figure 1 shows that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice. Representative immunofluorescence images and quantification are shown, demonstrating reduced levels of nuclear p65 in BMECs derived from CDH5-MAPK mice treated with SCGF. Error bars represent sample mean ± SEM. Statistical significance was determined using a two-tailed unpaired Student's t-test for pairwise comparisons and one-way ANOVA for multiple comparisons. *P<0.05; **P<0.01; ***P<0.001. [Figure 8L] Figure 1 shows that SCGF infusion resolves the hematopoietic and vascular defects in CDH5-MAPK mice. Representative immunofluorescence images and quantification are shown, demonstrating reduced levels of nuclear p65 in BMECs derived from CDH5-MAPK mice treated with SCGF. Error bars represent sample mean ± SEM. Statistical significance was determined using a two-tailed unpaired Student's t-test for pairwise comparisons and one-way ANOVA for multiple comparisons. *P<0.05; **P<0.01; ***P<0.001. [Figure 9A] We demonstrate that SCGF enhances hematopoietic regeneration after myelosuppressive injury. Results are shown for 650 Rad irradiation. Control and CDH5-MAPK mice were injected with 2 μg of SCGF every other day for a total of seven injections, starting on day +1, and hematopoietic recovery was assessed over 28 days. SCGF injection promoted significantly increased recovery of leukocytes, neutrophils, erythrocytes, and platelets at the indicated time points in a) control mice (n = 6-7 mice / cohort) and b) CDH5-MAPK mice (n = 7-8 mice / cohort). [Figure 9B] We demonstrate that SCGF enhances hematopoietic regeneration after myelosuppressive injury. Results are shown for 650 Rad irradiation. Control and CDH5-MAPK mice were injected with 2 μg of SCGF every other day for a total of seven injections, starting on day +1, and hematopoietic recovery was assessed over 28 days. SCGF injection promoted significantly increased recovery of leukocytes, neutrophils, erythrocytes, and platelets at the indicated time points in a) control mice (n = 6-7 mice / cohort) and b) CDH5-MAPK mice (n = 7-8 mice / cohort). [Figure 9C]SCGF enhances hematopoietic regeneration after myelosuppressive injury. Representative immunofluorescence images of femurs labeled in vivo with vascular-specific CD144 / VE-cadherin antibody (red) 28 days after irradiation show that SCGF injection improved vascular recovery in both c) control mice and d) CDH5-MAPK mice. [Figure 9D] SCGF enhances hematopoietic regeneration after myelosuppressive injury. Representative immunofluorescence images of femurs labeled in vivo with vascular-specific CD144 / VE-cadherin antibody (red) 28 days after irradiation show that SCGF injection improved vascular recovery in both c) control mice and d) CDH5-MAPK mice. [Figure 9E] SCGF enhances hematopoietic regeneration after myelosuppressive injury. Total cell numbers per femur are shown (n = 4–7 mice / cohort). [Figure 9F] Figure 1 shows that SCGF enhances hematopoietic regeneration after myelosuppressive injury. The frequency of phenotypic HSCs per 10 femoral cells assessed by flow cytometry is shown (n = 4–7 mice / cohort). [Figure 9G] We demonstrate that SCGF enhances hematopoietic regeneration after myelosuppressive injury. Competitive repopulation assays assessing total CD45.2+ cell engraftment and CD45.2+ lineage distribution of donor WBM cells are shown. Twenty-eight days after irradiation, donor cells were isolated from control and CDH5-MAPK mice treated with PBS / SCGF. 2.5 x 10 donor WBM cells (CD45.2) were transplanted into preconditioned CD45.1 recipient mice along with 5 x 10 competitor WBM cells (CD45.1). (n = 9-10 recipients / cohort; n = 5 donors / cohort). [Figure 9H]We demonstrate that SCGF enhances hematopoietic regeneration after myelosuppressive injury. Competitive repopulation assays assessing total CD45.2+ cell engraftment and CD45.2+ lineage distribution of donor WBM cells are shown. Twenty-eight days after irradiation, donor cells were isolated from control and CDH5-MAPK mice treated with PBS / SCGF. 2.5 x 10 donor WBM cells (CD45.2) were transplanted into preconditioned CD45.1 recipient mice along with 5 x 10 competitor WBM cells (CD45.1). (n = 9-10 recipients / cohort; n = 5 donors / cohort). [Figure 9I] These results demonstrate that SCGF enhances hematopoietic regeneration after myelosuppressive injury. Secondary transplantation assays show WBM cells from long-term engrafted primary recipients were isolated and transplanted into preconditioned CD45.1 recipient mice. Each secondary recipient received 2 x 10 donor WBM cells (n = 4-5 recipients / cohort; n = 5 donors / cohort). Error bars represent the sample mean ± SEM. Statistical significance was determined using a two-tailed, unpaired Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001). [Figure 9J] These results demonstrate that SCGF enhances hematopoietic regeneration after myelosuppressive injury. Secondary transplantation assays show WBM cells from long-term engrafted primary recipients were isolated and transplanted into preconditioned CD45.1 recipient mice. Each secondary recipient received 2 x 10 donor WBM cells (n = 4-5 recipients / cohort; n = 5 donors / cohort). Error bars represent the sample mean ± SEM. Statistical significance was determined using a two-tailed, unpaired Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001). [Figure 10]Schematic illustrating the effects of inflammation on BM niche cells and HSPCs. Endothelial MAPK activation triggers an NF-kB-dependent inflammatory stress response in the bone marrow, resulting in functional defects of the vascular niche and HSPCs. Suppression of inflammation by inhibiting endothelial NF-kB or injecting SCGF restores vascular integrity, resolves HSPC and niche defects, and enhances hematopoietic recovery after myelosuppression. [Figure 11] Under normal physiological conditions, HSCs reside in osteoblastic or vascular niches. Some HSC daughter cells leave the niche and begin mobilization and circulation in response to changes in SDF-1 levels in the BM. HSC homing is the reverse of recruitment and occurs in response to higher levels of SDF-1 in the BM. The osteoblastic niche may provide a quiescent microenvironment for HSC maintenance. In contrast, the vascular niche may promote HSC transendothelial migration during recruitment or homing, favoring HSC proliferation and further differentiation. The process of recruiting HSCs to the vascular niche may depend on endothelium-derived FGF-4 and SDF-1. As cells progress from the osteoblastic niche to the vascular niche, higher FGF-4 and oxygen gradients may play a role in HSC / HPC recruitment, proliferation, and differentiation. Under stress conditions such as thrombocytopenia, SDF-1 and VEGF activate MMP-9, which converts membrane-bound Kit ligand to soluble Kit ligand (sKitL), which in turn promotes HSC cell cycle entry, recruitment to the vascular niche, and differentiation. (Yin, T., & Li, L. (2006). The stem cell niches in bone. The Journal of clinical investigation, 116(5), 1195-1201. doi:10.1172 / JCI28568) [Figure 12A]Figure 12A. At steady state, platelet-biased HSCs are at the top of the hematopoietic hierarchy and can generate myeloid-biased HSCs and lymphoid-biased HSCs. Sequentially, myeloid-biased HSCs can generate both balanced-biased and lymphoid-biased HSCs, while lymphoid-biased HSCs do not generate their myeloid-biased counterparts. Platelet-biased HSCs have the potential to repopulate the platelet population faster than other HSC subsets. Myeloid-biased HSCs preferentially give rise to myeloid lineage cells via myeloid-committed progenitors. Balanced HSCs contribute equally to both myeloid and lymphoid lineages. Lymphoid-biased HSCs primarily generate lymphocytes over myeloid lineage cells via lymphoid-committed progenitors. Dashed lines represent the potential for one HSC subset to generate another HSC subset. Solid lines represent differentiation potential. [Figure 12B] FIG. 12B. Inflammation enhances the production of myeloid lineages, including myeloid progenitor cells and mature myeloid cells, resulting in a myeloid bias in hematopoiesis. [Figure 12C] Figure 12C. During aging, myeloid-biased HSCs increase, generating more myeloid than lymphoid cells. Red arrows indicate dominant differentiation pathways. Dashed lines represent potential pathways. Solid lines represent previously demonstrated differentiation potential. Line thickness reflects the relative contribution to each lineage commitment. (Adapted from Kovtonyuk, L.V., Fritsch, K., Feng, X., Manz, M.G. & Takizawa, H. Inflamm-Aging of Hematopoiesis, Hematopoietic Stem Cells, and the Bone Marrow Microenvironment. Frontiers in immunology 7, 502, doi:10.3389 / fimmu.2016.00502 (2016)). [Figure 13]This is a schematic diagram of the mitogen-activated protein kinase (MAPK) signaling pathway. MAPK signaling is activated by external stimuli, such as growth factors and cell stress, and leads to activation of a three-tiered cascade of MAPK kinase kinases (MAP3Ks), which then activate MAPK kinases (MAP2Ks), and finally MAPK. The major MAPK pathways involved in inflammatory diseases are ERK (extracellular-regulated kinase), p38 MAPK, and JNK (c-Jun NH2-terminal kinase). Downstream of p38 MAPK is MAPK-activated protein kinase 2 (MAPKAPK2 or MK2). (Adapted from Barnes, PJ (2016) "Kinases as Novel Therapeutic Targets in Asthma and Chronic Obstructive Pulmonary Disease," Pharmacological Revs. 68:788-815.) [Figure 14]Schematic diagram of the nuclear factor-κB (NF-κB) signaling pathway. The canonical (classical) pathway is activated by inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and lipopolysaccharide (LPS), leading to phosphorylation of inhibitors of κB kinase (IKKα, IKKβ in complex with essential modulator of NF-κB (NEMO)), which in turn phosphorylates inhibitors of κB (IκB-β), which are ubiquitinated and degraded by the proteasome, releasing p65 and p50, which then translocate to the nucleus. In the nucleus, they bind to κB DNA recognition sequences, leading to the activation of cytokine, chemokine, and protease genes. The non-canonical pathway is activated by CD40 and lymphotoxin (LT)-β, which activates NF-κB-inducing kinase (NIK), leading to activation of the IKKα homodimer, which phosphorylates RelB / p100, generating the RelB / p50 complex, which translocates to the nucleus and switches on immune genes (adapted from Barnes, PJ (2016) "Kinases as Novel Therapeutic Targets in Asthma and Chronic Obstructive Pulmonary Disease," Pharmacological Revs. 68:788-815). [Figure 1A-1]We show that CDH5-MAPK mice exhibited significantly reduced frequencies and absolute numbers of immunophenotypically defined hematopoietic stem and progenitor cells (HSPCs), including HSCs (defined as cKIT+LineageNegCD41-SCA1+CD150+CD48Neg), KLS cells (cKIT+LineageNegSCA1+), multipotent progenitor cells (MPPs; cKIT+LineageNeg, SCA1+CD150NegCD48Neg), and hematopoietic progenitor cell subsets (HPC-1 and HPC-2, defined as cKIT+LineageNegSCA1+CD150NegCD48+ and cKIT+LineageNegSCA1+CD150+CD48+, respectively), compared with littermate controls (Supplementary Figure 1A). Cell cycle analysis showed that HSCs and HSPCs from CDH5-MAPK mice exhibited loss of quiescence and increased apoptosis compared with littermate controls (Supplementary Figures 1B-1F). Together, these data suggest that chronic activation of endothelial MAPK negatively impacts niche activity, resulting in defects in steady-state hematopoiesis and HSC function. [Figure 1B-1]We show that CDH5-MAPK mice exhibited significantly reduced frequencies and absolute numbers of immunophenotypically defined hematopoietic stem and progenitor cells (HSPCs), including HSCs (defined as cKIT+LineageNegCD41-SCA1+CD150+CD48Neg), KLS cells (cKIT+LineageNegSCA1+), multipotent progenitor cells (MPPs; cKIT+LineageNeg, SCA1+CD150NegCD48Neg), and hematopoietic progenitor cell subsets (HPC-1 and HPC-2, defined as cKIT+LineageNegSCA1+CD150NegCD48+ and cKIT+LineageNegSCA1+CD150+CD48+, respectively), compared with littermate controls (Supplementary Figure 1A). Cell cycle analysis showed that HSCs and HSPCs from CDH5-MAPK mice exhibited loss of quiescence and increased apoptosis compared with littermate controls (Supplementary Figures 1B-1F). Together, these data suggest that chronic activation of endothelial MAPK negatively impacts niche activity, resulting in defects in steady-state hematopoiesis and HSC function. [Figure 1C-1]We show that CDH5-MAPK mice exhibited significantly reduced frequencies and absolute numbers of immunophenotypically defined hematopoietic stem and progenitor cells (HSPCs), including HSCs (defined as cKIT+LineageNegCD41-SCA1+CD150+CD48Neg), KLS cells (cKIT+LineageNegSCA1+), multipotent progenitor cells (MPPs; cKIT+LineageNeg, SCA1+CD150NegCD48Neg), and hematopoietic progenitor cell subsets (HPC-1 and HPC-2, defined as cKIT+LineageNegSCA1+CD150NegCD48+ and cKIT+LineageNegSCA1+CD150+CD48+, respectively), compared with littermate controls (Supplementary Figure 1A). Cell cycle analysis showed that HSCs and HSPCs from CDH5-MAPK mice exhibited loss of quiescence and increased apoptosis compared with littermate controls (Supplementary Figures 1B-1F). Together, these data suggest that chronic activation of endothelial MAPK negatively impacts niche activity, resulting in defects in steady-state hematopoiesis and HSC function. [Figure 1D-1]We show that CDH5-MAPK mice exhibited significantly reduced frequencies and absolute numbers of immunophenotypically defined hematopoietic stem and progenitor cells (HSPCs), including HSCs (defined as cKIT+LineageNegCD41-SCA1+CD150+CD48Neg), KLS cells (cKIT+LineageNegSCA1+), multipotent progenitor cells (MPPs; cKIT+LineageNeg, SCA1+CD150NegCD48Neg), and hematopoietic progenitor cell subsets (HPC-1 and HPC-2, defined as cKIT+LineageNegSCA1+CD150NegCD48+ and cKIT+LineageNegSCA1+CD150+CD48+, respectively), compared with littermate controls (Supplementary Figure 1A). Cell cycle analysis showed that HSCs and HSPCs from CDH5-MAPK mice exhibited loss of quiescence and increased apoptosis compared with littermate controls (Supplementary Figures 1B-1F). Together, these data suggest that chronic activation of endothelial MAPK negatively impacts niche activity, resulting in defects in steady-state hematopoiesis and HSC function. [Figure 1E-1]We show that CDH5-MAPK mice exhibited significantly reduced frequencies and absolute numbers of immunophenotypically defined hematopoietic stem and progenitor cells (HSPCs), including HSCs (defined as cKIT+LineageNegCD41-SCA1+CD150+CD48Neg), KLS cells (cKIT+LineageNegSCA1+), multipotent progenitor cells (MPPs; cKIT+LineageNeg, SCA1+CD150NegCD48Neg), and hematopoietic progenitor cell subsets (HPC-1 and HPC-2, defined as cKIT+LineageNegSCA1+CD150NegCD48+ and cKIT+LineageNegSCA1+CD150+CD48+, respectively), compared with littermate controls (Supplementary Figure 1A). Cell cycle analysis showed that HSCs and HSPCs from CDH5-MAPK mice exhibited loss of quiescence and increased apoptosis compared with littermate controls (Supplementary Figures 1B-1F). Together, these data suggest that chronic activation of endothelial MAPK negatively impacts niche activity, resulting in defects in steady-state hematopoiesis and HSC function. [Figure 1F-1]We show that CDH5-MAPK mice exhibited significantly reduced frequencies and absolute numbers of immunophenotypically defined hematopoietic stem and progenitor cells (HSPCs), including HSCs (defined as cKIT+LineageNegCD41-SCA1+CD150+CD48Neg), KLS cells (cKIT+LineageNegSCA1+), multipotent progenitor cells (MPPs; cKIT+LineageNeg, SCA1+CD150NegCD48Neg), and hematopoietic progenitor cell subsets (HPC-1 and HPC-2, defined as cKIT+LineageNegSCA1+CD150NegCD48+ and cKIT+LineageNegSCA1+CD150+CD48+, respectively), compared with littermate controls (Supplementary Figure 1A). Cell cycle analysis showed that HSCs and HSPCs from CDH5-MAPK mice exhibited loss of quiescence and increased apoptosis compared with littermate controls (Supplementary Figures 1B-1F). Together, these data suggest that chronic activation of endothelial MAPK negatively impacts niche activity, resulting in defects in steady-state hematopoiesis and HSC function. [Figure 2A-1] Hematopoietic analysis of CDH5-MAPK::IkB mice demonstrated restoration of BM cellularity and the frequency of phenotypic HSCs and HSPCs (Supplementary Figure 2A). HSC functionality, assayed by competitive BM transplantation, demonstrated complete restoration of long-term engraftment potential and reversal of myeloid-biased differentiation in CDH5-MAPK::IkB mice (Supplementary Figure 2B). WBM cells derived from CDH5-MAPK::IkB mice were also able to maintain continuous repopulation and multilineage reconstitution potential during secondary transplantation assays (Supplementary Figure 2C and Supplementary Figure 2D). CDH5-MAPK mice exhibited a reduction in immunophenotypically defined BM multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte / macrophage progenitors (GMPs), megakaryocytic / erythroid progenitors (MEPs), and B cell precursor subsets (sIgM-B220+ B cells, pre-pro B cells, pro B cells, and pre-B cells), which was functionally reflected in reduced peripheral blood counts (Supplementary Figure 2E and Supplementary Figure 2F). [Figure 2B-1]Hematopoietic analysis of CDH5-MAPK::IkB mice demonstrated restoration of BM cellularity and the frequency of phenotypic HSCs and HSPCs (Supplementary Figure 2A). HSC function, assayed by competitive BM transplantation, demonstrated complete restoration of long-term engraftment potential and reversal of myeloid-biased differentiation in CDH5-MAPK::IkB mice (Supplementary Figure 2B). WBM cells derived from CDH5-MAPK::IkB mice were also able to maintain continuous repopulation and multilineage reconstitution potential during secondary transplantation assays (Supplementary Figure 2C and Supplementary Figure 2D). CDH5-MAPK mice exhibited a reduction in immunophenotypically defined BM multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte / macrophage progenitors (GMPs), megakaryocytic / erythroid progenitors (MEPs), and B cell precursor subsets (sIgM-B220+ B cells, pre-pro B cells, pro B cells, and pre-B cells), which was functionally reflected in reduced peripheral blood counts (Supplementary Figure 2E and Supplementary Figure 2F). [Figure 2C-1] Hematopoietic analysis of CDH5-MAPK::IkB mice demonstrated restoration of BM cellularity and the frequency of phenotypic HSCs and HSPCs (Supplementary Figure 2A). HSC function, assayed by competitive BM transplantation, demonstrated complete restoration of long-term engraftment potential and reversal of myeloid-biased differentiation in CDH5-MAPK::IkB mice (Supplementary Figure 2B). WBM cells derived from CDH5-MAPK::IkB mice were also able to maintain continuous repopulation and multilineage reconstitution potential during secondary transplantation assays (Supplementary Figure 2C and Supplementary Figure 2D). CDH5-MAPK mice exhibited a reduction in immunophenotypically defined BM multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte / macrophage progenitors (GMPs), megakaryocytic / erythroid progenitors (MEPs), and B cell precursor subsets (sIgM-B220+ B cells, pre-pro B cells, pro B cells, and pre-B cells), which was functionally reflected in reduced peripheral blood counts (Supplementary Figure 2E and Supplementary Figure 2F). [Figure 2D-1]Hematopoietic analysis of CDH5-MAPK::IkB mice demonstrated restoration of BM cellularity and the frequency of phenotypic HSCs and HSPCs (Supplementary Figure 2A). HSC function, assayed by competitive BM transplantation, demonstrated complete restoration of long-term engraftment potential and reversal of myeloid-biased differentiation in CDH5-MAPK::IkB mice (Supplementary Figure 2B). WBM cells derived from CDH5-MAPK::IkB mice were also able to maintain continuous repopulation and multilineage reconstitution potential during secondary transplantation assays (Supplementary Figure 2C and Supplementary Figure 2D). CDH5-MAPK mice exhibited a reduction in immunophenotypically defined BM multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte / macrophage progenitors (GMPs), megakaryocytic / erythroid progenitors (MEPs), and B cell precursor subsets (sIgM-B220+ B cells, pre-pro B cells, pro B cells, and pre-B cells), which was functionally reflected in reduced peripheral blood counts (Supplementary Figure 2E and Supplementary Figure 2F). [Figure 2E-1] Hematopoietic analysis of CDH5-MAPK::IkB mice demonstrated restoration of BM cellularity and the frequency of phenotypic HSCs and HSPCs (Supplementary Figure 2A). HSC function, assayed by competitive BM transplantation, demonstrated complete restoration of long-term engraftment potential and reversal of myeloid-biased differentiation in CDH5-MAPK::IkB mice (Supplementary Figure 2B). WBM cells derived from CDH5-MAPK::IkB mice were also able to maintain continuous repopulation and multilineage reconstitution potential during secondary transplantation assays (Supplementary Figure 2C and Supplementary Figure 2D). CDH5-MAPK mice exhibited a reduction in immunophenotypically defined BM multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte / macrophage progenitors (GMPs), megakaryocytic / erythroid progenitors (MEPs), and B cell precursor subsets (sIgM-B220+ B cells, pre-pro B cells, pro B cells, and pre-B cells), which was functionally reflected in reduced peripheral blood counts (Supplementary Figure 2E and Supplementary Figure 2F). [Figure 2F-1]Hematopoietic analysis of CDH5-MAPK::IkB mice demonstrated restoration of BM cellularity and the frequency of phenotypic HSCs and HSPCs (Supplementary Figure 2A). HSC function, assayed by competitive BM transplantation, demonstrated complete restoration of long-term engraftment potential and reversal of myeloid-biased differentiation in CDH5-MAPK::IkB mice (Supplementary Figure 2B). WBM cells derived from CDH5-MAPK::IkB mice were also able to maintain continuous repopulation and multilineage reconstitution potential during secondary transplantation assays (Supplementary Figure 2C and Supplementary Figure 2D). CDH5-MAPK mice exhibited a reduction in immunophenotypically defined BM multipotent progenitors (MPPs), common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte / macrophage progenitors (GMPs), megakaryocytic / erythroid progenitors (MEPs), and B cell precursor subsets (sIgM-B220+ B cells, pre-pro B cells, pro B cells, and pre-B cells), which was functionally reflected in reduced peripheral blood counts (Supplementary Figure 2E and Supplementary Figure 2F). [Figure 3A-1] RT-qPCR analysis revealed a global upregulation of NF-kB target genes in hematopoietic cells (CD45+), stromal cells (CD45-Ter119-CD31-VE-cadherin-), and unfractionated whole bone marrow (WBM) cells from CDH5-MAPK mice (Supplementary Figures 3A-3F), indicating that activation of endothelial MAPK triggers a generalized inflammatory response within the BM. [Figure 3B-1] RT-qPCR analysis revealed a global upregulation of NF-kB target genes in hematopoietic cells (CD45+), stromal cells (CD45-Ter119-CD31-VE-cadherin-), and unfractionated whole bone marrow (WBM) cells from CDH5-MAPK mice (Supplementary Figures 3A-3F), indicating that activation of endothelial MAPK triggers a generalized inflammatory response within the BM. [Figure 3C-1]RT-qPCR analysis revealed a global upregulation of NF-kB target genes in hematopoietic cells (CD45+), stromal cells (CD45-Ter119-CD31-VE-cadherin-), and unfractionated whole bone marrow (WBM) cells from CDH5-MAPK mice (Supplementary Figures 3A-3F), indicating that activation of endothelial MAPK triggers a generalized inflammatory response within the BM. [Figure 3D-1] RT-qPCR analysis revealed a global upregulation of NF-kB target genes in hematopoietic cells (CD45+), stromal cells (CD45-Ter119-CD31-VE-cadherin-), and unfractionated whole bone marrow (WBM) cells from CDH5-MAPK mice (Supplementary Figures 3A-3F), indicating that activation of endothelial MAPK triggers a generalized inflammatory response within the BM. [Figure 3E-1] RT-qPCR analysis revealed a global upregulation of NF-kB target genes in hematopoietic cells (CD45+), stromal cells (CD45-Ter119-CD31-VE-cadherin-), and unfractionated whole bone marrow (WBM) cells from CDH5-MAPK mice (Supplementary Figures 3A-3F), indicating that activation of endothelial MAPK triggers a generalized inflammatory response within the BM. [Figure 3F-1] RT-qPCR analysis revealed a global upregulation of NF-kB target genes in hematopoietic cells (CD45+), stromal cells (CD45-Ter119-CD31-VE-cadherin-), and unfractionated whole bone marrow (WBM) cells from CDH5-MAPK mice (Supplementary Figures 3A-3F), indicating that activation of endothelial MAPK triggers a generalized inflammatory response within the BM. [Figure 4A-1] The fidelity of transgene expression in CDH5-MAPK mice was verified by tracking cre-mediated recombination using the endogenous Rosa26:eGFP reporter system. GFP expression was strictly restricted to endothelial cells within the bone marrow, with no detectable expression in any of the hematopoietic subsets analyzed, including HSCs, myeloid cells, B cells, and T cells (Supplementary Figure 4A and Supplementary Figure 4B). [Figure 4B-1] The fidelity of transgene expression in CDH5-MAPK mice was verified by tracking cre-mediated recombination using the endogenous Rosa26:eGFP reporter system. GFP expression was strictly restricted to endothelial cells within the bone marrow, with no detectable expression in any of the hematopoietic