Prevention and treatment of diseases by regulating mesenchymal stem cell activity with vectors encoding P62(SQSTM1) and pharmaceutical preparations containing P62(SQSTM1) protein

Abstract

Pharmaceutical preparations containing a vector encoding p62 / SQSTM1 and / or the p62 / SQSTM1 protein (collectively referred to as p62) can be used to regulate MSC activity, leading to disease prevention and / or treatment. p62 can be used to pulse isolated MSCs, which can then be administered to patients as a cell therapy, or p62 can be administered to patients to regulate MSCs in their bodies.

Description

[Brief explanation of the drawings]

[0001] [Figure 1] FIG. 1 shows the wild-type nucleic acid sequence of human p62 (SEQ ID NO: 1). [Figure 2] FIG. 2 shows the wild-type amino acid sequence of human p62 encoded by the nucleic acid sequence (SEQ ID NO: 2). [Figure 3] Figure 3 shows a schematic diagram of the domain structure of human p62. DETAILED DESCRIPTION OF THE INVENTION

[0002] Dysfunction of mesenchymal stem cells (MSCs) plays a key role in many diseases. Attempts to treat these diseases with MSC therapy have attracted attention. Here, we propose using vectors encoding p62 / SQSTM1 and / or pharmaceutical preparations containing the p62 / SQSTM1 protein (collectively referred to as p62) to modulate MSC activity, leading to disease prevention and / or treatment. p62 can be used to pulse isolated mesenchymal stem cells, which can then be administered to patients as cell therapy, or p62 can be administered to patients to regulate MSC activity in their own bodies. MSCs play an important role in aging and aging-related diseases. A specific application of p62 is the prevention and treatment of aging and aging-related diseases. Another application of p62 is the prevention and / or treatment of diseases associated with elevated reactive oxygen species (ROS) levels. Administration of p62 and / or p62-treated MSCs reduces ROS levels.

[0003] List of diseases treated with MSCs: acute respiratory disease syndrome (ARDS); COVID-19 and other virus-related pneumonia; graft-versus-host disease; spinal cord injury; liver fibrosis / cirrhosis; bronchopulmonary dysplasia; critical limb ischemia; solid organ transplantation; diabetic foot; stroke; fracture; perianal, rectovaginal, surgical leaking fistulas; Erectile dysfunction.

[0004] List of ROS-related diseases: inflammatory diseases Inflammatory bowel disease (Patlevic P, Vaskova J, Svorc P Jr, Vasko L, Svorc P, Reactive oxygen species and antioxidant defenses in human gastrointestinal diseases, Integr Med Res. 2016 Dec;5(4):250-258); Periodontitis (Tunstall Pedoe DS. Risk of sudden death in older athletes: increasing denominators. Br J Sports Med. 2004 Dec;38(6):671-2); Tendinopathy (Tunstall Pedoe DS. Sudden death risk in older athletes: increasing denominators. Br J Sports Med. 2004 Dec;38(6):671-2).

[0005] Neurodegeneration Neurodegeneration due to lipid droplet accumulation (Liu L, Zhang K, Sandoval H, Yamamoto S, Jaiswal M, Sanz E, Li Z, Hui J, Graham BH, Quintana A, Bellen HJ, Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration, Cell. 2015 Jan 15;160(1-2):177-90); adipogenesis (as a consequence of the above) (Kramer AH, Kadye R, Houseman PS, Prinsloo E, Mitochondrial STAT3 and reactive oxygen species: the fulcrum of adipogenesis?, JAKSTAT. 2015;4(2):e1084084); Increased ROS levels in the substantia nigra pars compacta cause neuronal apoptosis of dopaminergic neurons in Down syndrome and Parkinson's disease (Tieu K, Ischiropoulos H, Przedborski S, Nitric oxide and reactive oxygen species in Parkinson's disease, IUBMB Life. 2003 June;55(6):329-35); Delay (as a result of the above) (Tieu K, Ischiropoulos H, Przedborski S, Nitric oxide and reactive oxygen species in Parkinson's disease, IUBMB Life. 2003 June;55(6):329-35).

[0006] heart disease Myocardial ischemia (Sorescu D, Griendling KK, Congest Heart Fail, Reactive oxygen species, mitochondria, and NAD(P)H oxidase in the development and progression of heart failure, 2002 May-June;8(3):132-40); Cardiac hypertrophy (chemogenetic production of hydrogen peroxide in the heart induces severe cardiac dysfunction. Steinhorn B, Sorrentino A, Badole S, Bogdanova Y, Belousov V, Michel T, Nat Commun. 2018 Oct 2;9(1):4044); Heart failure (Mugoni V, Postel R, Catanzaro V, De Luca E, Turco E, Digilio G, Silengo L, Murphy MP, Medana C, Stainier DY, Bakkers J, Santoro MM, Ubiad1 is an antioxidant enzyme that regulates eNOS activity through CoQ10 synthesis. Cell. 2013 Jan 31;152(3):504-18).

[0007] vascular disease ischemia-reperfusion injury (Minutoli L, Puzzolo D, Rinaldi M, Irrera N, Marini H, Arcoraci V, Bitto A, Crea G, Pisani A, Squadrito F, Trichilo V, Bruschetta D, Micali A, Altavilla D, ROS-mediated NLRP3 inflammasome activation in brain, heart, kidney, and testicular ischemia / reperfusion injury, Oxid Med Cell Longev 2016:2183026); Atherosclerosis (Halliwell B, Free radicals, reactive oxygen species, and human disease: a critical appraisal with special reference to atherosclerosis. Br J Exp Pathol. 1989 Dec;70(6):737-57).

[0008] organ failure Kidney stones (Reactive oxygen species as molecular regulators of calcium oxalate kidney stone formation: evidence from clinical and experimental investigations, Khan SR. J Urol. 2013 Mar;189(3):803-11); Ischemic renal failure (Reactive oxygen species: Generation and role in the kidney, Baud L, Ardaillou R, Am J Physiol. 1986 Nov;251(5Pt2):F765-76); Muscular dystrophy (Allen DG, Whitehead NP, Froehner SC, Absence of dystrophin disrupts skeletal muscle signaling: Role of Ca2+, reactive oxygen species, and nitric oxide in the development of muscular dystrophy, Physiol Rev. 2016 January; 96(1):253 -305); Spermatogenesis (reactive oxygen species and spermatids, Sanocka D, Kurpisz M, Reprod Biol Endocrinol. March 23, 2004;2:12).

[0009] Aging Sarcopenia (Yang S, Lian G, ROS and disease: Role in metabolism and energy supply, Mol Cell Biochem. 2020;467(1):1-12); DNA damage (Yang S, Lian G, ROS and disease: Role in metabolism and energy supply, Mol Cell Biochem. 2020;467(1):1-12); hair loss (Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, JN, Rovio, AT, Bruder, CE et al. (2004). Premature aging in mice expressing a defective mitochondrial DNA polymerase. Nature 429, pp. 417-423); Osteoporosis (Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, JN, Rovio, AT, Bruder, CE et al. (2004). Premature aging in mice expressing a defective mitochondrial DNA polymerase. Nature 429, pp. 417-423); Dementia (Chung, HY, Cesari, M., Anton, S., Marzetti, E., Giovannini, S., Seo, AY et al. (2009). Molecular inflammation: a basis for aging and age-related diseases. Ageing Res. Rev. 8, pp. 18-30).

[0010] respiratory disease Asthma (Comhair, SA and Erzurum, SC (2010) Redox control of asthma: molecular mechanisms and therapeutic opportunities. Antiaxis. Redox Signal. 12, pp. 93-124); Chronic obstructive pulmonary disease (King, DA, Cordova, F. and Scharf, SM (2008). Nutritional aspects of chronic obstructive pulmonary disease. Proc. Am. Thorac. Soc. 5, 519-523).

[0011] List of adipose tissue-related diseases: inflammation macrophage infiltration (Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW, Obesity is associated with macrophage accumulation in adipose tissue, J Clin Invest. (2003) 112:1796-808); Increased IFN-γ activity → inflammation (O'Rourke RW, Metcalf MD, White AE, Madala A, Winters BR, Maizlin II et al., Depot-specific differences in inflammatory mediators and the role of nk cells and ifn-gamma in human adipose tissue inflammation. Int J Obes. (2009) 33:978-909); Increased CD4+ and CD8+ T cells in the obese state (Winer S, Chan Y, Paltser G, Truong D, Tsui H, Bahrami J et al. Normalization of obesity-associated insulin resistance by immunotherapy. Nat Med. (2009) 15:921-9); Insulin resistance (Uemura H, Katsuura-Kamano S, Yamaguchi M, Bahari T, Ishizu M, Fujioka M et al., Relationship between serum high-sensitivity c-reactive protein and body size and insulin resistance in a Japanese cohort, PLoS ONE. (2017) 12: e0178672); Cardiovascular disease (Cozlea DL, Farcas DM, Nagy A, Keresztesi AA, Tifrea R, Cozlea L et al., Impact of c-reactive protein on global cardiovascular risk in patients with coronary artery disease. Curr Health Sci J. (2013)).