subsets analyzed, including HSCs, myeloid cells, B cells, and T cells (Supplementary Figure 4A and Supplementary Figure 4B). [Figure 5A-1] We show that HSPCs from CDH5-MAPK mice exhibited significantly increased hypoxia and ROS levels, along with loss of quiescence and increased apoptosis. [Figure 5B-1] We show that HSPCs from CDH5-MAPK mice exhibited significantly increased hypoxia and ROS levels, along with loss of quiescence and increased apoptosis. [Figure 6A-1] Flow cytometry analysis of BM Lepr+ cells and osteoblasts did not reveal significant changes in the expression of cellular or HSC regulatory factors in CDH5-MAPK mice (Supplementary Figures 6A, 6E, 6G, and 6H). However, both Lepr+ cells and osteoblasts in CDH5-MAPK mice, like other BM cell subsets, showed increased expression of NF-κB-regulated target genes, which was suppressed upon inhibition of endothelial NF-κB signaling (Supplementary Figures 6F and 6I). [Figure 6B-1] Flow cytometry analysis of BM Lepr+ cells and osteoblasts did not reveal significant changes in the expression of cellular or HSC regulatory factors in CDH5-MAPK mice (Supplementary Figures 6A, 6E, 6G, and 6H). However, both Lepr+ cells and osteoblasts in CDH5-MAPK mice, like other BM cell subsets, showed increased expression of NF-κB-regulated target genes, which was suppressed upon inhibition of endothelial NF-κB signaling (Supplementary Figures 6F and 6I). [Figure 6C-1]Flow cytometry analysis of BM Lepr+ cells and osteoblasts did not reveal significant changes in the expression of cellular or HSC regulatory factors in CDH5-MAPK mice (Supplementary Figures 6A, 6E, 6G, and 6H). However, both Lepr+ cells and osteoblasts in CDH5-MAPK mice, like other BM cell subsets, showed increased expression of NF-κB-regulated target genes, which was suppressed upon inhibition of endothelial NF-κB signaling (Supplementary Figures 6F and 6I). [Figure 6D-1] Flow cytometry analysis of BM Lepr+ cells and osteoblasts did not reveal significant changes in the expression of cellular or HSC regulatory factors in CDH5-MAPK mice (Supplementary Figures 6A, 6E, 6G, and 6H). However, both Lepr+ cells and osteoblasts in CDH5-MAPK mice, like other BM cell subsets, showed increased expression of NF-κB-regulated target genes, which was suppressed upon inhibition of endothelial NF-κB signaling (Supplementary Figures 6F and 6I). [Figure 6E-1] Flow cytometry analysis of BM Lepr+ cells and osteoblasts did not reveal significant changes in the expression of cellular or HSC regulatory factors in CDH5-MAPK mice (Supplementary Figures 6A, 6E, 6G, and 6H). However, both Lepr+ cells and osteoblasts in CDH5-MAPK mice, like other BM cell subsets, showed increased expression of NF-κB-regulated target genes, which was suppressed upon inhibition of endothelial NF-κB signaling (Supplementary Figures 6F and 6I). [Figure 6F-1] Flow cytometry analysis of BM Lepr+ cells and osteoblasts did not reveal significant changes in the expression of cellular or HSC regulatory factors in CDH5-MAPK mice (Supplementary Figures 6A, 6E, 6G, and 6H). However, both Lepr+ cells and osteoblasts in CDH5-MAPK mice, like other BM cell subsets, showed increased expression of NF-κB-regulated target genes, which was suppressed upon inhibition of endothelial NF-κB signaling (Supplementary Figures 6F and 6I). [Figure 6G-1]Flow cytometry analysis of BM Lepr+ cells and osteoblasts did not reveal significant changes in the expression of cellular or HSC regulatory factors in CDH5-MAPK mice (Supplementary Figures 6A, 6E, 6G, and 6H). However, both Lepr+ cells and osteoblasts in CDH5-MAPK mice, like other BM cell subsets, showed increased expression of NF-κB-regulated target genes, which was suppressed upon inhibition of endothelial NF-κB signaling (Supplementary Figures 6F and 6I). [Figure 6H-1] Flow cytometry analysis of BM Lepr+ cells and osteoblasts did not reveal significant changes in the expression of cellular or HSC regulatory factors in CDH5-MAPK mice (Supplementary Figures 6A, 6E, 6G, and 6H). However, both Lepr+ cells and osteoblasts in CDH5-MAPK mice, like other BM cell subsets, showed increased expression of NF-κB-regulated target genes, which was suppressed upon inhibition of endothelial NF-κB signaling (Supplementary Figures 6F and 6I). [Figure 6I-1] Flow cytometry analysis of BM Lepr+ cells and osteoblasts did not reveal significant changes in the expression of cellular or HSC regulatory factors in CDH5-MAPK mice (Supplementary Figures 6A, 6E, 6G, and 6H). However, both Lepr+ cells and osteoblasts in CDH5-MAPK mice, like other BM cell subsets, showed increased expression of NF-κB-regulated target genes, which was suppressed upon inhibition of endothelial NF-κB signaling (Supplementary Figures 6F and 6I). [Figure 7A-1]To screen for novel candidate proteins that may regulate HSC function during inflammation, we performed proteomic analysis (SomaLogic) of plasma from Tie2.IkB-SS mice, which identified 82 proteins that were differentially expressed compared to littermate controls. Potential hematopoietic-promoting proteins were hypothesized to show an opposite trend in CDH5-MAPK mice compared to Tie2.IkB-SS mice. Using this approach, we identified 18 candidate factors that were significantly altered and inversely correlated (i.e., downregulated in CDH5-MAPK mice and upregulated in Tie2.IkB-SS mice, and vice versa) (Supplementary Figure 7, Figure 7A, and Supplementary Figure 7B). Among these, Clec11a / stem cell growth factor α (SCGF) was the most significantly downregulated protein in CDH5-MAPK mice (Supplementary Figure 7C). The specificity of the SCGF aptamer was confirmed, validating the reduction in plasma SCGF observed in CDH5-MAPK mice (Supplementary Figure 7D). Notably, CDH5-MAPK::IkB mice showed restored plasma SCGF levels, further indicating that SCGF may be a potential hematopoietic promoting factor that promotes recovery in CDH5-MAPK::IkB mice (Supplementary Figure 7E and Supplementary Figure 7F). [Figure 7B-1]To screen for novel candidate proteins that may regulate HSC function during inflammation, we performed proteomic analysis (SomaLogic) of plasma from Tie2.IkB-SS mice, which identified 82 proteins that were differentially expressed compared to littermate controls. Potential hematopoietic-promoting proteins were hypothesized to show an opposite trend in CDH5-MAPK mice compared to Tie2.IkB-SS mice. Using this approach, we identified 18 candidate factors that were significantly altered and inversely correlated (i.e., downregulated in CDH5-MAPK mice and upregulated in Tie2.IkB-SS mice, and vice versa) (Supplementary Figure 7, Figure 7A, and Supplementary Figure 7B). Among these, Clec11a / stem cell growth factor α (SCGF) was the most significantly downregulated protein in CDH5-MAPK mice (Supplementary Figure 7C). The specificity of the SCGF aptamer was confirmed, validating the reduction in plasma SCGF observed in CDH5-MAPK mice (Supplementary Figure 7D). Notably, CDH5-MAPK::IkB mice showed restored plasma SCGF levels, further indicating that SCGF may be a potential hematopoietic promoting factor that promotes recovery in CDH5-MAPK::IkB mice (Supplementary Figure 7E and Supplementary Figure 7F). [Figure 7C-1]To screen for novel candidate proteins that may regulate HSC function during inflammation, we performed proteomic analysis (SomaLogic) of plasma from Tie2.IkB-SS mice, which identified 82 proteins that were differentially expressed compared to littermate controls. Potential hematopoietic-promoting proteins were hypothesized to show an opposite trend in CDH5-MAPK mice compared to Tie2.IkB-SS mice. Using this approach, we identified 18 candidate factors that were significantly altered and inversely correlated (i.e., downregulated in CDH5-MAPK mice and upregulated in Tie2.IkB-SS mice, and vice versa) (Supplementary Figure 7, Figure 7A, and Supplementary Figure 7B). Among these, Clec11a / stem cell growth factor α (SCGF) was the most significantly downregulated protein in CDH5-MAPK mice (Supplementary Figure 7C). The specificity of the SCGF aptamer was confirmed, validating the reduction in plasma SCGF observed in CDH5-MAPK mice (Supplementary Figure 7D). Notably, CDH5-MAPK::IkB mice showed restored plasma SCGF levels, further indicating that SCGF may be a potential hematopoietic promoting factor that promotes recovery in CDH5-MAPK::IkB mice (Supplementary Figure 7E and Supplementary Figure 7F). [Figure 7D-1]To screen for novel candidate proteins that may regulate HSC function during inflammation, we performed proteomic analysis (SomaLogic) of plasma from Tie2.IkB-SS mice, which identified 82 proteins that were differentially expressed compared to littermate controls. Potential hematopoietic-promoting proteins were hypothesized to show an opposite trend in CDH5-MAPK mice compared to Tie2.IkB-SS mice. Using this approach, we identified 18 candidate factors that were significantly altered and inversely correlated (i.e., downregulated in CDH5-MAPK mice and upregulated in Tie2.IkB-SS mice, and vice versa) (Supplementary Figure 7, Figure 7A, and Supplementary Figure 7B). Among these, Clec11a / stem cell growth factor α (SCGF) was the most significantly downregulated protein in CDH5-MAPK mice (Supplementary Figure 7C). The specificity of the SCGF aptamer was confirmed, validating the reduction in plasma SCGF observed in CDH5-MAPK mice (Supplementary Figure 7D). Notably, CDH5-MAPK::IkB mice showed restored plasma SCGF levels, further indicating that SCGF may be a potential hematopoietic promoting factor that promotes recovery in CDH5-MAPK::IkB mice (Supplementary Figure 7E and Supplementary Figure 7F). [Figure 7E-1]To screen for novel candidate proteins that may regulate HSC function during inflammation, we performed proteomic analysis (SomaLogic) of plasma from Tie2.IkB-SS mice, which identified 82 proteins that were differentially expressed compared to littermate controls. Potential hematopoietic-promoting proteins were hypothesized to show an opposite trend in CDH5-MAPK mice compared to Tie2.IkB-SS mice. Using this approach, we identified 18 candidate factors that were significantly altered and inversely correlated (i.e., downregulated in CDH5-MAPK mice and upregulated in Tie2.IkB-SS mice, and vice versa) (Supplementary Figure 7, Figure 7A, and Supplementary Figure 7B). Among these, Clec11a / stem cell growth factor α (SCGF) was the most significantly downregulated protein in CDH5-MAPK mice (Supplementary Figure 7C). The specificity of the SCGF aptamer was confirmed, validating the reduction in plasma SCGF observed in CDH5-MAPK mice (Supplementary Figure 7D). Notably, CDH5-MAPK::IkB mice showed restored plasma SCGF levels, further indicating that SCGF may be a potential hematopoietic promoting factor that promotes recovery in CDH5-MAPK::IkB mice (Supplementary Figure 7E and Supplementary Figure 7F). [Figure 7F-1]To screen for novel candidate proteins that may regulate HSC function during inflammation, we performed proteomic analysis (SomaLogic) of plasma from Tie2.IkB-SS mice, which identified 82 proteins that were differentially expressed compared to littermate controls. Potential hematopoietic-promoting proteins were hypothesized to show an opposite trend in CDH5-MAPK mice compared to Tie2.IkB-SS mice. Using this approach, we identified 18 candidate factors that were significantly altered and inversely correlated (i.e., downregulated in CDH5-MAPK mice and upregulated in Tie2.IkB-SS mice, and vice versa) (Supplementary Figure 7, Figure 7A, and Supplementary Figure 7B). Among these, Clec11a / stem cell growth factor α (SCGF) was the most significantly downregulated protein in CDH5-MAPK mice (Supplementary Figure 7C). The specificity of the SCGF aptamer was confirmed, validating the reduction in plasma SCGF observed in CDH5-MAPK mice (Supplementary Figure 7D). Notably, CDH5-MAPK::IkB mice showed restored plasma SCGF levels, further indicating that SCGF may be a potential hematopoietic promoting factor that promotes recovery in CDH5-MAPK::IkB mice (Supplementary Figure 7E and Supplementary Figure 7F). [Figure 8A-1] To determine whether SCGF could reverse the hematopoietic defects in CDH5-MAPK mice, 4 μg of SCGF was subcutaneously injected for 5 consecutive days, and the phenotypic and functional attributes of the hematopoietic system were analyzed 24 h after the final injection (Supplementary Figure 8A). No significant differences were observed in the frequency and absolute number of cells (Supplementary Figure 8B). While SCGF injection into littermate control mice confirmed that SCGF had no effect on steady-state hematopoiesis, SCGF injection into CDH5-MAPK mice had a significant effect on hematopoiesis (Supplementary Figures 8C-8E). SCGF injection significantly increased the frequency of phenotypic HSCs and HSPCs in CDH5-MAPK mice (Supplementary Figure 8C). SCGF injection also reversed the peripheral blood myeloid bias and restored blood cell counts in CDH5-MAPK mice (Supplementary Figures 8D and 8E). [Figure 8B-1]To determine whether SCGF could reverse the hematopoietic defects in CDH5-MAPK mice, 4 μg of SCGF was subcutaneously injected for 5 consecutive days, and the phenotypic and functional attributes of the hematopoietic system were analyzed 24 h after the final injection (Supplementary Figure 8A). No significant differences were observed in the frequency and absolute number of cells (Supplementary Figure 8B). While SCGF injection into littermate control mice confirmed that SCGF had no effect on steady-state hematopoiesis, SCGF injection into CDH5-MAPK mice had a significant effect on hematopoiesis (Supplementary Figures 8C-8E). SCGF injection significantly increased the frequency of phenotypic HSCs and HSPCs in CDH5-MAPK mice (Supplementary Figure 8C). SCGF injection also reversed the peripheral blood myeloid bias and restored blood cell counts in CDH5-MAPK mice (Supplementary Figures 8D and 8E). [Figure 8C-1] To determine whether SCGF could reverse the hematopoietic defects in CDH5-MAPK mice, 4 μg of SCGF was subcutaneously injected for 5 consecutive days, and the phenotypic and functional attributes of the hematopoietic system were analyzed 24 h after the final injection (Supplementary Figure 8A). No significant differences were observed in the frequency and absolute number of cells (Supplementary Figure 8B). While SCGF injection into littermate control mice confirmed that SCGF had no effect on steady-state hematopoiesis, SCGF injection into CDH5-MAPK mice had a significant effect on hematopoiesis (Supplementary Figures 8C-8E). SCGF injection significantly increased the frequency of phenotypic HSCs and HSPCs in CDH5-MAPK mice (Supplementary Figure 8C). SCGF injection also reversed the peripheral blood myeloid bias and restored blood cell counts in CDH5-MAPK mice (Supplementary Figures 8D and 8E). [Figure 8D-1]To determine whether SCGF could reverse the hematopoietic defects in CDH5-MAPK mice, 4 μg of SCGF was subcutaneously injected for 5 consecutive days, and the phenotypic and functional attributes of the hematopoietic system were analyzed 24 h after the final injection (Supplementary Figure 8A). No significant differences were observed in the frequency and absolute number of cells (Supplementary Figure 8B). While SCGF injection into littermate control mice confirmed that SCGF had no effect on steady-state hematopoiesis, SCGF injection into CDH5-MAPK mice had a significant effect on hematopoiesis (Supplementary Figures 8C-8E). SCGF injection significantly increased the frequency of phenotypic HSCs and HSPCs in CDH5-MAPK mice (Supplementary Figure 8C). SCGF injection also reversed the peripheral blood myeloid bias and restored blood cell counts in CDH5-MAPK mice (Supplementary Figures 8D and 8E). [Figure 8E-1] To determine whether SCGF could reverse the hematopoietic defects in CDH5-MAPK mice, 4 μg of SCGF was subcutaneously injected for 5 consecutive days, and the phenotypic and functional attributes of the hematopoietic system were analyzed 24 h after the final injection (Supplementary Figure 8A). No significant differences were observed in the frequency and absolute number of cells (Supplementary Figure 8B). While SCGF injection into littermate control mice confirmed that SCGF had no effect on steady-state hematopoiesis, SCGF injection into CDH5-MAPK mice had a significant effect on hematopoiesis (Supplementary Figures 8C-8E). SCGF injection significantly increased the frequency of phenotypic HSCs and HSPCs in CDH5-MAPK mice (Supplementary Figure 8C). SCGF injection also reversed the peripheral blood myeloid bias and restored blood cell counts in CDH5-MAPK mice (Supplementary Figures 8D and 8E). [Figure 9A-1]Because SCGF has been shown to promote bone formation, it is possible that the decreased plasma SCGF levels associated with BM inflammation observed in CDH5-MAPK mice could lead to osteopenia. Indeed, CDH5-MAPK mice exhibited an overall decrease in trabecular volume, trabecular number, and thickness, indicating that endothelial MAPK activation had a detrimental effect on bone health (Supplementary Figures 9A–9D). While SCGF had no effect on bone formation in control mice, it caused a significant increase in trabecular volume, trabecular number, and thickness in CDH5-MAPK mice, confirming its role in promoting bone formation (Supplementary Figures 9A–9D). Notably, SCGF expression was absent in hematopoietic cells and BMECs and was primarily expressed in BM stromal cells, including the BM Lepr+ and osteoblastic stromal subsets (Supplementary Figure 9E). However, analysis of SCGF expression in total stromal cells, Lepr+ cells, and osteoblasts from the BM of Tie2-IkB-SS, CDH5-MAPK, and CDH5-MAPK::IkB mice revealed no significant changes in mRNA expression (Supplementary Figure 9F), indicating that the reduction in plasma SCGF in CDH5-MAPK mice is not due to transcriptional changes. [Figure 9B-1]Because SCGF has been shown to promote bone formation, it is possible that the decreased plasma SCGF levels associated with BM inflammation observed in CDH5-MAPK mice could lead to osteopenia. Indeed, CDH5-MAPK mice exhibited an overall decrease in trabecular volume, trabecular number, and thickness, indicating that endothelial MAPK activation had a detrimental effect on bone health (Supplementary Figures 9A–9D). While SCGF had no effect on bone formation in control mice, it caused a significant increase in trabecular volume, trabecular number, and thickness in CDH5-MAPK mice, confirming its role in promoting bone formation (Supplementary Figures 9A–9D). Notably, SCGF expression was absent in hematopoietic cells and BMECs and was primarily expressed in BM stromal cells, including the BM Lepr+ and osteoblastic stromal subsets (Supplementary Figure 9E). However, analysis of SCGF expression in total stromal cells, Lepr+ cells, and osteoblasts from the BM of Tie2-IkB-SS, CDH5-MAPK, and CDH5-MAPK::IkB mice revealed no significant changes in mRNA expression (Supplementary Figure 9F), indicating that the reduction in plasma SCGF in CDH5-MAPK mice is not due to transcriptional changes. [Figure 9C-1]Because SCGF has been shown to promote bone formation, it is possible that the decreased plasma SCGF levels associated with BM inflammation observed in CDH5-MAPK mice could lead to osteopenia. Indeed, CDH5-MAPK mice exhibited an overall decrease in trabecular volume, trabecular number, and thickness, indicating that endothelial MAPK activation had a detrimental effect on bone health (Supplementary Figures 9A–9D). While SCGF had no effect on bone formation in control mice, it caused a significant increase in trabecular volume, trabecular number, and thickness in CDH5-MAPK mice, confirming its role in promoting bone formation (Supplementary Figures 9A–9D). Notably, SCGF expression was absent in hematopoietic cells and BMECs and was primarily expressed in BM stromal cells, including the BM Lepr+ and osteoblastic stromal subsets (Supplementary Figure 9E). However, analysis of SCGF expression in total stromal cells, Lepr+ cells, and osteoblasts from the BM of Tie2-IkB-SS, CDH5-MAPK, and CDH5-MAPK::IkB mice revealed no significant changes in mRNA expression (Supplementary Figure 9F), indicating that the reduction in plasma SCGF in CDH5-MAPK mice is not due to transcriptional changes. [Figure 9D-1]Because SCGF has been shown to promote bone formation, it is possible that the decreased plasma SCGF levels associated with BM inflammation observed in CDH5-MAPK mice could lead to osteopenia. Indeed, CDH5-MAPK mice exhibited an overall decrease in trabecular volume, trabecular number, and thickness, indicating that endothelial MAPK activation had a detrimental effect on bone health (Supplementary Figures 9A–9D). While SCGF had no effect on bone formation in control mice, it caused a significant increase in trabecular volume, trabecular number, and thickness in CDH5-MAPK mice, confirming its role in promoting bone formation (Supplementary Figures 9A–9D). Notably, SCGF expression was absent in hematopoietic cells and BMECs and was primarily expressed in BM stromal cells, including the BM Lepr+ and osteoblastic stromal subsets (Supplementary Figure 9E). However, analysis of SCGF expression in total stromal cells, Lepr+ cells, and osteoblasts from the BM of Tie2-IkB-SS, CDH5-MAPK, and CDH5-MAPK::IkB mice revealed no significant changes in mRNA expression (Supplementary Figure 9F), indicating that the reduction in plasma SCGF in CDH5-MAPK mice is not due to transcriptional changes. [Figure 9E-1]Because SCGF has been shown to promote bone formation, it is possible that the decreased plasma SCGF levels associated with BM inflammation observed in CDH5-MAPK mice could lead to osteopenia. Indeed, CDH5-MAPK mice exhibited an overall decrease in trabecular volume, trabecular number, and thickness, indicating that endothelial MAPK activation had a detrimental effect on bone health (Supplementary Figures 9A–9D). While SCGF had no effect on bone formation in control mice, it caused a significant increase in trabecular volume, trabecular number, and thickness in CDH5-MAPK mice, confirming its role in promoting bone formation (Supplementary Figures 9A–9D). Notably, SCGF expression was absent in hematopoietic cells and BMECs and was primarily expressed in BM stromal cells, including the BM Lepr+ and osteoblastic stromal subsets (Supplementary Figure 9E). However, analysis of SCGF expression in total stromal cells, Lepr+ cells, and osteoblasts from the BM of Tie2-IkB-SS, CDH5-MAPK, and CDH5-MAPK::IkB mice revealed no significant changes in mRNA expression (Supplementary Figure 9F), indicating that the reduction in plasma SCGF in CDH5-MAPK mice is not due to transcriptional changes. [Figure 9F-1]Because SCGF has been shown to promote bone formation, it is possible that the decreased plasma SCGF levels associated with BM inflammation observed in CDH5-MAPK mice could lead to osteopenia. Indeed, CDH5-MAPK mice exhibited an overall decrease in trabecular volume, trabecular number, and thickness, indicating that endothelial MAPK activation had a detrimental effect on bone health (Supplementary Figures 9A–9D). While SCGF had no effect on bone formation in control mice, it caused a significant increase in trabecular volume, trabecular number, and thickness in CDH5-MAPK mice, confirming its role in promoting bone formation (Supplementary Figures 9A–9D). Notably, SCGF expression was absent in hematopoietic cells and BMECs and was primarily expressed in BM stromal cells, including the BM Lepr+ and osteoblastic stromal subsets (Supplementary Figure 9E). However, analysis of SCGF expression in total stromal cells, Lepr+ cells, and osteoblasts from the BM of Tie2-IkB-SS, CDH5-MAPK, and CDH5-MAPK::IkB mice revealed no significant changes in mRNA expression (Supplementary Figure 9F), indicating that the reduction in plasma SCGF in CDH5-MAPK mice is not due to transcriptional changes. [Figure 10A-1] Wild-type mice were subjected to a myelosuppressive dose of irradiation (650 Rad)410 and injected with either 0.5 μg, 1 μg, or 2 μg of SCGF every other day for a total of seven injections, beginning on day +1 post-irradiation. Hematopoietic recovery was assessed for 21 days (Supplementary Figure 10A). Dose-response experiments showed that injection of 2 μg of SCGF resulted in significantly enhanced recovery of white blood cells, red blood cells, and platelets, confirming that SCGF enhances hematopoietic recovery after myelosuppressive stress (Supplementary Figure 10A). Using this strategy, the schematic diagram in Figure B shows the protocol used to test whether SCGF could improve hematopoietic recovery and maintain HSPC activity in both control and CDH5-MAPK mice (Supplementary Figure 10B). [Figure 10B-1]Wild-type mice were subjected to a myelosuppressive dose of irradiation (650 Rad)410 and injected with either 0.5 μg, 1 μg, or 2 μg of SCGF every other day for a total of seven injections, beginning on day +1 post-irradiation. Hematopoietic recovery was assessed for 21 days (Supplementary Figure 10A). Dose-response experiments showed that injection of 2 μg of SCGF resulted in significantly enhanced recovery of white blood cells, red blood cells, and platelets, confirming that SCGF enhances hematopoietic recovery after myelosuppressive stress (Supplementary Figure 10A). Using this strategy, the schematic diagram in Figure B shows the protocol used to test whether SCGF could improve hematopoietic recovery and maintain HSPC activity in both control and CDH5-MAPK mice (Supplementary Figure 10B). DETAILED DESCRIPTION OF THE INVENTION