[0012] Ectopic lipid lipotoxicity insulin resistance (Rutkowski JM, Stern JH, Scherer PE, Cell biology of adipose expansion, J Cell Biol. (2015) 208:501-12); Decreased glucose uptake in skeletal muscle and liver (in addition to the above) (Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, et al., Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (irs-1)-associated phosphatidylinositol 3-kinase activity in muscle, J Biol Chem. (2002) 277:50230-6) - (Holl WL, Brozinick JT, Wang LP, Hawkins ED, Sargent KM, Liu Y, et al., Inhibition of ceramide synthesis ameliorates insulin resistance induced by glucocorticoids, saturated fat, and obesity, Cell Metab. (2007) 5:167-79); cardiovascular homeostasis (Costa RM, Neves KB, Tostes RC, Labato NS, Perivascular adipose tissue as a fat depot associated with cardiovascular risk in obesity, Front Physiol. (2018) 9:253); Pancreatic fatty / metabolic disorders (Uygun A, Kadayifci A, Demirci H, Saglam M, Sakin YS, Ozturk K et al., Effect of fatty pancreas on serum glucose parameters in patients with nonalcoholic steatohepatitis, Eur J Intern Med. (2015) 26:37-41); Hepatic lipid accumulation and inflammation (Kabir M, Catalano KJ, Ananthnarayan S, Kim SP, Van Citters GW, Dea MK, et al. Molecular evidence supporting the portal vein theory: a causal relationship between visceral fat accumulation and hepatic insulin resistance. Am J Physiol Endocrinol Metab. (2005) 288:E454-61).

[0013] Aging Age-related increase in bone marrow adipose tissue and decrease in trabecular bone volume (Justesen J, Stenderup K, Ebbesen EN, Mosekilde L, Steiniche T, Kassem M, Bone marrow adipose tissue volume increases with age and in osteoporotic patients, Biogerontology (2001) 2:165-71); osteoporosis (as a consequence of the above) (Justesen J, Stenderup K, Ebbesen EN, Mosekilde L, Steiniche T, Kassem M, Adipocyte tissue volume in bone marrow increases with age and in osteoporotic patients, Biogerontology (2001) 2:165-71); Oxidative stress / inflammation / telomere shortening (Mundstock E, Sarria EE, Zatti H, Mattos Louzada F, Kich Grun L, Herbert Jones M, Guma FT, Mazzola In Memoriam J, Epifanio M, Stein RT, Barbe-Tuana FM, Mattiello R, The effect of obesity on telomere length: a systematic review and meta-analysis, Obesity (Silver Spring). 2015 November;23(11):2165-74); Mitochondrial dysfunction (Hernandez-Aguilera A, Rull A, Rodriguez-Gallego E, Riera-Borrull M, Luciano-Mateo F, Camps J, Menendez JA, Joven J. Mitochondrial dysfunction: inflammation-associated non-infectious diseases and therapeutic opportunities. Mediators Inflamm. 2013:135698); Cellular senescence (Obesity promotes T cell senescence in mouse visceral adipose tissue, Shirakawa K, Yan X, Shinmura K, Endo J, Kataoka M, Katsumata Y, Yamamoto T, Anzai A, Isobe S, Yoshida N, Itoh H, Manabe I, Sekai M, Hamazaki Y, Fukuda K, Minato N, Sano M, J Clin Invest. 2016 Dec 1, 126(12):4626-4639); Stem cell function (Subcutaneous adipose tissue depots of functionally active stem cells are reduced in obese patients, Onate B, Vilahur G, Ferrer-Lorente R, Ybarra J, Diez-Caballero A, Ballesta-Lopez C, Moscatiello F, Herrero J, Badimon L, FASEB J. 2012 Oct;26(10):4327-36).

[0014] Embodiment 1 MSCs are isolated from patients diagnosed with and / or at high risk for a disease listed in the MSC-treated disease list, transfected with a vector encoding p62 / SQSTM1, and then administered to the patient. p62 is either a gene encoding the full-length protein or an isoform lacking one or more domains. Alternatively, the gene can be modified by codon optimization. Alternatively, p62 can be a p62 with one or more amino acid substitutions or deletions. This treatment reduces the severity of disease symptoms.

[0015] Embodiment 2 Patients diagnosed with and / or at high risk for a disease listed as treatable with MSCs are administered a vector encoding p62 / SQSTM1. p62 is either a gene encoding the complete protein or an isoform lacking one or more domains. Alternatively, the gene can be modified by codon optimization. Alternatively, p62 may contain one or more amino acid substitutions or deletions. This treatment reduces the severity of disease symptoms.

[0016] Embodiment 3 The vector of any of embodiments 1-2 is utilized, which is a plasmid, RNA, cosmid, virus, viral particle, or bacterium containing a p62 plasmid or p62 RNA.

[0017] Embodiment 4 MSCs are isolated from patients diagnosed with and / or at high risk for a disease listed in the MSC-treated disease list, transfected with the protein p62 / SQSTM1, expanded, and administered to the patient. p62 may be either a full-sequence protein or an isoform lacking one or more domains. Alternatively, p62 may be a form in which one or more amino acids have been substituted or deleted. This treatment reduces the severity of disease symptoms.

[0018] Embodiment 5 Patients diagnosed with and / or at high risk for a disease listed as a candidate for treatment with MSCs are administered p62 / SQSTM1 protein. p62 may be either a full-sequence protein or an isoform lacking one or more domains. Alternatively, p62 may be a p62 with one or more amino acid substitutions or deletions. This treatment reduces the severity of disease symptoms.

[0019] Embodiment 6 Any of embodiments 1-5 is practiced using a pharmaceutical carrier that facilitates delivery of p62 to the body, penetration of p62 into cells, and / or stability of p62. This treatment reduces the severity of disease symptoms.

[0020] Embodiment 7 Any of the embodiments 1-6 may be practiced in conjunction with other therapeutic methods, including drugs, and procedures applied to the prevention and treatment of diseases listed in the list of diseases treated with MSCs, which treatments reduce the severity of the symptoms of the disease.

[0021] Embodiment 8 For patients diagnosed with a disease not listed as treatable with MSCs or at high risk for a disease not listed as treatable with MSCs, the patient's MSCs are tested to determine whether they demonstrate low or insufficient differentiation potential. If insufficient disease prevention or treatment, secretion of signaling molecules, or a gene expression profile indicative of disease is demonstrated, the same procedures as in embodiments 1 to 7 are carried out. This treatment reduces the severity of the disease symptoms.

[0022] Embodiment 9 For patients exhibiting high levels of ROS or diagnosed with a disease associated with high levels of ROS, including but not limited to those listed in the ROS-associated disease list, or at high risk for a disease associated with elevated ROS levels, including but not limited to those listed in the ROS-associated disease list, p62 is administered in the same manner as in embodiments 1 to 8. This treatment reduces the severity of the disease symptoms.

[0023] Embodiment 10 For patients receiving MSC cell therapy or prescribed MSC cell therapy for the prevention or treatment of disease, p62 is administered in place of the MSC cell therapy to achieve the preventive or therapeutic effect.

[0024] Example 11 Patients diagnosed with or at high risk of developing a disease on the list of adipose tissue-associated diseases are administered p62 as in embodiments 1 to 8. This treatment reduces the severity of the disease symptoms.

[0025] Overview of the plasmid DNA encoding p62 / SQSTM1 After demonstrating potent antitumor and antimetastatic effects in experimental rodent models and spontaneous canine tumors, this plasmid was tested in a Phase I / IIa clinical trial, demonstrating a favorable safety profile and initial signs of clinical benefit for patients. Notably, this plasmid restored chemotherapy sensitivity in ovarian and breast cancer patients who had become unresponsive to chemotherapy prior to study enrollment. This product also demonstrated favorable results when used as an adjuvant to CMF chemotherapy (cyclophosphamide, methotrexate, and fluorouracil) in patients with triple-negative breast cancer. This may be because chemotherapy, radiation therapy, and other cancer treatments act (at least in part) through the immune response. The p62 plasmid altered the intratumor environment, making it more favorable for immune attack. The p62 plasmid altered the extracellular matrix and even reversed tumor grade and increased the number of tumor-infiltrating lymphocytes (TILs) in canine breast cancer. It also improved the efficiency of adaptive immunotherapy in mice. Tumors employ multiple mechanisms to suppress immune responses. Chronic inflammation is one of these factors, and therefore the anti-cancer effects of p62 plasmid may be mediated (at least in part) by reducing cancer-associated chronic inflammation.