[0100] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art.

[0101] As used herein, the term "about" means ±20% of the numerical value of the number with which it is being used. Thus, about 50% means a range of 40% to 60%, inclusive.

[0102] As used herein, the term "adaptive immunity" refers to the protection of a host organism from pathogens or toxins that is mediated by B cells and T cells and characterized by immunological memory. Adaptive immunity is highly specific to a given antigen and is highly adaptive.

[0103] "Administering," when used in conjunction with a therapeutic procedure, means providing or applying a therapeutic agent directly into or onto a target organ, tissue, or cell, or administering a therapeutic agent to a control, thereby causing the therapeutic agent to have a positive effect on the targeted organ, tissue, cell, or subject. Thus, as used herein, the term "administering," when used in conjunction with a composition comprising an angiocrine factor, can include, but is not limited to, providing the composition into or onto a target organ, tissue, or cell, or providing the composition systemically to a patient, for example, by intravenous injection, so that the therapeutic agent reaches the target organ, tissue, or cell. "Administering" may be accomplished by parenteral, oral, or topical administration, by inhalation, or by such methods in combination with other known techniques.

[0104] The term "agonist" as used herein refers to a chemical substance that can activate a receptor and induce a full or partial pharmacological response. Receptors can be activated or inactivated by either endogenous or exogenous agonists and antagonists, resulting in stimulation or inhibition of a biological response. A physiological agonist is a substance that produces the same physical response but does not bind to the same receptor. An endogenous agonist for a specific receptor is a compound naturally produced by the body that binds to and activates the receptor. A superagonist is a compound that can produce a maximum response greater than the endogenous agonist of the target receptor, and therefore has an efficiency greater than 100%. This does not necessarily mean that it is more potent than an endogenous agonist, but rather a comparison of the maximum possible response that can be generated within a cell after receptor binding. A full agonist binds to and activates a receptor, thereby demonstrating full efficacy at that receptor. A partial agonist also binds to and activates a given receptor, but has only partial efficacy at the receptor compared to a full agonist. An inverse agonist is a drug that binds to the same receptor binding site as an agonist and reverses the constitutive activity of the receptor. Inverse agonists exert the opposite pharmacological effect to receptor agonists. Irreversible agonists are a type of agonist that permanently bind to the receptor in such a way that the receptor is permanently activated. They differ from simple agonists in that the binding of an agonist to the receptor is reversible, while the binding of an irreversible agonist to the receptor is considered irreversible. This allows the compound to produce a short burst of agonist activity, followed by desensitization and receptor internalization, which, with prolonged treatment, produces a more antagonist-like effect. Selective agonists are specific to a particular type of receptor. The term "allogeneic" as used herein means that the donor and recipient are of different genetic makeups but the same species. T

[0105] As used herein, the term "autologous" means derived from the same individual.

[0106] The term "amino acid" is used to refer to an organic molecule that contains both an amino group and a carboxyl group. Those that function as the building blocks of naturally occurring proteins are alpha amino acids, in which both the amino group and the carboxyl group are attached to the same carbon atom. The terms "amino acid residue" or "residue" are used interchangeably to refer to amino acids that are incorporated into proteins, polypeptides, or peptides, including, but not limited to, naturally occurring amino acids and known analogs of natural amino acids that can function in a manner similar to the naturally occurring amino acids.

[0107] The abbreviations used herein for amino acids are the conventional abbreviations: A = Ala = alanine; R = Arg = arginine; N = Asn = asparagine; D = Asp = aspartic acid; C = Cys = cysteine; Q = Gln = glutamine; E = Glu = glutamic acid; G = Gly = glycine; H = His = histidine; I = Ile = isoleucine; L = Leu = leucine; K = Lys = lysine; M = Met = methionine; F = Phe = phenylalanine; P = Pro = proline; S = Ser = serine; T = Thr = threonine; W = Trp = tryptophan; Y = Tyr = tyrosine; V = Val = valine. Amino acids may be L-amino acids or D-amino acids. Amino acids may be replaced with modified synthetic amino acids to increase the half-life of the peptide, or to increase the potency of the peptide, or to increase the bioavailability of the peptide.

[0108] The following represent groups of amino acids that are conservatively substituted for one another: Alanine (A), serine (S), threonine (T), Aspartic acid (D), glutamic acid (E), Asparagine (N), Glutamine (Q), Arginine (R), Lysine (K), Isoleucine (I), leucine (L), methionine (M), valine (V), and Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0109] As used herein, the term "angiogenesis" refers to the process by which new blood vessels are formed from pre-existing vessels by the "sprouting" of endothelial cells, thereby growing the vascular tree.

[0110] As used herein, the term "angiocrine factors" refers to vascular niche-derived paracrine factors produced by endothelial cells that maintain organ homeostasis, balance stem cell self-renewal and differentiation, and coordinate organ regeneration and tumor growth. Angiocrine factors include secreted and membrane-bound inhibitory and stimulatory growth factors, trophogens, chemokines, cytokines, extracellular matrix components, exosomes, and other cellular products delivered by tissue-specific ECs that help regulate homeostatic and regenerative processes in a paracrine or juxtacrine manner. These factors also participate in adaptive healing and fibrotic remodeling. A subset of angiocrine factors can function as morphogens to determine the shape, structure, size, and pattern of regenerating organs. The angiocrine profile of each tissue-specific EC bed differs, reflecting the diversity of cell types found adjacent to ECs in an organ. While a subset of angiocrine factors is constitutively produced, some angiogenic factors can regulate the production of other tissue-specific angiocrine factors. For example, VEGF-A induces the expression of defined angiocrine factors through its interaction with VEGFR-1 and VEGFR-2 (Figure 1e). Similarly, FGF-2 (through activation of FGFR-1) and angiopoietin (through interaction with the receptor Tie2) trigger the expression of unique clusters of angiocrine factors. TSP-1 functions in a complex manner, not only directly influencing the differentiation of stem and progenitor cells but also acting as an inhibitory angiogenic factor. The molecular program governing the context-dependent production of angiocrine factors from organ-specific ECs remains undefined. (Rafii, S., et al., “Angiocrine functions of organ-specific endothelial cells,” Nature (2016) 529(7586):316-325)

[0111] As used herein, the terms "animal," "patient," and "subject" include, but are not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals. In some embodiments, the terms "animal," "patient," and "subject" may refer to mammals, including humans.

[0112] The term "antibody" as used herein refers to a polypeptide or group of polypeptides that are comprised of at least one binding domain formed from the folding of a polypeptide chain that has a three-dimensional binding space with an internal surface shape and charge distribution that is complementary to the characteristics of an antigenic determinant of an antigen.

[0113] The basic structural unit of an entire antibody molecule consists of four polypeptide chains: two identical light (L) chains (each containing approximately 220 amino acids) and two identical heavy (H) chains (each usually containing approximately 440 amino acids). The two heavy chains and two light chains are linked by a combination of non-covalent and covalent (disulfide) bonds. The molecule consists of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of the light chain and the N-terminal region of the heavy chain. Usually, both the light and heavy chains cooperate to form the antigen-binding surface. Human antibodies exhibit two types of light chains, kappa and lambda, and individual immunoglobulin molecules generally have only one or the other.

[0114] Antibodies may be oligoclonal, polyclonal, monoclonal, chimeric, CDR-grafted, multispecific, bispecific, catalytic, chimeric, humanized, fully human, anti-idiotypic, and labeled antibodies in soluble or conjugated form, as well as fragments, variants, or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques. Monoclonal antibodies (mAbs) can be produced by fusing mouse spleen cells from immunized donors with mouse myeloma cell lines to obtain established mouse hybridoma clones that grow in selective media. Hybridoma cells are immortalized hybrid cells resulting from the in vitro fusion of antibody-secreting B cells and myeloma cells. In vitro immunization, which refers to the primary activation of antigen-specific B cells in culture, is another established means of producing mouse monoclonal antibodies. Diverse libraries of immunoglobulin heavy (VH) and light (Vκ and Vλ) chain variable genes from peripheral blood lymphocytes can also be amplified by polymerase chain reaction (PCR) amplification. Genes encoding single polypeptide chains in which the heavy and light chain variable domains are linked by a polypeptide spacer (single-chain Fv or scFv) can be generated by randomly combining heavy and light chain V genes using PCR. The combinatorial library can then be cloned for display on the surface of filamentous bacteriophage by fusing it to a minor coat protein at the tip of the phage. The guided selection approach is based on shuffling human immunoglobulin V genes with rodent immunoglobulin V genes.The method involves (i) shuffling a repertoire of human λ light chains with the heavy chain variable region (VH) domain of a mouse monoclonal antibody that reacts with the antigen of interest; (ii) selecting a semi-human Fab on that antigen; (iii) using the selected λ light chain genes as "docking domains" for a library of human heavy chains in a second round of shuffling to isolate clonal Fab fragments bearing human light chain genes; (v) transfecting mouse myeloma cells by electroporation with a mammalian cell expression vector containing the genes; and (vi) expressing the Fab V genes that react with the antigen as intact IgG1, λ antibody molecules in the mouse myeloma. The antibody can be of any species. The term antibody also includes binding fragments of the antibodies of the invention; exemplary fragments include Fv, Fab, Fab', single-chain antibodies (svFC), dimeric variable regions (diabodies), and disulfide-stabilized variable regions (dsFv). Structural and functional domains can be identified by comparing the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. For example, computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. See, e.g., Bowie et al., Science 253:164 (1991) (incorporated by reference in its entirety).

[0115] As used herein, the terms "antigen" and "immunogen" are used interchangeably to refer to a substance that elicits an immune response. An "antigenic determinant" or "epitope" is an antigenic site on a molecule. A continuous antigenic determinant / epitope is essentially a linear chain. In an ordered structure such as a helical polymer or protein, an antigenic determinant / epitope will essentially be a limited region or patch within or on the surface of the structure that contains amino acid side chains from different parts of the molecule that are accessible to each other. These are conformational determinants.

[0116] The term "apoptosis" or "programmed cell death" refers to a highly regulated, active process that contributes to biological homeostasis, consisting of a series of biochemical events that result in various morphological changes, including blebbing, changes to the cell membrane, e.g., loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation, without causing damage to the organism.

[0117] Apoptotic cell death is induced by many different factors and involves many signaling pathways, some dependent on caspase proteases (a class of cysteine ​​proteases) and others independent of caspase. It can be triggered by many different cellular stimuli, including cell surface receptors, mitochondrial responses to stress, and cytotoxic T cells, which result in the activation of apoptotic signaling pathways.

[0118] Caspases involved in apoptosis transmit the apoptotic signal in a proteolytic cascade, where caspases cleave and activate other caspases, which then degrade other cellular targets, causing cell death. Caspases at the top end of the cascade include caspase 8 and caspase 9. Caspase 8 is the first caspase to engage in response to receptors containing a death domain (DD), such as Fas.

[0119] Receptors in the TNF receptor family are involved in the induction of apoptosis and inflammatory signaling. The Fas receptor (CD95) mediates apoptotic signaling via Fas ligand expressed on the surface of other cells. Fas-FasL interaction plays an important role in the immune system, and deficiencies in this system lead to autoimmunity, whereby Fas-mediated apoptosis eliminates autoreactive lymphocytes. Fas signaling is also involved in immune surveillance to eliminate transformed and virus-infected cells. Binding of Fas to oligomerized FasL on another cell activates apoptotic signaling via a cytoplasmic domain called the death domain (DD), which interacts with signaling adaptors including FAF, FADD, and DAX to activate the caspase proteolytic cascade. Caspase 8 and caspase 10 are activated first, then cleave and activate various cellular substrates, leading to downstream caspases and cell death.

[0120] Mitochondria participate in the apoptosis signaling pathway through the release of mitochondrial proteins into the cytoplasm. Cytochrome c, a key protein in electron transport, is released from mitochondria in response to apoptotic signals and activates the mitochondrially released protease Apaf-1. Activated Apaf-1 activates caspase-9 and the remaining caspase pathways. Smac / DIABLO is released from mitochondria and inhibits IAP proteins, which normally interact with caspase-9 to inhibit apoptosis. Bcl-2 family proteins regulate apoptosis when family members form complexes that enter the mitochondrial membrane, thereby regulating the release of cytochrome c and other proteins. TNF family receptors that trigger apoptosis directly activate the caspase cascade but can also activate Bid, a Bcl-2 family member that activates mitochondria-mediated apoptosis. Another Bcl-2 family member, Bax, is activated by this pathway and localizes to the mitochondrial membrane, increasing its permeability, thereby releasing cytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation and block apoptosis. Similar to cytochrome c, AIF (apoptosis-inducing factor) is a protein found in mitochondria, from which it is released upon apoptotic stimuli. While cytochrome c is involved in caspase-dependent apoptosis signaling, AIF release stimulates caspase-independent apoptosis, translocating to the nucleus and binding to DNA. DNA binding by AIF stimulates chromatin condensation and DNA fragmentation, likely through the recruitment of nucleases.

[0121] The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which then interacts with Apaf-1, which leads to the autocleavage and activation of caspase 9. Caspases 3, 6, and 7 are downstream caspases that are activated by upstream proteases and act to cleave cellular targets.

[0122] Granzyme B and perforin proteins released by cytotoxic T cells induce apoptosis in target cells, leading to the formation of transmembrane pores and possibly through caspase cleavage, although a caspase-independent mechanism of granzyme B-mediated apoptosis has been suggested.

[0123] Fragmentation of the nuclear genome by multiple nucleases, activated by apoptotic signaling pathways to create nucleosome ladders, is a characteristic cellular response of apoptosis. One nuclease involved in apoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse (CAD). DFF / CAD is activated during apoptosis by cleavage of its associated inhibitor, ICAD, by caspase proteases. DFF / CAD interacts with chromatin components such as topoisomerase II and histone H1 to condense chromatin structure and likely recruit CAD to chromatin. Another apoptosis-activating protease is endonuclease G (EndoG). EndoG is encoded by the nuclear genome but localizes to mitochondria in normal cells. EndoG may play a role in mitochondrial genome replication and apoptosis. Apoptotic signaling triggers the release of EndoG from mitochondria. The EndoG and DFF / CAD pathways are independent, as the EndoG pathway still occurs in cells lacking DFF.

[0124] Hypoxia and hypoxia followed by reoxygenation can cause cytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3), a serine-threonine kinase ubiquitously expressed in most cell types, appears to mediate or enhance apoptosis through many stimuli that activate the mitochondrial cell death pathway. (Loberg, RD, et al., J. Biol. Chem. 277(44):41667-673 (2002)) It has been demonstrated to induce caspase-3 activation and activate the proapoptotic tumor suppressor gene p53. GSK-3 has also been suggested to promote the activation and translocation of Bax, a proapoptotic Bcl-2 family member, and induce cytochrome c release upon aggregation and mitochondrial localization. Akt is a key regulator of GSK-3, and its phosphorylation and inactivation may mediate some of Akt's antiapoptotic effects.

[0125] As used herein, the term "autocrine signaling" refers to a type of cell signaling in which cells secrete signaling molecules that act on themselves or other neighboring cells of the same type.

[0126] The terms "autologous" or "self," used interchangeably herein, mean derived from the same organism.

[0127] As used herein, the term "bond" and other grammatical forms refer to a permanent force of attraction between chemical substances.

[0128] "Binding fragments" of antibodies can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies.

[0129] A "bispecific" or "bifunctional" antibody is an antibody in which each of its binding sites is not identical. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical.

[0130] As used herein, the term "binding specificity" includes both binding to a specific partner and not binding to other molecules. Functionally important binding can occur with a range of low to high affinity, and design elements can suppress undesired interactions. Post-translational modifications can also alter the chemical nature and structure of the interaction. "Promiscuous binding" may involve a degree of structural plasticity, which can result in different subsets of residues being important for binding to different partners. "Relative binding specificity" is a characteristic in biochemical systems in which a molecule interacts differentially with its target or partner, thereby affecting them distinctly depending on the identity of each target or partner.

[0131] As used herein, the term "biomarker" (or "biosignature") refers to a peptide, protein, nucleic acid, antibody, gene, metabolite, or any other substance used as an indicator of a biological state. It is a characteristic that can be objectively measured and evaluated as a cellular or molecular indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention. As used herein, the term "indicator" refers to any substance, number, or ratio derived from a set of observed facts whose relative change over time or whose presence or absence may reveal a visible or telltale signal, sign, mark, note, or symptom. Once a proposed biomarker has been validated, it may be used to diagnose disease risk, the presence of disease, or to tailor an individual's treatment for disease (selection of drug treatment or administration regime). When evaluating potential drug therapies, biomarkers may be used as surrogates for natural endpoints such as survival or irreversible morbidity. If a treatment alters the biomarker and that alteration directly correlates with improved health, the biomarker can serve as a surrogate endpoint for assessing clinical benefit. Clinical endpoints are variables that can be used to measure how a patient feels, functions, or survives. Surrogate endpoints are biomarkers that are intended to stand in for clinical endpoints, and these biomarkers are demonstrated to predict the clinical endpoint with a level of confidence acceptable to regulatory authorities and the clinical community.

[0132] Bone Cells. Four cell types of bone are involved in its formation and maintenance: 1) osteoprogenitor cells, 2) osteoblasts, 3) osteocytes, and 4) osteoclasts.

[0133] Osteoprogenitor cells. Osteoprogenitor cells arise from mesenchymal cells and occur within the periosteum and the endosteum of mature bone. They are found in the region of the embryonic mesenchymal compartment where bone formation begins and in the region near the surface of developing bone. Structurally, osteoprogenitor cells differ from the mesenchymal cells from which they originate. They are irregularly shaped, elongated cells with pale-staining cytoplasm and pale-staining nuclei. Osteoprogenitor cells proliferate by mitosis and are identified primarily by their location and association with osteoblasts. Some osteoprogenitor cells differentiate into osteocytes. Although osteoblasts and osteocytes are no longer mitotic, a population of osteoprogenitor cells has been shown to persist throughout lifespan.

[0134] Osteoblasts. Located at the surface of the osteoid joint (a narrow area at the surface of bone where the newly formed organic matrix has not yet mineralized), osteoblasts originate from osteoprogenitor cells. They are immature, mononuclear, bone-forming cells that synthesize collagen and control mineralization. Morphologically, osteoblasts are distinguishable from osteoprogenitor cells; they generally have larger, more rounded nuclei, more prominent nucleoli, and a much more basophilic cytoplasm. Osteoblasts produce a protein mixture known as osteoid, primarily composed of type I collagen, which mineralizes into bone. Osteoblasts also manufacture hormones, such as prostaglandins, alkaline phosphatase, enzymes involved in bone mineralization, and matrix proteins.

[0135] Osteocytes. Osteocytes are star-shaped, mature bone cells derived from osteoblasts. They are the most abundant cells found in compact bone and maintain bone structure. Like osteoblasts, bone cells are incapable of mitosis. They actively participate in the daily turnover of bone matrix and reside in small spaces, cavities, gaps, or depressions in the bone matrix called lacunae. Osteocytes are thought to be part of a cellular feedback mechanism that maintains the bone matrix, regulates calcium homeostasis, and directs bone formation where it is most needed. Bone adapts by becoming stronger to withstand applied forces, and osteocytes can detect mechanical deformation and mediate bone formation by osteoblasts.

[0136] Osteoclasts. Osteoclasts are derived from a monocyte stem cell lineage, possess a phagocytic mechanism similar to macrophages, and are commonly found in bone cavities called Howship's lacunae. They are large, multinucleated cells specialized for bone resorption. During resorption, osteoclasts seal off areas of the bone surface and then, when activated, excrete hydrogen ions, creating a highly acidic environment that dissolves hydroxyapatite components. Osteoclast number and activity increase when calcium absorption is stimulated by injection of parathyroid hormone (PTH), whereas osteoclast activity is suppressed by injection of calcitonin, a hormone produced by thyroid parafollicular cells.

[0137] Bone Matrix. Bone matrix accounts for approximately 90% of the total weight of compact bone and is composed of microcrystalline calcium phosphate similar to hydroxyapatite (60%) and fibrous type I collagen (27%). The remaining 3% is composed of minor collagen types and other proteins, such as osteocalcin, osteonectin, osteopontin, and bone sialoprotein, as well as proteoglycans, glycosaminoglycans, and lipids. Bone extracellular matrix glycoproteins and proteoglycans bind various growth factors and cytokines and serve as a repository of conserved signals that act on osteoblasts and osteoclasts. Examples of growth factors and cytokines found in bone matrix include, but are not limited to, bone morphogenetic proteins (BMPs), epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-I), transforming growth factor (TGF), bone-derived growth factor (BDGF), cartilage-derived growth factor (CDGF), skeletal growth factor (hSGF), interleukin 1 (IL-I), and macrophage-derived factors. There is an emerging understanding that extracellular matrix molecules themselves can play regulatory roles, providing both direct biological effects on cells as well as important spatial and contextual information.

[0138] Periosteum and Endosteum. The periosteum is the outer layer of fibrous connective tissue of bone, except at the articular surface of the bone. Its attachment to bone varies with location and age. In infant bones, the periosteum peels easily. In adult bones, it adheres more firmly, especially at the insertions of tendons and ligaments, and more periosteal fibers penetrate the bone as Sharpey's perforators (bundles of collagen fibers that enter the bone's peripheral lamellae). The periosteum consists of two layers: the outer layer is composed of coarse fibrous connective tissue that contains few cells and many blood vessels and nerves. The inner layer, which is less vascular but more cellular, contains many elastic fibers. During growth, a primitive connective tissue osteogenic layer forms the inner layer of the periosteum. In adults, this is represented only by scattered rows of flat cells closely attached to the bone. The periosteum serves as a support bed for blood vessels and nerves to the bone, as well as for the anchorage of tendons and ligaments. The osteogenic layer, considered part of the periosteum, is known to supply osteoblasts for growth and repair and serves as an important limiting layer that controls and limits the spread of bone formation. Both the periosteum and the bone it contains are regions of the connective tissue compartment and are not separated from each other or from other connective tissues by a basal lamella or basement membrane. Periosteal stem cells have been shown to be important for bone regeneration and repair. (Zhang et al., 2005, J. Musculoskelet. Neuronal. Interact. 5(4):360-362).

[0139] The endosteum lines the surfaces of the intrabone cavities (the medullary cavity and central canal) and also the trabecular surfaces of the medullary cavity. In growing bone, it is composed of delicate striae of medullary reticular connective tissue, beneath which is a layer of osteoblasts. In adults, the bone-forming cells are flattened and indistinguishable as a separate layer. They can transform into osteogenic cells when there is a stimulus for bone formation, such as after a fracture.

[0140] Bone Components. Bone is composed of cells and an intercellular matrix of organic and inorganic material. The organic fraction consists of collagen, glycosaminoglycans, proteoglycans, and glycoproteins. The protein matrix of bone is primarily composed of collagen, a family of fibrous proteins capable of forming insoluble, rigid fibers. The predominant collagen in bone is type I collagen. The inorganic component of bone, which is responsible for stiffness and can account for up to two-thirds of the fat-free dry weight, is primarily composed of calcium phosphate and calcium carbonate, with small amounts of magnesium hydroxide, magnesium fluoride, and magnesium sulfate in the form of calcium hydroxyapatite. Composition varies with age and multiple dietary factors. Bone minerals form long, fine crystals that add strength and rigidity to collagen fibers; the process by which they are laid down is called mineralization.

[0141] As used herein, the term "bone marrow" refers to the soft hematopoietic tissue that fills bone cavities and contains fat and immature and mature blood cells, including white blood cells, red blood cells, and platelets. Bone marrow contains a variety of progenitor and mature cell types, such as hematopoietic cells, which are precursors to a wide range of connective tissue cells, including mature blood cells and mesenchymal stem cells (also called stromal cells), both of which are capable of differentiating into other cell types. Hematopoietic stem cells (HSCs) in the bone marrow give rise to two major types of cells: the myeloid lineage (including monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, and megakaryocytes or platelets) and the lymphoid lineage (including T cells, B cells, and natural killer cells).

[0142] Bone Remodeling. In adults, bone is constantly being broken down by osteoclasts and remodeled by osteoblasts. It has been reported that as much as 18% of bone is recycled each year through a regenerative process known as bone remodeling, which maintains bone rigidity. The balance of this dynamic process changes as people age. In younger people, it favors bone formation, whereas in older people, it favors resorption. As new bone material is added peripherally from the inner surface of the periosteum, there is hollowing of the interior area, forming the marrow cavity. This destruction of bone tissue is caused by osteoclasts, which enter the bone through blood vessels. Osteoclasts dissolve both the inorganic and protein portions of the bone matrix. Each osteoclast extends numerous cellular processes into the matrix, pumping hydrogen ions into the surrounding material, thereby acidifying and solubilizing it. Blood vessels also import hematopoietic cells, which reside in the bone marrow throughout the organism's lifespan.

[0143] The number and activity of osteoclasts must be tightly regulated. If there are too many active osteoclasts, too much bone will dissolve, leading to osteoporosis. Conversely, if not enough osteoclasts are produced, the bone will not hollow out for marrow, leading to osteopetrosis (also known as stone bone disease, a disorder in which bone hardens and becomes dense).

[0144] The terms "bone marrow transplant" (BMT) or "hematopoietic stem cell transplant" (HSCT) are used interchangeably to refer to a procedure in which bone marrow stem cells are harvested from one individual (donor) and administered to another individual (recipient). Stem cells can be harvested directly from the bone marrow or from the blood by leukapheresis. Bone marrow transplants can be autologous (using the patient's own stem cells harvested from the bone marrow and stored prior to the procedure), allogeneic (using stem cells donated by a non-identical twin), or syngeneic (using stem cells donated by an identical twin).

[0145] As used herein, the term "CD34" is a marker found on the surface of bone marrow stem cells.

[0146] As used herein, the term "CD45" refers to lymphocyte common antigen.

[0147] The term "Clec11a / stem cell growth factor-α" or "SCGF" refers to a secreted sulfated glycoprotein that functions as a key regulator of bone health and has been suggested as a growth factor for primitive hematopoietic progenitor cells.

[0148] The term "cancellous bone tissue," also known as trabeculae or spongy bone, refers to the open, cellular, porous network of bone. It is composed of a network of rod- and plate-like elements that fill the interior of bone, lightening the overall structure and providing room for blood vessels and bone marrow, allowing the blood supply to surround the bone. Trabecular bone accounts for 20% of the total bone mass but has nearly 10 times the surface area of ​​cortical bone. It does not contain Haversian sites or osteons and has a porosity of approximately 30% to 90%. In cancellous bone, the marrow cavities are relatively large and irregularly arranged, and the bone material takes the form of elongated, interlocking trabeculae and sharp spicules. The head of the bone, called the epiphysis, has a spongy appearance and is composed of elongated, irregular trabeculae or rods that interlock to form a lattice. The interstitial spaces contain bone marrow, while the thin outer shell appears dense. The irregular medullary cavity of the epiphysis becomes continuous with the central medullary cavity of the bone shaft, called the diaphysis, the walls of which are formed by thin plates of cortical bone.