[0026] Inflammation is a low-grade pro-inflammatory phenotype that causes age-related inflammatory diseases (Franceschi et al., 2000), such as osteopenia (Pacifici, 2012). Previous publications demonstrated that intramuscular application of p62 plasmid reduces the severity of age-related inflammatory diseases in rodent models, including metabolic disorders and / or age-related macular degeneration. Osteoporosis is a major age-related disease affecting the postmenopausal female population (Riggs et al., 2002). Menopause triggers a series of events, including a decline in estrogen levels, an increase in lymphocyte counts, and the release of pro-inflammatory cytokines and chemokines by bone marrow stromal cells that disrupt bone and bone marrow homeostasis (Nakashima et al., 2009; Agas et al., 2015). The list of these cytokines includes, but is not limited to, IFNγ, TNFα, IL-1α, IL1β, IL6, and RANKL. Intramuscular administration of p62 plasmid inhibited skeletal deterioration in ovariectomized (OVX) / osteoporotic mice, increasing key bone formation markers such as Runx2 and Osterix, as well as reducing bone resorption factors such as TNFα and RANKL in protein extracts of bone marrow cell populations (Sabbieti et al., 2015). Notably, decreased secretion of cytokines IL6, IL1b, and IL17, known to be essential inducers of inflammation and bone loss, was also observed in bone marrow stromal cells (BMCs) from OVXp62-treated mice (Sabbieti et al., 2015).

[0027] Anti-inflammatory / anti-osteoporotic agents that reduce bone degeneration include hormone therapy such as parathyroid hormone (PTH), calcium phosphate (CaP) and tricalcium phosphate (TCP)-reinforced ceramic biomaterials ( Perez et al., 2018 ), and plasmid-based gene therapy, e.g., bone morphogenetic protein-4 (BMP-4)-plasmid DNA ( Huang et al., 2005 ) or BMP-2-plasmid DNA ( Wegman et al., 2013 ). Some of these therapies are hypothesized to function through the regulation of mesenchymal stem cells (Stem Cells Dev. 2004 Jun;13(3):273-80. doi:10.1089 / 154732804323099208, Role of BMP-6, IL-6, and BMP-4 in Mesenchymal Stem Cell-Dependent Bone Development: Impact on Osteoblast Differentiation Induced by Parathyroid Hormone and Vitamin D (3), J Sammons, N Ahmed, M El-Sheemy, HT Hassan). MSCs can be considered "guardians" of the inflammatory and immune system overreactions that underlie many disease processes (Prockop et al., 2012). Therefore, we decided to test whether p62 plasmids function by modulating MSCs in vitro and / or in vivo and reestablishing the function of bone marrow MSC pools and MSC subgroups.

[0028] Endogenous p62, transcribed and translated from BMC chromosomes, is important for BMC differentiation schedules, osteoblast maturation, and the effects of PTH, a well-known osteogenic regulator (Agas et al., 2020; Agas et al., 2021). However, this does not mean that p62 plasmid acts directly on MSCs (transfecting and expressing plasmid-encoded p62 within affected MSCs) or indirectly without invading affected MSCs. To evaluate the second option, we incubated BMCs not exposed to p62 plasmid in medium collected from p62 plasmid-transfected cells and monitored their differentiation / homeostasis status. After observing the indirect effect of p62 plasmid on MSCs in vitro, we tested whether administration of p62 plasmid had any effect on MSCs in ovariectomized (OVX) rats. OVX rats, as a model more similar to human disease than mice, have been used to study bone physiology and in various anti-osteoporosis experimental protocols (Frost et al., 1992; Jee et al., 2001; Bagi et al., 2011). BMCs play an important role in osteoporosis in OVX rats (Bone Mesenchymal Stem Cell Therapy for Ovariectomized Osteoporotic Rats: A Systematic Review and Meta-Analysis, Zhenxiong Jin, Jinman Chen, Bing Shu, Yanhua Xiao, and Dezhi Tang, BMC Musculoskeletal Disorders, Volume 20, Article Number: 556 (2019)). In addition to the scientific interest in addressing the potential mechanism of action of p62 plasmids, this study may have translational value because hip fractures result in significant morbidity and mortality with significant socioeconomic impacts (Kanis et al., 2005). p62 plasmid DNA may be a potential therapeutic agent to reverse bone loss and combat the development of bone inflammation.