[0149] The term "cell cycle" refers to the progression of cells through four phases: G1 (interphase), S (DNA synthesis phase), G2 (interphase), and M (mitosis phase). (Ibid., citing Nakamura-Ishizu, A., et al., Development (2014) 141:4656-4666; Sisken, JE and Morasca, L., J. Cell Biol. (1965) 25:179-189). Cells that progress beyond the restriction point of G1 enter the S phase, while cells that do not progress beyond the restriction point remain undivided. These undivided cells can exit the cell cycle and enter the G0 phase, a state in which cells are called quiescent or resting. (Ibid., citing Pardee, AB, Proc. Natl. Acad. Sci. USA (1974) 71:1286-90). Such non-cycling cells in the G0 phase can either reversibly re-enter the cell cycle and divide (ibid., citing Cheung, TH and Rando, TA, Nat. Rev. Mol. Cell Biol. (2013) 14:329-340) or remain quiescent, lose cycling potential, and in some cases, senesce (ibid., citing Campisi, J. Cell (2005) 120:513-22).

[0150] As used herein, the term "cell lineage" or "lineage" refers to the developmental history of a differentiated cell as traced back to the cell from which it arises.

[0151] The term "chemokine" as used herein refers to a family of low molecular weight (8-11 kDa) structurally related proteins with diverse immune and neurological functions (Mackay CRNat Immunol., Vol. 2:95-101, (2001); Youn B. et al. Immunol Rev. (2000) Vol. 177:150-174), which can be classified into four subfamilies (C, CC, CXC, and CX3C) based on the relative positions of conserved cysteine ​​residues (Rossi D. et al. Annu Rev Immunol. (2000) 18:217-242). Chemokines are essential molecules for directing leukocyte migration between blood, lymph nodes, and tissues. Because they are not always restricted to a single type of receptor, they constitute a complex signaling network (Loetscher P. et al. J. Biol. Chem. (2001) 276:2986-2991). Chemokines affect cells by activating surface receptors, which are seven-transmembrane domain G protein-coupled receptors. The response of leukocytes to a particular chemokine is determined by the expression of the chemokine receptor. Binding of chemokines to receptors activates various signal transduction cascades, similar to the action of cytokines, which achieve the activation of biological responses. Upon activation, the secretion of ligands for the CCR5 receptor regulates normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α / , and MIP-1β (Schrum S. et al. J Immunol. (1996) 157:3598-3604), while the ligand for CXC chemokine receptor 3 (CXCR3), inducible protein (IP)-10 (Taub D.D. et al. J Exp Med. (1993) 177:1809-1814), promotes undesirable elevated T cell proliferation. H1 Furthermore, elevated levels of the harmful pro-inflammatory cytokines IL-2 and IFN-γ correlate with type 1 diabetes (T1D) (Rabinovitch A. et al. Cell Biochem Biophys. (2007) 48(2-3):159-63). Chemokines are involved in the T H1It has been observed in pancreatic infiltration and other inflammatory lesions characterized by T cell infiltration (Bradley LM et al. J Immunol. (1999). 162:2511-2520).

[0152] As used herein, the term "chemotherapy" refers to treatment using drugs to destroy cancer cells, but is also used in cancer-free bone marrow transplant patients to ensure successful engraftment.

[0153] As used herein, the term "conditioning" refers to a combination of chemotherapy drugs and, optionally, radiation given several days before transplantation that collectively prepare the body for transplantation.

[0154] As used herein, the term "contact" and its various grammatical forms refer to the state or condition of contact or of immediate or local proximity.

[0155] The term "cortical bone tissue" (also called compact bone or compact substance) refers to the hard outer layer of bone, which has minimal gaps and spaces. This tissue gives bone its smooth, white, and solid appearance. Cortical bone is composed of Haversian sites (canals through which blood vessels and connective tissue pass through bone) and osteons (the basic structural units of cortical bone, including Haversian canals and their concentrically arranged lamellae). Thus, in cortical bone, bone is surrounded by a blood supply. Cortical bone has a porosity of approximately 5% to approximately 30% (inclusive) and accounts for approximately 80% of the total bone mass in the adult skeleton. In cortical bone, the spaces or channels are narrow and the bone material is densely packed.

[0156] As used herein, the term "cytokine" refers to small, soluble protein substances secreted by cells that exert various effects on other cells. Cytokines mediate many important physiological functions, including growth, development, wound healing, and immune responses. They act by binding to cell-specific receptors on the cell membrane. This initiates different signaling cascades within the cell, ultimately leading to biochemical and phenotypic changes in the target cell. Generally, cytokines act locally. They include type I cytokines, including many interleukins, as well as several hematopoietic growth factors; type II cytokines, including interferons and interleukin-10; tumor necrosis factor (TNF)-related molecules, including TNFα and lymphotoxin; members of the immunoglobulin superfamily, including interleukin-1 (IL-1); and chemokines, a family of molecules that play important roles in various immune and inflammatory functions. The same cytokine can affect cells differently depending on their state. Cytokines often regulate the expression of other cytokines, triggering their cascades.

[0157] As used herein, the term "damage-associated molecular patterns" (DAMPs) refers to endogenous danger molecules released from damaged or dying cells that activate the innate immune system by interacting with pattern recognition receptors (PRRs).

[0158] As used herein, the term "derived" is meant to encompass any method for receiving, obtaining, or modifying something from a source of origin.

[0159] As used herein, the terms "detecting," "determining," and other grammatical forms thereof are used to refer to methods performed to identify or quantify a biomarker, e.g., the presence or level of an miRNA, or the presence or absence of a condition in a biological sample. The amount of biomarker expression or activity detected in a sample can be at or below the detection level of the assay or method.

[0160] As used herein, the term "differentiation" refers to a developmental process involving an increasing level of organization or complexity of cells or tissues with more specialized functions.

[0161] As used herein, the term "disease" or "disorder" refers to an impairment of health or a state of abnormal function.

[0162] The term "endogenous" as used herein refers to something that is naturally occurring, incorporated within, contained within, attached to, attached to, or present within.

[0163] As used herein, the term "engraftment" refers to the process by which normal growth of transplanted (donor) stem cells and production of blood cells in the patient's (recipient's) bone marrow space resumes after transplantation.

[0164] As used herein, the term "enrich" refers to increasing the proportion of a desired substance, for example, to increase the relative frequency of a cell or cellular component subtype compared to its natural frequency in a cell population. Positive selection, negative selection, or both are generally considered necessary for any enrichment scheme. Selection methods include, but are not limited to, magnetic separation and fluorescence-activated cell sorting (FACS).

[0165] As used herein, the term "erythropoiesis" refers to the formation of red blood cells in hematopoietic tissues. During early fetal development, erythropoiesis occurs in the yolk sac, spleen, and liver. After birth, all erythropoiesis occurs in the bone marrow. The erythroid lineage in the bone marrow and spleen is initiated with early progenitor cells, proerythroblasts, derived from pluripotent stem cells. In adult bone marrow, definitive erythropoiesis is initiated when HSC-derived common myeloid progenitors (pluripotent stem cells) commit to the erythroid lineage. The appearance of pronormoblasts (also called proerythroblasts or ribriblasts) marks the first differentiation step. This is followed by early, intermediate, and late normoblast (erythroid) stages, at which point the nucleus is expelled and the cell becomes a reticulocyte. Upon leaving the bone marrow, reticulocytes enter the circulation and become fully mature RBCs.

[0166] As used herein, the term "exogenous" refers to something that is not naturally occurring or that originates or is produced outside of a particular cell, organism, or species.

[0167] As used herein, the term "growing" and its various grammatical forms refers to the process by which dispersed living cells multiply in vitro in a culture medium resulting in an increase in the number or quantity of living cells.

[0168] As used herein, the term "expression" and its various grammatical forms refer to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polynucleotide, or protein. The transcript and the encoded polypeptide are sometimes collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may also include splicing of the mRNA in a eukaryotic cell. Expression may also refer to post-translational modification of a polypeptide or protein.

[0169] As used herein, the term "extracellular matrix" (or "ECM") refers to the scaffolding in a cell's external environment with which cells interact through specific cell surface receptors. The extracellular matrix serves many functions, including, but not limited to, providing support and anchorage for cells, separating one tissue from another, and regulating intracellular communication. The extracellular matrix is ​​composed of an interlocking mesh of fibrous proteins and glycosaminoglycans (GAGs). Examples of fibrous proteins found in the extracellular matrix include collagen, elastin, fibronectin, and laminin. Examples of GAGs found in the extracellular matrix include proteoglycans (e.g., heparin sulfate), chondroitin sulfate, keratan sulfate, and non-proteoglycan polysaccharides (e.g., hyaluronic acid). The term "proteoglycan" refers to a group of glycoproteins containing a core protein attached to one or more glycosaminoglycans.

[0170] As used herein, the term "fragment" or "peptide fragment" refers to a smaller portion derived, cleaved, or broken from a larger peptide, polypeptide, or protein, which retains the desired biological activity of the larger peptide, polypeptide, or protein.

[0171] As used herein, the term "graft" refers to tissue or organs infused or transplanted from a donor to a recipient, including, but not limited to, self-tissue transplanted from one body site to another in the same individual (an "autograft"), tissue transplanted between genetically identical individuals or tissues sufficiently immunologically compatible to permit tissue transplantation (a "syngeneic graft"), tissue transplanted between genetically different members of the same species (an "allogeneic graft" or "allograft"), and tissue transplanted between different species (a "xenograft").

[0172] As used herein, the term "growth factor" refers to an extracellular polypeptide molecule that binds to a cell surface receptor and triggers an intracellular signaling pathway, resulting in proliferation, differentiation, or other cellular response, stimulating the accumulation of proteins and other macromolecules, e.g., by increasing the rate of synthesis, decreasing the rate of degradation, or both. Exemplary growth factors include fibroblast growth factor (FGF), insulin-like growth factor (IGF-I), transforming growth factor-beta (TGF-β), and vascular endothelial growth factor (VEGF).

[0173] Fibroblast Growth Factors (FGFs). The fibroblast growth factor (FGF) family currently has more than 12 structurally related members. FGF1 is also known as acidic FGF; FGF2 is sometimes called basic FGF (bFGF); and FGF7 is sometimes referred to as keratinocyte growth factor. In vertebrates, more than 12 different FGF genes are known; they can generate hundreds of protein isoforms through RNA splicing or start codon variation in various tissues. FGFs can activate a series of receptor tyrosine kinases called fibroblast growth factor receptors (FGFRs). Receptor tyrosine kinases are proteins that extend across the cell membrane. The portion of the protein that binds to paracrine factors is located on the extracellular side, while the resting tyrosine kinase (i.e., the protein that can phosphorylate other proteins by sequestering ATP) is located on the intracellular side. When an FGF receptor binds to an FGF (and only when it binds to an FGF), the dormant kinase is activated and phosphorylates and activates specific proteins in the responding cell.

[0174] FGFs are involved in several developmental functions, including angiogenesis (blood vessel formation), mesoderm formation, and axon outgrowth. Although FGFs can often substitute for one another, their expression patterns confer distinct functions to them. For example, FGF2 is particularly important for angiogenesis, while FGF8 is involved in midbrain and limb development.

[0175] Insulin-like growth factor 1 (IGF-1). IGF-1, a hormone similar in molecular structure to insulin, has growth-promoting effects on nearly all cells in the body, particularly skeletal muscle, cartilage, bone, liver, kidney, nerves, skin, hematopoietic cells, and lungs. It plays a key role in childhood growth and continues to have anabolic effects in adults. IGF-1 is produced primarily as an endocrine hormone by the liver and in a paracrine / autocrine manner in target tissues. Its production is stimulated by growth hormone (GH) and can be delayed by nutritional deficiency, growth hormone insensitivity, absence of growth hormone receptors, or impaired downstream signaling molecules, including tyrosine protein phosphatase type 11 (also known as SHP2, encoded by the PTPN11 gene in humans) and signal transducer and activator of transcription 5B (STAT5B), a member of the STAT family of transcription factors. Its primary action is mediated by binding to its specific receptor, the insulin-like growth factor 1 receptor (IGF1R), which is present in many cell types in many tissues. Upon binding to the receptor tyrosine kinase IGF1R, intracellular signaling is initiated; IGF-1 is one of the most potent natural activators of the AKT signaling pathway, a stimulator of cell growth and proliferation, and a potent inhibitor of programmed cell death. IGF-1 is the primary mediator of the effects of growth hormone (GH). Growth hormone is produced in the pituitary gland and released into the bloodstream, which subsequently stimulates the liver to produce IGF-1. IGF-1, in turn, stimulates whole-body growth. In addition to its insulin-like effects, IGF-1 can also regulate cell growth and development, particularly in nerve cells, as well as cellular DNA synthesis.

[0176] IGF-1 has been shown to increase the expression level of the chemokine receptor CXCR4 (a receptor for stromal cell-derived factor-1, SDF-1) and significantly enhance the migratory response of MSCs to SDF-1 (Li, Y, et al. 2007 Biochem. Biophys. Res. Communic. 356(3):780-784). The increase in IGF-1-induced MSC migration in response to SDF-1 was attenuated by PI3 kinase inhibitors (LY294002 and wortmannin), but not by the mitogen-activated protein / ERK kinase inhibitor PD98059. Without being limited to a particular theory, these data suggest that IGF-1 enhances the migratory response of MSCs through CXCR4 chemokine receptor signaling, which is PI3 / Akt-dependent.

[0177] Transforming growth factor beta (TGF-β). The TGF-β superfamily includes over 30 structurally related members that mediate some of the most important interactions in development. Proteins encoded by TGF-β superfamily genes are processed to contain mature peptides at the carboxy-terminal region. These peptides dimerize into homodimers (with themselves) or heterodimers (with other TGF-β peptides) and are secreted from cells. The TGF-β superfamily includes the TGF-β family, the activin family, bone morphogenetic proteins (BMPs), the Vg-1 family, and other proteins, including glial-derived neurotrophic factor (GDNF, required for the differentiation of renal and enteric neurons) and Müllerian inhibitory factor, which is involved in mammalian sex determination. TGF-β family members TGF-β1, 2, 3, and 5 are important in regulating the formation of extracellular matrix between cells and in regulating cell division (both positively and negatively). TGF-β1 increases the amount of extracellular matrix epithelial cells make by stimulating collagen and fibronectin synthesis and inhibiting matrix degradation. TGF-β may be important in controlling where and when epithelia can branch to form ducts in the kidney, lung, and salivary glands.

[0178] Vascular endothelial growth factor (VEGF). VEGF is a growth factor that mediates many functions of endothelial cells, including proliferation, migration, invasion, survival, and permeability. VEGF and its corresponding receptors are key regulators in the cascade of molecular and cellular events that ultimately lead to the development of the vascular system, either through vasculogenesis, angiogenesis, or the formation of the lymphatic vasculature. VEGF is a key regulator in physiological angiogenesis and also plays an important role in skeletal growth and repair.

[0179] The normal function of VEGF is to create new blood vessels during embryonic development, after injury, and to bypass blocked blood vessels. In the established mature vasculature, the endothelium plays a key role in maintaining the homeostasis of surrounding tissues by providing a communication network to neighboring tissues to respond to their needs as needed. Furthermore, the vasculature provides growth factors, hormones, cytokines, chemokines, and metabolites required by surrounding tissues and acts as a barrier to restrict the movement of molecules and cells.

[0180] As used herein, the term "immune reconstitution" or "reconstitution" refers to the process of reconstituting the immune system from transplanted HSCs following HSCT.

[0181] The terms "immune response" and "immune-mediated" are used interchangeably herein to refer to any functional manifestation of a subject's immune system against either foreign or self antigens, regardless of whether the outcome of these responses is beneficial or harmful to the subject.

[0182] The term "immune system" as used herein refers to the body's defense system against disease. The innate immune system provides a non-specific first line of defense against pathogens. It includes physical barriers (e.g., skin) and both cellular (granulocytes, natural killer cells) and humoral (complement system) defense mechanisms. The response of the innate immune system is immediate, but unlike the adaptive immune system, it does not provide lasting immunity to pathogens.

[0183] As used herein, the term "innate immunity" refers herein to various natural resistance mechanisms, e.g., anatomical barriers, antimicrobial peptides, the complement system, and macrophages and neutrophils containing nonspecific pathogen-recognition receptors, by which adaptive immunity is induced following an initial encounter with a pathogen. Innate immunity is always present in all individuals, does not increase with repeated exposure to a given pathogen, and distinguishes between groups of similar pathogens rather than responding to a specific pathogen.

[0184] The terms "immunomodulatory," "immunomodulatory drug," and "immunomodulatory" are used interchangeably herein to refer to substances, agents, or cells capable of directly or indirectly enhancing or attenuating an immune response, for example, by expressing chemokines, cytokines, and other mediators of the immune response.

[0185] As used herein, the term "immunosuppressant" refers to an agent that reduces the body's immune response.

[0186] As used herein, the term "immunosuppression" refers to a state of reduced immunity or a decreased immune response of the body. As used herein, the term "immunosuppressant therapy" refers to a treatment that reduces the activity of the body's immune system.

[0187] The term "inflammation" as used herein refers to the physiological process by which vascularized tissue responds to injury. See, e.g., FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers, Philadelphia (1999) at pp. 1051-1053 (incorporated herein by reference). During the inflammatory process, cells involved in detoxification and repair are recruited to sites damaged by inflammatory mediators. Inflammation is often characterized by intense infiltration of leukocytes, particularly neutrophils (polymorphonuclear cells), at the site of inflammation. These cells promote tissue damage by releasing toxic substances at the blood vessel wall or intact tissue. Traditionally, inflammation has been divided into acute and chronic responses. The term "acute inflammation" as used herein refers to a rapid, transient (minutes to days), and relatively uniform response to acute injury characterized by the accumulation of fluid, plasma proteins, and neutrophil leukocytes. The term "chronic inflammation," as used herein, refers to inflammation that is more long-term and has an ambiguous, indefinite termination. Chronic inflammation takes over when acute inflammation persists due to incomplete clearance of the initial inflammatory agent or as a result of multiple acute events occurring at the same location. Chronic inflammation, which involves the influx of lymphocytes and macrophages and the growth of fibroblasts, can lead to tissue scarring at sites of prolonged or repeated inflammatory activity.

[0188] As used herein, the terms "inflammatory mediator" or "inflammatory cytokine" refer to molecular mediators of the inflammatory process. These soluble, diffusible molecules act both locally at the site of tissue injury and infection as well as at more distant sites. Some inflammatory mediators are activated by the inflammatory process, while others are synthesized and / or released from cellular sources in response to acute inflammation or by other soluble inflammatory mediators. Examples of inflammatory mediators of the inflammatory response include, but are not limited to, histamine, serotonin, and neuropeptides, plasma proteases, complement, kinins, coagulation and fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, platelet-activating factors (PAFs), peptides, and amines, including proinflammatory cytokines, including, but not limited to, interleukin-1-beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IF-γ), and interleukin-12 (IL-12).

[0189] As used herein, the term "infusion" and other grammatical forms refer to the introduction of a fluid other than blood into a vein.

[0190] The terms "inhibiting," "inhibit," or "inhibiting" are used herein to refer to reducing the amount or rate of a process, stopping a process entirely, or decreasing, limiting, or blocking its action or function. Inhibition may include a reduction or decrease of the amount, rate, action function, or process of a substance by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%.

[0191] As used herein, the term "inhibitor" refers to a second molecule that binds to, contacts, or otherwise interferes with the activity of a first molecule, thereby reducing the activity of the first molecule.

[0192] The term "injury" as used herein refers to damage or harm to a bodily structure or function caused by an external agent or force, which may be physical or chemical, or an internal condition.