[0029] Materials and Methods DNA plasmids Human p62 (SQSTM, isoform 1) cloned into the pcDNA3.1 vector was provided by CureLab Oncology (USA) ( Sabbieti et al., 2015 ).

[0030] Preparation and culture of mouse bone marrow mesenchymal cells (MSCs) Three-month-old C57BL / 6J (Envigo, Udine, Italy) mice were used and sacrificed by CO2 narcosis and cervical dislocation. MSCs were collected from the femur and tibia of mice and cultured as described by Soleimani and Nadri (2009). Cultures were grown in α-MEM containing 10% HIFCS, penicillin, and streptomycin until they reached 80% confluence.

[0031] Cytokine and chemokine release by mouse MSCs MSCs from C57BL / 6J p62+ / + (WT) mice were transiently transfected with hp62 DNA or pcDNA3.1 (vector control) for 24 hours as previously described (Sabbieti et al., 2020). The medium was then changed and replaced with fresh medium. Two days later, medium from both p62 DNA and pcDNA3.1-transfected cultures was collected, and small aliquots of these supernatants were used to assess cytokine / chemokine release using the Mouse Cytokine Array Panel A Kit (R&D Systems, Milan, Italy) (Sabbieti et al., 2015).

[0032] The remaining supernatant, so-called "conditioned media," was filtered through a 0.22 μm filter to remove cells and used to culture untransfected MSCs for 24 hours. The conditioned media was then discarded, and the cultures were washed and incubated with fresh media. After 2 days, the media was collected and tested for cytokine / chemokine release using a Mouse Cytokine Array Panel A kit (R&D Systems, Milan, Italy).

[0033] Ovariectomy and treatment of rats Virus-free 4-month-old female Wistar rats (Envigo, San Pietro al Natison Udine, Italy) were housed in individual cages in a temperature (22°C) and humidity (50%) controlled room with a 12-h light / 12-h dark cycle. Rats were randomly divided into two groups: a sham-operated group (SO, n = 18) and an ovariectomized group (OVX, n = 18). Three months later, each rat group was further randomly assigned to three subgroups (G1–G3): a saline-only subgroup (G1, n = 6) and subgroups (G2, n = 6, G3, n = 6) that received intramuscular injections of either pcDNA3.1 or hp62 DNA into the hind limb once a week for three weeks. Three months after the last injection, all mice were anesthetized with a mixture of isoflurane and air, and X-ray analysis was performed (see below for details). All animals were then sacrificed by CO2 narcosis. The Italian Committee on Ethics of Animal Experiments has approved the procedures described in this study (approval number: 225 / 2018-PR).

[0034] ROI (Region of Interest) and Quantitative X-ray Analysis Anesthetized rats were placed in a dorsal position, and radiographs were taken to ensure that the pelvis, femur, and tibia were included in the images (Agas et al., 2017). A portable X-ray generator (Gierth HF 80 / 15 plus ULTRA LEICH, Gierth X-Ray International GmbH, Germany) mounted on a fixed stage with a 60 cm focal length was used. The X-ray exposure dose was 54 kV for 0.04 seconds. Radiographs were acquired in DICOM format using a Fujifilm FCR Capsule X (Fujifilm Corporation, Japan) and processed using both Osirix (Pixmeo SARL, Switzerland) and ImageJ (http: / / rsb.info.nih.gov / ij / ) software. Image analysis protocols have been previously reported (McManus and Grill, 2011; Waung et al., 2014). Bone mineral density (BMD) was assessed in the femur using OsiriX software. The following regions of interest (ROI) were selected and defined: the proximal and distal femoral epiphysis (5,600 µm x 7,100 µm); and the femoral diaphysis (3,500 µm x 2,400 µm). Subsequently, the DICOM images were converted to TIFF images using ImageJ, and a 16-interval pseudocolor scale was applied to the grayscale. This scale represents a gradual increase in mineral density in 16 equal intervals, starting from black pixels (value 0) and ending with white pixels (value 255). The distribution of pixels within the same ROI defined above was then calculated and displayed in a histogram.

[0035] Histological bone marrow analysis and immunohistochemical staining Femurs from rats in all groups were dissected free of adhering tissue, fixed in 4% paraformaldehyde, and decalcified as previously described (Agas et al., 2017). Briefly, dehydrated samples were embedded in paraffin and histological sections were obtained using a microtome (Leica Reichert-Jung 2040; Leica Microsystems Srl, All Microscopy and Histology Buccinasco, MI, Italy). Sections were stained with toluidine blue, hematoxylin and eosin (HE), or Masson's trichrome stain.

[0036] To analyze bone marrow fat accumulation, other sections were stained with freshly prepared Oil Red O. Statistical analysis was performed on 12 sections from each sample and on the area of interest under the femoral metaphysis (area, 0.4 × 0.4 mm). 2 ) adipocyte count / mm 2Sections were obtained from the oocytes. Bone marrow adipocytes were quantified as previously described (Wang et al., 2015; Zhu et al., 2013). Other sections were rinsed with phosphate-buffered saline (PBS) and incubated with 0.3% HO. They were then incubated with blocking buffer (PBS containing 0.3% Triton X-100 and 1% bovine serum albumin) containing 10% normal serum for 30–60 min at room temperature (RT) in a humidified chamber. Next, sections were incubated for 1 h at room temperature with the following primary antibodies diluted 1:80 in blocking buffer: rabbit anti-Osterix and rabbit anti-Osteocalcin (Santa Cruz Biotechnology, DBA, Milan, Italy); rabbit anti-CD90, rabbit anti-LepR, rabbit anti-Sca1, and mouse anti-Nestin (Abcam, Prodotti Gianni, Milan, Italy). After washing with PBS, sections were incubated for 30 min at room temperature with biotinylated goat anti-mouse IgG (Vector Laboratories, DBA Milan, Italy) or biotinylated goat anti-rabbit IgG (Bether Laboratories, Aurogene srl, Rome, Italy), both diluted 1:200 in blocking buffer. Control experiments were performed omitting the primary antibody. Slides were imaged using a Leica DM 2500 microscope (Leica).

[0037] Quantitative immunohistochemistry analysis High-resolution, 36-megapixel digital photomicrographs (12 per experimental group) of the defined bone marrow were taken using a Leica DM 2500 light microscope. Pixel intensity was quantified using freely available ImageJ software (version 1.34j, National Institutes of Health, Bethesda, MD).

[0038] Rat cytokine and chemokine assays Long bones (femur, tibia, and humerus) from all rat groups were dissected and cleaned of adhering tissue. The ends were removed and the marrow cavity was flushed with α-MEM (Minimum Essential Medium) as previously described ( Sabbieti et al., 2015 ). Cytokine / chemokine profiles in supernatants of 2-day cultured bone marrow cell (BMC) populations were assessed using the Rat Cytokine Array Panel A Kit (R&D Systems, Milan, Italy) according to the manufacturer's instructions. Immunoreactive dots were visualized using LiteAblot Turbo luminol reagent (Euroclone, Milan, Italy) and Hyperfilm-ECL film (Euroclone, Milan, Italy) and quantified densitometrically.

[0039] P62-transfected cells secrete soluble factors that bias MSCs toward an anti-inflammatory phenotype. A major question was whether the p62 plasmid administered to the body acts directly or through some kind of messenger secreted by cells that take up the injected plasmid. In particular, this question remains important in understanding the ability of p62-encoding plasmids to reverse osteoporosis in mice. To test this question, we transfected cultured MSCs with the p62 plasmid. Directly transfected MSCs showed increased release of anti-inflammatory cytokines and chemokines. However, this may be because MSCs released cytokines in response to factors secreted by the plasmid-pulsed cells that act in an endocrine, paracrine, or autocrine manner, rather than the plasmid itself. We collected conditioned medium from plasmid-transfected MSCs and added it to MSCs that had not been in direct contact with the plasmid. In untransfected cultures, levels of anti-inflammatory IL-1ra and IL-10 increased, while pro-inflammatory IL-1α, IL-1β, IL-6, IL-7, IL-17, and IL-27 molecules decreased (Figure 1). These results strongly suggest that cells transfected with the p62 plasmid secrete a stable soluble signal that regulates the functional activity of distant MSCs.