[0193] The term "isolated" is used herein to refer to a material, such as, but not limited to, a nucleic acid, peptide, polypeptide, or protein, that is (1) substantially or essentially free from components that normally accompany or interact with it as found in a naturally occurring environment. The terms "substantially free" or "essentially free" are used herein to refer to being substantially or significantly free, or greater than about 95%, 96%, 97%, 98%, 99%, or 100% free. Isolated material optionally contains material not found with the material in its natural environment, or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention in its composition and / or located in a subcellular location (e.g., the genome or subcellular organelles) that is not native to the material found in that environment. The alterations to generate synthetic material may be performed on or removed from the material in its natural state.

[0194] As used herein, the term "Lineage positive (Lin+)" refers to a mixture of all cells that express mature cell lineage markers. The remaining cells are lineage negative (Lin-), meaning they do not stain with lineage antibodies. All step and progenitor cell activities were identified within the Lin- population.

[0195] The term "lymphocyte common antigen" or CD45 refers to a receptor-binding protein tyrosine phosphatase expressed on all leukocytes.

[0196] The terms "major histocompatibility complex" and "MHC" are used herein to refer to molecular compartments known as epitopes or antigens, cell surface molecules that mediate leukocyte interactions with other leukocytes or somatic cells. The MHC is encoded by a large group of genes and can be organized into three subgroups: class I, class II, and class III. In humans, the MHC gene complex is called HLA (for "human leukocyte antigen"), and in mice, it is called H-2 (for "histocompatibility"). Both species have three major MHC class I genes, called HLA-A, HLA-B, and HLA-C in humans and H2-K, H2-D, and H2-L in mice. These encode the α chain of each MHC class I protein. The other subunit of the MHC class I molecule is β2-microglobulin. The class II region contains the genes for the α and β chains (designated A and B) of the human MHC class II molecules HLA-DR, HLA-DP, and HLA-DQ. The MHC class II region also contains the genes for the TAP1:TAP2 peptide transporter, the PSMB (or LMP) gene encoding a proteasome subunit, the genes encoding the DM α and BM β chains (DMA and DMB), the genes encoding the α and β chains of the DO molecule (DOA and DOB, respectively), and the gene encoding tapasin (TAPBP). Class II genes encode various other proteins with immune functions. The DMA and DMB genes, which encode the subunits of the HLA-DM molecule that catalyze peptide binding to MHC class II molecules, are related to the MHC class II genes, as are the DOA and DOB genes, which encode the subunits of the regulatory HLA-DO molecule. Janeways Immunobiology, 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, pp. 232-233.

[0197] The term "matrix metalloproteinase" as used herein refers to a collection of zinc-dependent proteases involved in the degradation and remodeling of extracellular matrix components (Guiot, J. et al. Lung (2017) 195(3):273-280, citing Oikonomidi et al. Curr Med Chem. 2009;16(10):1214-1228). For example, the MMP2 gene provides instructions for making matrix metallopeptidase 2. This enzyme is produced by cells throughout the body and becomes part of the extracellular matrix, a complex lattice of proteins and other molecules that forms in the spaces between cells. One of the primary known functions of MMP-2 is to cleave type IV collagen, the main structural component of basement membranes. Basement membranes are thin, sheet-like structures that separate and support cells as part of the extracellular matrix.

[0198] The term "mimetic" as used herein refers to a compound or substance that is chemically similar to a parent compound or substance and retains at least some of the desired function of the parent compound or substance. The term "mimetic" is used interchangeably with "mimetics," which refers to a chemical substance that contains chemical moieties that mimic the function of a peptide. For example, if a peptide contains two charged chemical moieties that have functional activity, the mimetic arranges the two charged chemical moieties in a spatial orientation and constrained structure, so that the charged chemical function is maintained in three-dimensional space.

[0199] As used herein, the term "modify" or "modulate" means to regulate, change, adapt, or adjust to a particular measure or proportion. As used herein in the context of a cell type, the term "modified" or "modulated" refers to changing the morphology or characteristics of the cell type.

[0200] The term "myeloid cells" refers collectively to granulocytes and monocytes, which are differentiated progeny of a common progenitor cell derived from hematopoietic stem cells in the bone marrow. Commitment of myeloid cells to one lineage or another is regulated by distinct transcription factors and subsequent terminal differentiation and release into the circulation in response to specific colony-stimulating factors. [Kawamoto, H., Minato, N. Intl J. Biochem. Cell Biol. (2004) 36(8):1374-70]

[0201] As used herein, the term "myeloablative therapy" refers to a treatment regimen (e.g., high-dose chemotherapy or high-dose irradiation) used to kill surviving cells in the bone marrow, including cancer cells, which reduces the number of normal hematopoietic cells in the bone marrow, resulting in a decrease in red blood cells, white blood cells, and platelets. As used herein, the term "non-myeloablative" refers to a pre-transplant conditioning regimen in which limited amounts of chemotherapy are administered to prevent rejection of donor bone marrow stem cells without destroying the recipient's bone marrow.

[0202] As used herein, the term "myelosuppression" refers to a condition in which bone marrow activity is decreased, resulting in a decrease in red blood cells, white blood cells, and platelets. When myelosuppression is severe, it is called myeloablation.

[0203] The abbreviation "MAPK" used herein refers to mitogen-activated protein kinase (MAPK) signaling, which activates a three-tiered cascade with MAPK kinase kinase (MAP3K), which then activates MAPAK kinase (MAP2K), and finally MAPK. MAPKs are protein Ser / Thr kinases that convert extracellular stimuli into a wide range of cellular responses. (Cargnello, M. and Roux, PP, Microbiol. Mol. Biol. Rev. (2011) 75(1):50-83) The major MAPK pathways involved in inflammatory diseases are extracellular-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Upstream kinases include TGFβ-activated kinase 1 (TAK1) and apoptosis signal-regulating kinase 1 (ASK1). Downstream of p38 MAPK is MAPK-activated protein kinase 2 (MAPKAPK2 or MK2). (See Figure 11 in Barnes, PJ (2016) Pharmacological Revs. 68:788-815).

[0204] A diverse group of MAPK-activated protein kinases (MAPKAPKs) has been defined downstream of mitogen-activated protein kinases (MAPKs). These enzymes transduce signals to target proteins that are not direct substrates of MAPKs and thus play a role in relaying phosphorylation-dependent signaling by the MAPK cascade to diverse cellular functions. One of these groups is formed by three MAPKAPKs: MK2, MK3 (also known as 3pK), and MK5 (also known as PRAK). Mitogen-activated protein kinase-activated protein kinase 2 (also known as "MAPKAPK2," "MAPKAP-K2," or "MK2") is a kinase of the serine / threonine (Ser / Thr) protein kinase family. MK2 is highly homologous to MK3 (approximately 75% amino acid identity). The kinase domains of MK2 and MK3 are most similar (approximately 35%-40% identity) to the C-terminal kinase domains (CTKDs) of calcium / calmodulin-dependent protein kinase (CaMK), phosphorylase b kinase, and ribosomal S6 kinase (RSK) isoforms. The MK2 gene encodes two alternatively spliced ​​transcripts of 370 amino acids (MK2A) and 400 amino acids (MK2B). The MK3 gene encodes a single transcript of 382 amino acids. The MK2 and MK3 proteins are highly homologous, but MK2A has a shorter C-terminal region. The C-terminus of MK2B contains a functional bipartite nuclear localization sequence (NLS) (Lys-Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Arg-Arg-Lys-Lys; SEQ ID NO: 1), which is absent in the shorter MK2A isoform, indicating that alternative splicing determines the cellular localization of MK2 isoforms. MK3 has a similar nuclear localization sequence. The nuclear localization sequence found in both MK2B and MK3 encompasses the D domain (Leu-Leu-Lys-Arg-Arg-Lys-Lys; SEQ ID NO: 2), which has been shown to mediate the specific interaction of MK2B and MK3 with p38α and p38β. MK2B and MK3 also have a functional nuclear export signal (NES) located N-terminal to the NLS and D domain.The NES of MK2B is sufficient to induce nuclear export after stimulation, a process that can be inhibited by leptomycin B. The N-terminal sequences of the catalytic domains of MK2 and MK3 are proline-rich and contain one (MK3) or two (MK2) putative Src homology 3 (SH3) domain binding sites (as shown in the case of MK2) to mediate binding to the SH3 domain of c-Abl in vitro (Cargnello, M. and Roux, PP, Microbiol. Mol. Biol. Rev. (2011) 75(1):50-83).

[0205] MK2B and MK3 are primarily located in the nucleus of quiescent cells, whereas MK2A resides in the cytoplasm. Both MK2B and MK3 are rapidly exported to the cytoplasm via a chromosome region maintenance protein (CRM1)-dependent mechanism upon stress stimulation. Nuclear export of MK2B appears to be mediated by kinase activation, as a phosphomimetic mutation of Thr334 within the activation loop of the kinase enhances cytoplasmic localization of MK2B. Without being limited by theory, it is believed that MK2B and MK3 may contain a constitutively active nuclear localization signal (NLS) and a phosphorylation-regulated nuclear export signal (NES) (Id.).

[0206] MK2 and MK3 are ubiquitously expressed, with relative expression appearing to be increased in heart, lung, kidney, reproductive organs (mammary gland and testis), skin and skeletal muscle tissue, as well as immune-related cells such as white blood cells / leukocytes and dendritic cells.

[0207] Activation of MK2 and MK3 Kinase Activity. Various activators of p38α and p38β potently stimulate MK2 and MK3 activity. p38 mediates the in vitro and in vivo phosphorylation of MK2 at four proline-directed sites: Thr25, Thr222, Ser272, and Thr334. Of these sites, only Thr25 is not conserved in MK3. Without being limited by theory, the function of phosphorylated Thr25 is unknown, but its location between two SH3 domain binding sites suggests that it may regulate protein-protein interactions. Thr222 in MK2 (Thr201 in MK3) is located in the activation loop of the kinase domain and has been shown to be essential for MK2 and MK3 kinase activity. Thr334 in MK2 (Thr313 in MK3) is located at the C-terminus of the catalytic domain and is essential for kinase activity. The crystal structure of MK2 has been determined, and without being limited by theory, it is suggested that phosphorylation of Thr334 may function as a switch for nuclear import and export of MK2. Phosphorylation of Thr334 may also weaken or prevent binding to the C-terminal catalytic domain of MK2, expose the NES, and promote nuclear export. (Ibid.)

[0208] Studies have shown that p38 can activate MK2 and MK3 in the nucleus, but that the activation and nuclear export of MK2 and MK3 are coupled by a phosphorylation-dependent conformational switch that also determines the stabilization and localization of p38, and experimental evidence suggests that the cellular location of p38 itself is controlled by MK2 and possibly MK3. Further studies have shown that nuclear p38 is exported to the cytoplasm in a complex with MK2 after MK2 phosphorylation and activation. Studies have shown that p38 levels are low in MK2-deficient cells and that expression of a catalytically inactive MK2 protein restores p38 levels, suggesting that the interaction between p38 and MK2 may be important for p38 stabilization. (Menon, MB, et al., J. Biol. Chem. (2010) 285:33242-251Z).

[0209] Studies using MK2 knockout mice or MK2-deficient cells have shown that MK2 increases the production of inflammatory cytokines, including TNF-α, IL-1, and IL-6, by increasing the rate of mRNA translation. In MK2-deficient mice, no significant reduction in TNF-α transcription, processing, or shedding could be detected. The p38 pathway is known to play an important role in regulating mRNA stability, and MK2 represents a potential target through which p38 mediates this function. Studies using MK2-deficient mice have shown that the catalytic activity of MK2 is required for cytokine production and its effects on migration. Without being limited by theory, this suggests that MK2 phosphorylates targets involved in mRNA stability. Consistent with this, MK2 has been shown to bind and / or phosphorylate heterogeneous nuclear ribonucleoprotein (hnRNP) A0, tristetraprolin (TTP), the poly(A)-binding protein PABP1, and HuR, a ubiquitously expressed member of the ELAV (embryonic lethal abnormal visual aberration) family of RNA-binding proteins. These substrates are known to bind or copurify with mRNAs containing AU-rich elements in their 3' untranslated regions, suggesting that MK2 may regulate the stability of AU-rich mRNAs such as TNF-α. While it is currently unclear whether MK3 plays a similar role, LPS treatment of MK2-deficient fibroblasts completely abolishes hnRNP A0 phosphorylation, suggesting that MK3 cannot compensate for the loss of MK2. (Cargnello, M. and Roux, PP, Microbiol. Mol. Biol. Rev. (2011) 75(1):50-83)).

[0210] MK3, together with MK2, is involved in the phosphorylation of eukaryotic elongation factor 2 (eEF2) kinase. eEF2 kinase phosphorylates and inactivates eEF2. eEF2 activity is important for the elongation of mRNA during translation, and phosphorylation of eEF2 at Thr56 leads to the termination of mRNA translation. MK2 and MK3 phosphorylation of eEF2 kinase at Ser377 suggests that these enzymes may regulate eEF2 kinase activity, thereby regulating mRNA translation elongation. (Roux, PP, Blennis, J., Microbiol. & Molec. Biol. Revs. (2004) 68(2):320-344).

[0211] Transcriptional regulation by MK2 and MK3. Nuclear MK2, like many MKs, contributes to the phosphorylation of cAMP response element-binding (CREB), activating transcription factor 1 (ATF-1), serum response factor (SRF), and the transcription factor ER81. Comparison of wild-type and MK2-deficient cells revealed that MK2 is the major SRF kinase induced by stress, suggesting a role for MK2 in the immediate early stress-mediated response. Both MK2 and MK3 interact with the basic helix-loop-helix transcription factor E47 in vivo and phosphorylate E47 in vitro. MK2-mediated phosphorylation of E47 was found to suppress the transcriptional activity of E47, thereby inhibiting E47-dependent gene expression, suggesting that MK2 and MK3 may regulate tissue-specific gene expression and cell differentiation (ibid.).

[0212] Other Targets of MK2 and MK3. Several other MK2 and MK3 substrates have also been identified, reflecting the diverse functions of MK2 and MK3 in several biological processes. The scaffolding protein 14-3-3ζ is a physiological MK2 substrate. Studies have shown that 14-3-3ζ interacts with many components of cell signaling pathways, including protein kinases, phosphatases, and transcription factors. Further studies have shown that MK2-mediated phosphorylation of 14-3-3ζ at Ser58 impairs its binding activity, suggesting that MK2 may affect the regulation of several signaling molecules normally regulated by 14-3-3ζ. (Cargnello, M. and Roux, PP, Microbiol. Mol. Biol. Rev. (2011) 75(1):50-83)).

[0213] Additional studies have shown that MK2 also interacts with and phosphorylates the p16 subunit of the seven-member Arp2 and Arp3 complex (p16-Arc) on Ser77. p16-Arc plays a role in regulating the actin cytoskeleton, suggesting that MK2 may be involved in this process (Id.).

[0214] Further studies have shown that the small heat shock protein HSP27 (also known as HSPB1), the lymphocyte-specific protein LSP-1, and vimentin are phosphorylated by MK2. HSPB1, also known as HSP27, forms large oligomers that may function as molecular chaperones and protect cells from heat shock and oxidative stress. Upon phosphorylation, HSPB1 loses its ability to form large oligomers and is unable to block actin polymerization, suggesting that MK2-mediated phosphorylation of HSPB1 serves a homeostatic function aimed at regulating otherwise unstable actin dynamics during stress. MK3 has also been shown to phosphorylate HSPB1 in vitro and in vivo. (Gurgis, FMS, et al., Molecular Pharmacol. 2014) 85:345-56; Guay, J. et al., (1997) J. Cell Sci. 110(pt.3):357-68).

[0215] HSPB1 has also been shown to bind to polyubiquitin chains and the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of the transcription factor NF-kappa B (NF-κB) by degrading its main inhibitor, Ikappa B-alpha (IκB-alpha). Overexpression of HSPB1 has been shown to increase NF-αkappa B (NF-κB) nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, TNF-alpha, and interleukin-1 beta (IL-1β). Furthermore, previous studies have suggested that HSPB1 prefers the degradation of ubiquitinated proteins, such as phosphorylated Ikappa B-alpha (IκB-alpha), under stress conditions, and that this function of HSPB1 explains its anti-apoptotic properties through the enhancement of NF-kappa B (NF-κB) activity (Parcellier, A. et al., (2003) Mol Cell Biol, 23(16):5790-5802).

[0216] NF-κB signaling pathway. The abbreviation "NFκB" as used herein refers to a proinflammatory transcription factor. It switches on multiple inflammatory genes, including cytokines, chemokines, proteases, and apoptosis inhibitors, resulting in an amplified inflammatory response (Barnes, PJ, (2016) Pharmacol. Rev. 68:788-815). The molecular pathway involved in NF-κB activation involves several kinases. The typical (canonical) pathway by which inflammatory stimuli and infections activate NF-κB signaling involves the IKK (inhibitor of κB kinase) complex, which is composed of two catalytic subunits, IKK-α and IKK-β, and a regulatory subunit, IKK-γ (or NFκB essential modulator). (Ibid. Hayden, MS and Ghosh, S (2012) Genes Dev. 26:203-234). The IKK complex phosphorylates NF-κB-bound IκBs (targeting them for degradation by the proteasome, thereby releasing NF-κB dimers composed of p65 and p50 subunits), which translocate to the nucleus and bind to κB recognition sites in the promoter regions of inflammatory and immune genes, resulting in transcriptional activation (Figure 12). This response is primarily dependent on the catalytic subunit IKK-β (also known as IKK2), which carries out IκB phosphorylation. The non-canonical (alternative) pathway is activated by IKK-β in response to certain members of the TNF family, e.g., lymphotoxin β. The noncanonical pathway involves the upstream kinase NF-κB-inducing kinase (NIK), which phosphorylates KK-α homodimers, releasing RelB and processing p100 to p52 (ibid., citing Sun, SC. ​​(2012) Immunol. Rev. 246:125-140). This pathway may switch on a diverse set of genes and mediate immune functions distinct from those of the canonical pathway. Dominant-negative IKK-β inhibits most of the proinflammatory functions of NF-κB, whereas inhibiting IKK-α only plays a role in response to limited stimuli and in certain cells, such as B lymphocytes. The noncanonical pathway is involved in immune system development and adaptive immune responses.The coactivator molecule CD40, expressed on antigen-presenting cells such as dendritic cells and macrophages, activates the non-canonical pathway when it interacts with CD40L expressed on lymphocytes (ibid., citing Lombardi, V et al. (2010) Int. Arch. Allergy Immnol. 151:179-89).

[0217] As used herein, the term "NOD-like receptor" or NLR refers to a large family of proteins containing a nucleotide oligomerization domain (NOD) associated with various other domains, whose general function is the detection of microbial and cellular stress. The NOD subfamily is a subgroup of NLR proteins that contain a caspase activation and recruitment (CARD) domain, which is used to activate downstream signaling.

[0218] The term "nucleic acid" is used herein to refer to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues which possess the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids (e.g., peptide nucleic acids) in a manner similar to naturally occurring nucleotides.

[0219] The term "nucleotide" is used herein to refer to a chemical compound consisting of a heterocyclic base, a sugar, and one or more phosphate groups. In the most common nucleotides, the base is a derivative of purine or pyrimidine, and the sugar is the pentose deoxyribose or ribose. Nucleotides are nucleic acid monomers with three or more bonds that join together to form nucleic acids. Nucleotides are the structural units of RNA, DNA, and several cofactors, including, but not limited to, CoA, FAD, DMN, NAD, and NADP. Purines include adenine (A) and guanine (G), and pyrimidines include cytosine (C), thymine (T), and uracil (U).

[0220] The term "osteogenesis" as used herein refers to the process by which bone or bony tissue is formed. Bone tissue is a rigid form of connective tissue that is usually organized into distinct structures called bones. There are two main modes of bone formation, both of which involve the conversion of existing mesenchymal tissue into bone tissue. The direct conversion of mesenchymal tissue into bone is called intramembranous ossification. This process occurs primarily in the bones of the skull. In other cases, mesenchymal cells differentiate into cartilage, which is later replaced by bone. The process by which a cartilage intermediate is formed and replaced by bone cells is called endochondral ossification.

[0221] Intramembranous ossification is the characteristic method by which the flat bones of the scapula, skull, and turtle shell form. In intramembranous ossification, bone develops a sheet of fibrous connective tissue. During intramembranous ossification of the skull, neural crest-derived mesenchymal cells proliferate and condense into compact nodules. Some of these cells develop into canaliculi, while others change shape and become osteoblasts, committed to becoming osteoprogenitor cells. Osteoblasts secrete a collagen-proteoglycan matrix that can bind calcium salts. This binding allows the prebone (osteoid) matrix to mineralize. In most cases, osteoblasts are separated from the area of ​​mineralization by a layer of the osteoid matrix they secrete. Occasionally, osteoblasts become trapped in the mineralized matrix and become osteocytes. As mineralization progresses, bone spicules radiate from the area where ossification has begun, and the entire area of ​​the mineralized spicules is surrounded by dense mesenchymal cells that form the periosteum. The cells lining the periosteum also become osteoblasts and deposit osteoid matrix parallel to that of the existing spicules. In this way, many layers of bone are formed.

[0222] Intramembranous ossification is characterized by the invasion of capillaries into the mesenchymal zone and the emergence and differentiation of mesenchymal cells into mature osteoblasts, which constitutively deposit bone matrix, resulting in the formation of bony spines, which grow and develop, eventually fusing with other spines to form trabeculae. As the trabeculae increase in size and number, they interconnect to form woven bone (a disorganized, weak structure with a high proportion of osteocytes), which is eventually replaced by more organized, stronger, and lamellar bone.

[0223] The molecular mechanism of intramembranous ossification involves the activation of bone morphogenetic proteins (BMPs) and a transcription factor called CBFA1. Head epidermis-derived bone morphogenetic proteins, such as BMP2, BMP4, and BMP7, are thought to direct neural crest-derived mesenchymal cells to become bone cells. BMPs activate the Cbfa1 gene in mesenchymal cells. CBFA1 transcription factors are known to convert mesenchymal cells into osteoblasts. Studies have shown that mouse CBFA1 mRNA is primarily restricted to bone-forming mesenchymal condensations and is therefore restricted to the osteoblast lineage. CBFA1 is known to activate genes for osteocalcin, osteopontin, and other bone-specific extracellular matrix proteins.

[0224] Endochondral Ossification (Intracartilaginous Ossification). Endochondral ossification, which involves the in vivo formation of cartilage tissue from aggregated mesenchymal cells and its subsequent replacement by bone, can be divided into five stages. The skeletal components of the spine, pelvis, and limbs are initially formed of cartilage and later become bone.

[0225] First, mesenchymal cells are committed to becoming chondrocytes. This commitment is triggered by paracrine factors that induce nearby mesodermal cells to express two transcription factors, Pax1 and Scleraxis. These transcription factors are known to activate cartilage-specific genes. For example, Scleraxis is expressed in the mesenchyme from the sclerotome, facial mesenchyme that forms the cartilage precursor to bone, and limb mesenchyme.

[0226] In the second stage of endochondral ossification, committed mesenchymal cells condense into compact nodules and differentiate into chondrocytes (cartilage cells that generate and maintain the cartilage matrix, which is primarily composed of collagen and proteoglycans). Studies have shown that N-cadherin is important for initiating these condensations and N-CAM is important for maintaining them. In humans, the SOX9 gene, which encodes a DNA-binding protein, is expressed in prechondral condensations.

[0227] In the third stage of endochondral ossification, chondrocytes rapidly proliferate to form the model bone. As they divide, chondrocytes secrete a cartilage-specific extracellular matrix.

[0228] In the fourth stage, chondrocytes stop dividing and dramatically increase in volume to become hypertrophic chondrocytes. These large chondrocytes alter the matrix they produce (by adding collagen X and more fibronectin) so that it becomes mineralized with calcium carbonate.

[0229] The fifth stage involves the invasion of blood vessels into the cartilage model. Hypertrophic chondrocytes die by apoptosis, and this space becomes bone marrow. When the chondrocytes die, a group of cells surrounding the cartilage model differentiates into osteoblasts, which begin to form a bone matrix on the partially degraded cartilage. Eventually, all of the cartilage is replaced by bone. Thus, the cartilage tissue serves as a model for subsequent bone.

[0230] The replacement of chondrocytes by bone cells depends on the mineralization of the extracellular matrix. Many events, including an initial switch from aerobic to anaerobic respiration, lead to chondrocyte hypertrophy and mineralization. This alters cellular metabolism and mitochondrial energy potential. Hypertrophic chondrocytes secrete numerous small membrane-bound vesicles into the extracellular matrix. These vesicles are active in the production of calcium and phosphate ions and contain enzymes that initiate the mineralization process within the cartilage matrix. Hypertrophic chondrocytes, their metabolism and mitochondrial membranes change and then die by apoptosis.

[0231] In the long bones of many mammals (including humans), endochondral ossification spreads outward from the center of the bone in both directions. As the ossification front approaches the edge of the cartilage model, chondrocytes near the ossification front hypertrophy and proliferate, pushing the cartilaginous end of the bone. The cartilaginous region at the end of a long bone is called the epiphyseal growth plate. These plates contain three regions: a region of chondrocyte proliferation, a region of mature chondrocytes, and a region of hypertrophic chondrocytes. As the medial cartilage hypertrophy and ossification front extend further outward, the remaining cartilage at the epiphyseal growth plate proliferates. As long as the epiphyseal growth plate can produce chondrocytes, the bone continues to grow.

[0232] As used herein, the term "osteopenia" refers to a loss of bone mass that is less severe than osteoporosis, defined as a T-score of -1 to -2.5 on bone mineral density measurement.

[0233] As used herein, the term "osteoporosis" refers to a decrease in bone density, causing bones to become more porous and fragile, increasing the risk of fracture, defined as a T-score of -2.5 or less.

[0234] As used herein, the term "organ" refers to a specialized structure made up of cells and tissues that performs some specific function in an organism.

[0235] As used herein, the term "paracrine signaling" refers to short-range intercellular communication via secreted signal molecules that act on neighboring cells.

[0236] As used herein, the term "pathogen-associated molecular patterns" (PAMPs) refers to molecules specifically associated with groups of pathogens that are recognized by cells of the innate immune system.

[0237] The terms "polypeptide" and "protein" are used herein in their broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. Except where noted, the subunits are linked by peptide bonds. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. These terms also include modifications, including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. As is well known and discussed above, it will be understood that polypeptides may not be entirely linear. For example, polypeptides may generally be branched as a result of post-translational events, whether by natural processing, by human manipulation that does not occur in nature, or as a result of ubiquitination, or may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may also be synthesized by entirely synthetic methods.

[0238] The term "pharmaceutical composition" is used herein to refer to a composition used to prevent, reduce the intensity of, cure, or otherwise treat a targeted condition or disease. The terms "formulation" and "composition" are used interchangeably herein to refer to the described product of the invention, including all active and inactive ingredients.

[0239] The term "pharmaceutically acceptable" is used to refer to a carrier, diluent, or excipient that is compatible with the other ingredients of a formulation or composition (meaning capable of being combined with each other under normal conditions of use in a manner such that there is no interaction which would substantially reduce the effectiveness of the composition) and not harmful to the recipient thereof. A carrier must be of sufficiently high purity and sufficiently low toxicity to render it suitable for administration to the subject being treated. A carrier must also maintain the stability and bioavailability of the active agent. For example, the term "pharmaceutically acceptable" can mean approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use.

[0240] The term "progenitor cells" as used herein refers to immature cells in bone marrow that can be isolated by growing a suspension of bone marrow cells in a culture dish supplemented with growth factors. Progenitor cells mature into precursor cells that mature into blood cells. Progenitor cells are called colony-forming units (CFUs) or colony-forming cells (CFCs). Specific lineages of progenitor cells are designated by suffixes such as, but not limited to, CFU-E (erythroid), CFU-GM (granulocyte / macrophage), and CFU-GEMM (multipotent hematopoietic precursor cells).

[0241] As used herein, the term "purification" and its various grammatical forms refers to a process of separating or removing foreign, extraneous, or undesirable elements.

[0242] The term "quiescence" as used herein is often used to characterize stem cells present in tissues, allowing them to function as a dormant reserve that can replenish tissues during homeostasis. Quiescence is considered a fundamental feature of hematopoietic stem cells (HSCs), which have the potential for multilineage differentiation and self-renewal and can give rise to all cell types within the blood lineage (Nakamura-Ischizu, A. et al., Development (2014) 141:4656-66, citing Pietras, E. M. et al., J. Cell Biol. (2011) 195:709-720). Precise regulation of the cell cycle of quiescent HSCs is required for the effective production of mature hematopoietic cells with minimal stem cell attrition (ibid., citing Orford, K. W. and Scadden, D. T., Nature Rev. Genet. (2008) 9:115-128). Because proliferating cells are more susceptible to genetic mutations and undergo senescence once maximum turnover (known as the Hayflick limit) is reached (ibid., citing Hayflick, L. and Moorhead, P.S., Expl Cell Res., (1961) 25:585-621), quiescence likely protects HSCs from malignant transformation and dysfunction (ibid., citing Wang, J.C.Y. and Dick, J.E., Trends Cell Biol. (2005) 15:494-501). Both cell-intrinsic and -extrinsic signals induced in response to various stresses, such as inflammation or blood loss, enable quiescent HSCs to re-enter the cell cycle and proliferate and differentiate (ibid., citing Morrison, SJ and Weissman, IL Immunity (1994) 1:661-673; Suda, T. et al., Proc. Nat. Acad. Sci. USA (1983) 80:6689-93).

[0243] The term "reference sequence" refers to a sequence used as a basis for sequence comparison. The reference sequence can be a subset or the entirety of a specified sequence.

[0244] As used herein, the term "relapse" refers to the reappearance of a disease after a period of remission.

[0245] As used herein, the term "remission" refers to the reduction or elimination of a disease and its symptoms.

[0246] As used herein, the term "splice site variant" refers to genetic changes in DNA sequences that occur at the boundaries of exons and introns (splice sites) that can result in changes in the protein-coding sequence.

[0247] As used herein, the term "steady state" refers to a state of dynamic equilibrium where the rate of loss equals the rate of gain.

[0248] The term "stem cell" as used herein refers to an undifferentiated cell with high proliferative potential that has the ability to self-renew and generate daughter cells that can undergo terminal differentiation into multiple distinct cell phenotypes. Stem cells are distinguished from other cell types by two characteristics. First, they are undifferentiated cells that can regenerate themselves by cell division, possibly after a long period of inactivity. Second, under specific physiological or experimental conditions, they can be induced to become tissue- or organ-specific cells with specialized functions. In some organs, such as the intestine and bone marrow, stem cells divide regularly to repair and replace worn or damaged tissues. However, in other organs, such as the pancreas and heart, stem cells divide only under special conditions.

[0249] Adult (somatic) stem cells are undifferentiated cells found among the differentiated cells of a tissue or organ. Their primary role in vivo is to maintain and repair the tissue in which they are found. Adult stem cells have been identified in many organs and tissues, including the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, gastrointestinal tract, liver, ovarian epithelium, and testes. Adult stem cells are thought to reside in specific regions of each tissue known as stem cell niches, where they can remain quiescent (non-dividing) for long periods of time until activated by the normal need for more cells to maintain the tissue or by disease or tissue injury.

[0250] Bone marrow stem cells. As used herein, the term "bone marrow stem cells" refers to stem cells derived from bone marrow, including HSCs and MSCs. The mononuclear fraction of bone marrow contains stromal cells, hematopoietic progenitors, and endothelial progenitors.

[0251] Peripheral blood stem cells. As used herein, the term "peripheral blood stem cells" refers to stem cells derived from peripheral blood. Peripheral blood contains adult (somatic) stem cells, which are undifferentiated cells found among differentiated cells of tissues or organs. Examples of peripheral blood stem cells include, but are not limited to, hematopoietic stem cells and mesenchymal stem cells [Dzierzak E. et al., "Of lineage and legacy: the development of mammalian hematopoietic stem cells", Nature Immunol., Vol. 9(2): 129-136, (2008)].

[0252] Hematopoietic stem cells. As used herein, "hematopoietic stem cells" (colony forming units of myeloid and lymphoid cells (CFU-M, L), or CD34 + HSCs (also known as hematopoietic stem cells) are rare multipotent cells within hematopoietic organs that are responsible for the continuous production of blood cells throughout life [Li Y. et al., “Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production”, Genes Dev., Vol. 28(23):2596-2612, (2014)]. HSCs can generate various cell types, including erythrocytes, neutrophils, basophils, eosinophils, platelets, mast cells, monocytes, tissue macrophages, osteoclasts, and T and B lymphocytes. Regulation of hematopoietic stem cells is a complex process involving self-renewal, survival and proliferation, lineage commitment, and differentiation, and is orchestrated by diverse mechanisms, including intrinsic cellular programming and external stimuli such as adhesive interactions with the microenvironmental stroma and the action of cytokines.

[0253] Various paracrine factors (cytokines) are important in directing hematopoietic stem cells (HSCs) to differentiate along specific pathways. Cytokines can be produced by several cell types but are collected and concentrated by the extracellular matrix of stromal (mesenchymal) cells at the site of hematopoiesis. For example, granulocyte-macrophage colony-stimulating factor (GM-CSF) and multilineage growth factor IL-3 (IL-3) both bind to heparan sulfate glycosaminoglycans in the bone marrow stroma. The extracellular matrix then presents these factors to stem cells at concentrations high enough to bind to their receptors [Alvarez S. et al., "GM-CSF and IL-3 activities in schistosomal liver granulomas are controlled by stroma-associated heparan sulfate proteoglycans," J. Leukoc. Biol., Vol. 59(3):435-441, (1996)].

[0254] Mesenchymal stem cells. Mesenchymal stem cells (MSCs) (also known as bone marrow mesenchymal stem cells or skeletal stem cells) are non-blood adult stem cells found in various tissues. They are characterized by a spindle-shaped morphology, the expression of specific markers on the cell surface, and the ability to differentiate along at least three lineages (osteogenesis, chondrogenesis, and adipogenesis) under appropriate conditions [Najar M. et al., "Mesenchymal stromal cells and immunomodulation: A gathering of regulatory immune cells," Cytotherapy, Vol. 18(2):160-171, (2016)]. Although a single marker that clearly delineates MSCs in vivo has not been identified due to a lack of consensus regarding MSC phenotype, MSCs are generally positive for the cell surface markers CD105, CD166, CD90, and CD44, and negative for representative hematopoietic antigens, such as CD45, CD34, and CD14. Regarding the differentiation potential of MSCs, studies have reported that bone marrow-derived MSC populations have the ability to develop into terminally differentiated mesenchymal phenotypes both in vitro and in vivo, including bone, cartilage, tendon, muscle, adipose tissue, and hematopoiesis supporting the stroma. Studies using transgenic and knockout mice and human musculoskeletal disorders have reported that MSCs differentiate into multiple lineages during embryonic development and adult homeostasis. [Najar M. et al., "Mesenchymal stromal cells and immunomodulation: A gathering of regulatory immune cells," Cytotherapy, Vol. 18(2):160-171, (2016)]

[0255] Analysis of in vitro differentiation of MSCs under appropriate conditions that mimic in vivo processes has led to the identification of various factors essential for stem cell commitment. Among them, secreted molecules and their receptors (e.g., transforming growth factor-(β)), extracellular matrix molecules (e.g., collagen and proteoglycans), the actin cytoskeleton, and intracellular transcription factors (e.g., Cbfal / Runx2, PPARy, Sox9, and MEF2) have been shown to play important roles in triggering the commitment of pluripotent MSCs to specific lineages and maintaining their differentiated phenotype [Davis LA et al., "Mesodermal fate decisions of a stem cell: the Wnt switch," Cell Mol Life Sci., Vol. 65(17):2568-2574, (2008)].

[0256] As used herein, the term "stem cell niche" refers to the specific region of each tissue where adult stem cells reside, which may remain quiescent (non-dividing) for long periods of time until activated by the normal need for more cells to maintain the tissue or by disease or tissue injury. Cells of the stem cell niche interact with stem cells to maintain them or promote their differentiation.

[0257] As used herein, the term "stem cell rescue" or "rescue transplant" refers to a method of replacing hematopoietic stem cells destroyed by treatment with high doses of anticancer drugs or radiation therapy. This is usually done using the patient's own stem cells that were stored before treatment. The stem cells help the bone marrow recover and produce healthy blood cells. Stem cell rescue may allow more chemotherapy or radiation therapy to be administered so that more cancer cells are killed.

[0258] As used herein, a "subject in need" of treatment for a particular condition refers to a subject who has the condition, has been diagnosed with the condition, or is at risk for developing the condition. According to some embodiments, the phrase "subject in need" of such treatment also refers to (i) a patient who will be administered a composition of the described invention; (ii) a patient who has received a composition of the described invention; or (iii) a patient who has received at least one composition of the described invention, unless otherwise indicated by context and usage.

[0259] The term "suspension" as used herein refers to a dispersion (mixture) in which a finely divided species is combined with another species such that the former is finely divided and mixed so that it does not settle rapidly.

[0260] The term "target" as used herein refers to a biological entity, such as, but not limited to, a protein, cell, organ, or nucleic acid, whose activity can be altered by an external stimulus. Depending on the nature of the stimulus, there may be no direct change in the target, or a conformational change in the target may be induced.

[0261] As used herein, the term "therapeutic agent" or "active agent" refers to the ingredient, constituent, or component of the composition of the described invention that is responsible for the intended therapeutic effect.

[0262] As used herein, the term "therapeutic component" refers to a therapeutically effective dosage (i.e., dose and frequency of administration) that eliminates, reduces, or prevents the progression of a particular disease symptom in a percentage of the population.

[0263] As used herein, the term "therapeutic effect" refers to an outcome of treatment, which outcome is deemed desirable and beneficial. A therapeutic effect may include, directly or indirectly, the prevention, reduction, or elimination of a disease symptom. A therapeutic effect may also include, directly or indirectly, the prevention, reduction, or elimination of the progression of a disease symptom.

[0264] As used herein, the term "tissue" refers to a collection of similar cells and the intercellular material that surrounds them. For example, connective tissue is the supportive or framework tissue of the body, formed of a fibrous and granular material containing numerous cells of various types. It is derived from the mesenchyme, which in turn is derived from the mesoderm. Types of connective tissue include, but are not limited to, areolar or loose; adipose; sense, regular or irregular, white fiber; elastic; mucous; lymphatic tissue; cartilage; and bone.

[0265] As used herein, the term "Toll-like receptor" refers to innate receptors on macrophages, dendritic cells, and some other cells that recognize pathogens and their products. Recognition stimulates the receptor-bearing cells to produce cytokines that help mount an immune response.

[0266] As used herein, the term "transplantation" and its various grammatical forms refer to a surgical procedure in which tissue or organs are transferred from one area of ​​the human body to another area, or from one human (donor) to another human (recipient).

[0267] As used herein, the terms "treat," "treated," or "treating" refer to both therapeutic treatment and / or prophylactic or preventative measures, the purpose being to prevent or slow (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. For purposes of this invention, a beneficial or desired clinical result includes, but is not limited to, alleviation of symptoms; a decrease in the extent of the condition, disorder, or disease; stabilization (i.e., not worsening) of the condition, disorder, or disease state; a delay in the onset or slowing of progression of the condition, disorder, or disease; an improvement in the condition, disorder, or disease state; and remission (whether partial or total), or improvement or reversal of the condition, disorder, or disease, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0268] As used herein, the term "angiogenesis" refers to the process of new blood vessel formation.

[0269] The term "vol / vol percent" is a measure of the concentration of a substance in a solution. It is expressed as the ratio of the volume of the solute to the total volume of the solution times 100. Whenever a solution is prepared by mixing pure liquid solutions, volume percent (vol / vol% or v / v%) should be used.

[0270] The abbreviation "WBM" stands for whole bone marrow.

[0271] The term "weight by weight percent" or wt / wt % is used herein to refer to the ratio of the weight of the solute to the total weight of the solution.

[0272] As used herein, the terms "wild-type," "naturally-occurring," or their grammatical equivalents are meant to refer to an amino acid sequence or nucleotide sequence that is found in nature and includes allelic variation, i.e., an amino acid sequence or nucleotide sequence that has not typically been intentionally modified. Thus, the terms "non-naturally occurring," "synthetic," and "recombinant," or their grammatical equivalents, are used interchangeably to refer to an amino acid sequence or nucleotide sequence that is not found in nature, i.e., an amino acid sequence or nucleotide sequence that has typically been intentionally modified. It is understood that once a recombinant nucleic acid is generated and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than through in vitro manipulation. However, such a nucleic acid, once produced recombinantly, may subsequently be replicated non-recombinantly, yet still be considered recombinant for purposes of the described invention.

[0273] method According to one aspect, the described invention provides a method for improving hematopoietic reconstitution of BM following myelosuppressive insult, comprising inhibiting endothelial cell-specific NF-κB in the bone marrow.

[0274] According to some embodiments, the myelosuppressive insult comprises sublethal radiation, chemotherapy, or both. According to some embodiments, the myelosuppressive insult comprises sublethal irradiation. According to some embodiments, the myelosuppressive insult comprises total body irradiation. According to some embodiments, the myelosuppressive insult comprises total lymph node irradiation. According to some embodiments, the myelosuppressive insult comprises exposure to radiation. According to some embodiments, the radiation can be derived from any suitable source, such as an anti-Cobalt-60 source.

[0275] According to some embodiments, the myelosuppressive insult is myeloablative.

[0276] According to some embodiments, the myelosuppressive insult comprises about 1 Joule of energy (Gy) absorbed per kilogram of material to about 30 Gy. According to some embodiments, the myelosuppressive insult comprises about 1 Gy, about 2 Gy, about 3 Gy, about 4 Gy, about 5 Gy, about 6 Gy, about 7 Gy, about 8 Gy, about 9 Gy, about 10 Gy, about 11 Gy, about 12 Gy, about 13 Gy, about 14 Gy, about 15 Gy, about 16 Gy, about 17 Gy, about 18 Gy, about 19 Gy, about 20 Gy, about 21 Gy, about 22 Gy, about 23 Gy, about 24 Gy, about 25 Gy, about 26 Gy, about 27 Gy, about 28 Gy, about 29 Gy, or about 30 Gy. According to some embodiments, the myelosuppressive insult comprises about 1 Gy to about 16 Gy.

[0277] According to some embodiments, the myelosuppressive insult comprises a single dose of total body irradiation. According to some embodiments, the myelosuppressive insult comprises fractionated doses of total body irradiation. According to some embodiments, the myelosuppressive insult comprising irradiation is delivered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. According to some embodiments, the myelosuppressive insult comprising irradiation is delivered over 2 to about 6 days. According to some embodiments, the myelosuppressive insult occurs after the final delivery of radiation.

[0278] According to some embodiments, when the myelosuppressive insult includes irradiation, the irradiation dose is about 1 Gy to about 16 Gy. According to some embodiments, when the myelosuppressive insult includes irradiation, the total irradiation dose is about 1 Gy to about 16 Gy. According to some embodiments, when the myelosuppressive insult includes irradiation, the total irradiation dose is about 1 Gy to about 16 Gy delivered over about 2 days to about 6 days. According to some embodiments, the irradiation further includes lung shielding.

[0279] According to some embodiments, the myelosuppressive insult comprises chemotherapy. According to some embodiments, the myelosuppressive insult comprises high-dose chemotherapy. According to some embodiments, the myelosuppressive insult comprises chemotherapy with an alkylating agent. According to some embodiments, the myelosuppressive insult comprises high-dose chemotherapy with an alkylating agent.

[0280] According to some embodiments, following the myelosuppressive insult, the subject's bone marrow comprises inflammation in the HSC niche. According to some embodiments, the myelosuppressive insult comprises myeloablation. According to some embodiments, the myelosuppressive insult results in an inability to achieve autologous hematological recovery.

[0281] According to some embodiments, NF-κB inhibition in endothelial cells within the bone marrow (BM) is effective in suppressing downstream NF-κB signaling in the BM. According to some embodiments, NF-κB inhibition in endothelial cells within the BM is effective in downregulating target NFκB genes in the BM. According to some embodiments, NF-κB inhibition in endothelial cells within the BM is effective in suppressing downstream NF-κB signaling in the BM and downregulating target NFκB genes in endothelial cells within the BM. According to some embodiments, NF-κB inhibition in endothelial cells within the BM protects the hematopoietic compartment and enhances recovery after myelosuppressive injury.

[0282] According to some embodiments, the hematopoietic cell population in the bone marrow comprises bone marrow endothelial cells (BMECs), hematopoietic stem cells (HSCs), and stromal cells (MSCs).

[0283] According to some embodiments, BMECs include bone marrow (BM) stromal cells, BM Lepr+ cells, and BM osteoblasts. According to some embodiments, the immunophenotype of BMECs is CD45-Ter119-CD31+VE-Cadherin+. According to some embodiments, BM stromal cells include BM Lepr+ and BM osteoblast stromal subsets. According to some embodiments, the immunophenotype of BM stromal cells is CD45-Ter119-CD31-VE-Cadherin-. According to some embodiments, the immunophenotype of BM Lepr+ cells within the BM stromal population is CD45-Ter119-CD31-Lepr+. According to some embodiments, the immunophenotype of BM osteoblasts is CD45-Ter119-CD31-SCA1-CD51+.

[0284] According to some embodiments, hematopoietic stem and progenitor cells (HSPCs) include hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). According to some embodiments, the immunophenotype of the HSCs comprises Lineage-CD48-CD150 Bright. According to some embodiments, the immunophenotype of the HSCs is Lineage-(Ter119 / CD11b / GR1 / B220 / CD3)-CD41-cKIT+SCA1+CD48-CD150+.

[0285] According to some embodiments, the immunophenotype of KLS HSPCs is Lin-cKIT+SCA1+. According to some embodiments, the KLS compartment is enriched for HSCs.

[0286] According to some embodiments, markers of the mature hematopoietic stem cell lineage include B220, CD4, CD8, Gr-1, Mac-1, and Ter-119.

[0287] According to some embodiments, BMECs affect BM function and hematopoiesis during inflammatory stress within the BM. According to some embodiments, endothelial MAPK can be constitutively activated in adult endothelium. According to some embodiments, BM endothelial niche activity is reduced by chronic activation of endothelial MAPK. According to some embodiments, reduced BM endothelial niche activity results in impaired steady-state hematopoiesis and HSC function.

[0288] According to some embodiments, chronic activation of endothelial MAPK results in an inflammatory stress response that disrupts the endothelial network. According to some embodiments, the inflammatory stress response includes one or more of increased vasodilation, decreased vascular integrity, including increased BM vascular leakage, and increased levels of inflammatory mediators, including sICAM, VCAM, and IL1b. According to some embodiments, chronic activation of endothelial MAPK results in inflammation through downstream activation of canonical NF-κB signaling. According to some embodiments, downstream activation of canonical NF-κB signaling includes one or more of increased levels of expression of NF-κB signaling targets, including pro-inflammatory cytokines and chemokines IL-1a, IL-1b, Cxcl1, Cxcl3, Ccl12, and Ccl22, including increased p65 phosphorylation within the endothelium without significant changes in total iκBα levels, increased MEK1DD-driven ERK1 / 2 phosphorylation, or increased levels.

[0289] According to some embodiments, constitutive activation of endothelial MAPK reduces bone marrow cellularity and decreases the frequency and absolute numbers of hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), including KLS cells, multipotent progenitor cells (MPPs), and hematopoietic progenitor cell subsets (HPC-1 and HPC-2), compared to littermate controls. According to some embodiments, the immunophenotype of MMPs is cKIT+Lin-SCA1+CD150-CD48-. According to some embodiments, the immunophenotype of HPC-1 is cKIT+Lin-SCA1+CD150-CD48+. According to some embodiments, the immunophenotype of HPC-2 is cKIT+Lin-SCA1+CD150+CD48+.

[0290] According to some embodiments, BM endothelial cells comprising constitutively activated endothelial MAPK comprise reduced long-term HSC engraftment potential compared to controls. According to some embodiments, HSCs and HSPCs from animals with constitutively activated endothelial MAPK comprise loss of quiescence and increased apoptosis compared to littermate controls.

[0291] According to some embodiments, inhibition of endothelial NF-κB signaling is effective in restoring BM vascular integrity in chronically MAPK-activated endothelium. According to some embodiments, increasing expression of IkB-SS suppresses p65 nuclear translocation in chronically MAPK-activated endothelium.

[0292] According to some embodiments, restoration of BM endothelial niche integrity in chronically activated MAPK epithelium suppressed by IkBSS affects functional restoration of HSCs and the hematopoietic system. According to some embodiments, functional restoration of HSCs includes restoration of BM cellularity. According to some embodiments, functional restoration of HSCs includes restoration of BM cellularity and frequency of phenotypic HSCs and HSPCs. According to some embodiments, functional restoration of HSCs includes restoration of long-term engraftment potential and reversal of myeloid-biased differentiation. According to some embodiments, functional restoration of HSCs includes restoration of BM cellularity and frequency of phenotypic HSCs and HSPCs, restoration of long-term engraftment potential, and reversal of myeloid-biased differentiation.

[0293] According to some embodiments, hematopoietic reconstitution after myelosuppression by endothelium, including sublethal myelosuppressive injury and subsequent downstream NF-κB activation, delays hematopoietic recovery. According to some embodiments, the analysis of hematopoietic recovery is by peripheral blood analysis. According to some embodiments, endothelial-specific inhibition of NF-κB protects the hematopoietic compartment and enhances recovery after myelosuppressive injury. According to some embodiments, the endothelial-specific inhibition of NFκB is by IkBSS. According to some embodime...

Claims

1. 1. A method for reducing vascular inflammation within a hematopoietic bone marrow microenvironment comprising bone marrow endothelial cells (BMEC), hematopoietic stem cells (HSC), and bone marrow stromal cells following myelosuppressive insult, wherein reduced BMEC activity results in defects in steady-state hematopoiesis and HSC function, said method comprising: a. administering to the subject a pharmaceutical composition comprising a recombinant or synthetic angiocrine factor and a pharmaceutically acceptable carrier; and b. a. reducing inflammation in the hematopoietic microenvironment of the bone marrow; b. maintaining vascular integrity in the hematopoietic microenvironment of the bone marrow; c. Increase the frequency and number of cell types in the hematopoietic compartment, including one or more of hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), multipotent progenitor cells (MPPs), and hematopoietic progenitor cell subsets, to achieve multilineage reconstitution; enhancing hematopoietic recovery in said hematopoietic bone marrow microenvironment following said myelosuppressive insult by one or more of: Including, The method, wherein the vascular inflammation comprises one or more of increased vasodilation, decreased vascular integrity including increased bone marrow vascular leakage, and increased levels of inflammatory mediators.

2. 2. The method of claim 1, wherein the angiocrine factor is one or more recombinant or synthetic proteins selected from the group consisting of Clec11a, HapIn1, Hspd1, Igfbp1, Bgn, Wnt7a, Sparc, RP53, Bmpr1a, Ighm, Thbs4, Camk2d, Sirt2, Camk2b, Slitrk5, Dctpp1, Hnrnpa2b, and Erap1.

3. The method of claim 2, wherein the angiocrine factor is recombinant or synthetic Clec11α (stem cell growth factor).

4. 2. The method of claim 1, wherein the inflammation in the hematopoietic microenvironment of the bone marrow comprises vascular inflammation, inflammation of BM stromal cells, and inflammation of hematopoietic cells.

5. The method of claim 1 , wherein the defect in HSC function comprises impaired HSC quiescence and increased HSC apoptosis.

6. The method of claim 1, wherein reducing vascular inflammation comprises suppressing downstream NFkB signaling in the BMECs within the bone marrow, downregulating target NFkB genes in endothelial cells of the bone marrow, or both.

7. 10. The method of claim 1, wherein the myelosuppressive insult comprises exposure to radiation, chemotherapy, or both.

8. 8. The method of claim 7, wherein the radiation is sublethal radiation, total body irradiation, or total lymph node irradiation.

9. The method of claim 7 , wherein the myelosuppressive insult comprises chemotherapy.

10. 8. The method of claim 7, wherein the myelosuppressive insult is myeloablative.

11. 2. The method of claim 1, wherein the bone marrow (BM) microenvironment comprises BMECs, BM stromal cells, BM Lepr+ cells, and BM osteoblasts.

12. The method of claim 11 , wherein the BMECs are sinusoidal and arteriolar BMECs.

13. The method of claim 1, wherein the immunophenotype of BMEC is CD45-Ter119-CD31+VE-cadherin+.

14. The method of claim 1, wherein the immunophenotype of BM stromal cells is CD45-Ter119-CD31-VE-cadherin-.

15. The method of claim 11, wherein the immunophenotype of BM Lepr+ cells within the BM stromal population is CD45-Ter119-CD31-Lepr+.

16. 2. The method of claim 1, wherein the immunophenotype of mouse HSC comprises lin-Ter119-CD11b-GR1-B220-CD3-CD41-ckit+SCA1+CD48-CD150+.

17. The method of claim 1, wherein the immunophenotype of human HSC comprises CD45RA-CD38-CD34+CD90+.

18. 2. The method of claim 1, wherein the reduced BMEC activity following the myeloablative insult results in impaired steady-state hematopoiesis and HSC function.

19. 1. A method for improving hematopoietic homing, engraftment, reconstitution, and regeneration of bone marrow following myelosuppressive insult in a subject in need thereof, comprising: a. administering to the subject a pharmaceutical composition comprising a recombinant or synthetic angiocrine factor and a pharmaceutically acceptable carrier; and b. administering a stem cell combination therapy comprising transplantation of a therapeutic amount of multipotent self-renewing hematopoietic stem cells (HSCs) effective to regenerate the blood system and promote hematopoietic reconstitution of the bone marrow; and c) administering a vascular endothelial combination therapy comprising transplantation of a therapeutic amount of bone marrow endothelial cells (BMECs) effective to regenerate the blood system and promote hematopoietic reconstitution of the bone marrow; d. Reducing vascular inflammation within the hematopoietic bone marrow microenvironment, including bone marrow endothelial cells (BMEC), hematopoietic stem cells (HSC), and bone marrow stromal cells, following the myelosuppressive insult, wherein reduced BMEC activity results in impaired steady-state hematopoiesis and HSC function; e. a. reducing inflammation in the hematopoietic microenvironment of the bone marrow; b. maintaining vascular integrity in the hematopoietic microenvironment of the bone marrow; c. Increase the frequency and number of cell types in the hematopoietic compartment, including one or more of hematopoietic stem cells (HSCs), hematopoietic stem and progenitor cells (HSPCs), multipotent progenitor cells (MPPs), and hematopoietic progenitor cell subsets, to achieve multilineage reconstitution; enhancing hematopoietic recovery in said hematopoietic bone marrow microenvironment following said myelosuppressive insult by one or more of: Including, The method, wherein the vascular inflammation comprises one or more of increased vasodilation, decreased vascular integrity including increased bone marrow vascular leakage, and increased levels of inflammatory mediators.

20. 20. The method of claim 19, wherein the angiocrine factor is one or more recombinant or synthetic proteins selected from the group consisting of Clec11a, HapIn1, Hspd1, Igfbp1, Bgn, Wnt7a, Sparc, RP53, Bmpr1a, Ighm, Thbs4, Camk2d, Sirt2, Camk2b, Slitrk5, Dctpp1, Hnrnpa2b, and Erap1.

21. 21. The method of claim 20, wherein the angiocrine factor is recombinant or synthetic Clec11α (stem cell growth factor).

22. 20. The method of claim 19, wherein the defect in HSC function comprises impaired HSC quiescence and increased HSC apoptosis.

23. The stem cell combination therapy is a. isolating hematopoietic stem cells from a population of mononuclear cells isolated from a tissue source; b. Enriching said isolated population of mononuclear cells of hematopoietic stem cells by positive or negative selection; and c. administering to the subject the enriched isolated population of hematopoietic stem cells; 20. The method of claim 19, comprising:

24. The vascular endothelial cell combination therapy is a. isolating endothelial cells from human umbilical cord; b. Enriching the isolated population for vascular endothelial cells by positive or negative selection; and c. administering to the subject the enriched isolated population of vascular endothelial cells; 20. The method of claim 19, comprising:

25. 24. The method of claim 23, wherein the tissue source is autologous.

26. 24. The method of claim 23, wherein the tissue source is allogeneic.

27. 20. The method of claim 19, wherein reducing vascular inflammation comprises suppressing downstream NFkB signaling in the BMECs within the bone marrow, downregulating target NFkB genes in endothelial cells of the bone marrow, or both.

28. 20. The method of claim 19, wherein the myelosuppressive insult comprises exposure to radiation, chemotherapy, or both.

29. 29. The method of claim 28, wherein the radiation is sublethal radiation, total body irradiation, or total lymph node irradiation.

30. 29. The method of claim 28, wherein the myelosuppressive insult is chemotherapy.

31. 29. The method of claim 28, wherein the myelosuppressive insult is myeloablative.

32. 20. The method of claim 19, wherein the bone marrow (BM) microenvironment comprises BMECs, BM stromal cells, BM Lepr+ cells, and BM osteoblasts.

33. 33. The method of claim 32, wherein the BMECs are sinusoidal and arteriolar BMECs.

34. The method of claim 32, wherein the immunophenotype of BMEC is CD45-Ter119-CD31+VE-cadherin+.

35. The method of claim 32, wherein the immunophenotype of BM stromal cells is CD45-Ter119-CD31-VE-cadherin-.

36. 33. The method of claim 32, wherein the immunophenotype of BM Lepr+ cells within the BM stromal population is CD45-Ter119-CD31-Lepr+.

37. 33. The method of claim 32, wherein the immunophenotype of mouse HSC comprises lin-Ter119-CD11b-GR1-B220-CD3-CD41-ckit+SCA1+CD48-CD150+.

38. 33. The method of claim 32, wherein the immunophenotype of human HSC comprises CD45RA-CD38-CD34+CD90+.

39. 20. The method of claim 19, wherein the method enhances stable long-term engraftment of the bone marrow, reduces myeloid bias in the peripheral blood, or both.

40. The method of claim 19, wherein the pharmaceutical composition is administered before, after, or simultaneously with the administration of the stem cell combination therapy.

41. 20. The method of claim 19, wherein the inflammation in the hematopoietic microenvironment of the bone marrow comprises vascular inflammation, inflammation of BM stromal cells, and inflammation of hematopoietic cells.

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