[0040] Administration of a plasmid encoding p62 reverses bone loss in OVX rats Following in vitro experiments, we tested the efficacy of p62 plasmid as an anti-inflammatory and anti-osteoporotic agent, as well as an MSC differentiation factor, in OVX rats. X-ray analysis and pseudocolor image evaluation of the proximal and distal femoral epiphysis and femoral diaphysis were performed in sham-operated (SO) and OVX rats injected with p62 DNA or pcDNA3.1 plasmid (empty vector). A strong decrease in bone mineral density (BMD) was observed in OVX rats compared with the SO group, confirming the effectiveness of ovariectomy in inducing osteoporosis in rats. Treatment with p62 DNA dramatically increased BMD in OVX rats, but no significant effect was found in the SO group. The pcDNA3.1 plasmid did not alter BMD in either OVX or SO rats (Figure 2A, B).

[0041] The results of X-ray analysis were further confirmed by histological observation. Femurs from rats treated with p62 DNA exhibited a bone-protective environment, corresponding to clinical improvement and / or alleviation of pathological symptoms. In OVX bone marrow, toluidine blue staining at the femoral submetaphyseal and epiphyseal levels revealed decreased structural cohesion and niche disaggregation due to adipocyte infiltration. A decrease in adipocytes and reorganization of bone marrow microregions were observed in OVX rats treated with p62 (Figure 3A). Furthermore, HE and trichrome staining were applied to evaluate the cellularity and tissue integrity of the bone marrow at the submetaphyseal and diaphyseal levels. Osteoporotic rats showed disruption of bone marrow cellularity, partially due to a decrease in the stem / progenitor cell pool and concomitant diffusion of adipocytes into the perivascular and intraosseous spaces. p62 treatment restored bone marrow niche morphology in the OVX group, limiting adipocyte expansion (Figure 3B-C). Oil red staining further confirmed that administration of p62 plasmid significantly reduced adipocyte "invasion" into the bone marrow of OVX rats (Figure 3D, E). Overall, our observations support the notion that administration of p62 plasmid stimulates stem / progenitor cells to restore the finely tuned homeostatic equilibrium within the bone marrow niche, the cellularity and organizational integrity of the niche disrupted by "inflammatory senescence."

[0042] Restoration of osteopenic bone marrow profile by administration of p62 plasmid The integrity of the bone marrow niche is directly related to the functions of MSCs, HSCs, progenitor cells, and mature cells. Adipocyte expansion within the bone marrow impairs hematopoiesis, MSC-HSC interactions, and the release of soluble molecules. We tested whether ovariectomy alters bone marrow cell subpopulations and whether these effects can be reversed by administration of p62 plasmid. The results demonstrated that osteogenic lineage cells, including mesenchymal stem cells, progenitor cells, and mature cell populations such as CD90+, Nestin+, LepR+, Sca1+, Osx+, and OC+ subgroups, were reduced in the bone marrow of OVX rats compared with SO littermates. Administered p62 plasmid was able to slightly increase the above subpopulations in SO rats, but not significantly. In contrast, in OVX rats, p62 plasmid injection promoted the expansion of diaphyseal / progenitor cells and mature osteocytes, while simultaneously suppressing adipocytes in the subepiphyseal and diaphyseal compartments (Figure 4). These results further support the notion that p62 DNA treatment exerts a regulatory effect on rat mesenchymal bone marrow subpopulations that reformats their differentiation schedule toward osteogenesis. Importantly, the effect of p62-encoding vectors on mesenchymal cells is context-dependent; p62 vectors act differently under inflammatory and non-inflammatory conditions, which may be important for drug selectivity.

[0043] Intramuscular administration of a plasmid encoding p62 exerts anti-inflammatory effects in the bone marrow We investigated whether IM-injected p62 plasmid modulates the ability of MSCs to release osteoinductive and anti-inflammatory molecules. Supernatants collected from cells obtained from p62-treated OVX rats contained reduced levels of pro-inflammatory IL-1α, IL-1β, IL-3, IL-17, CCL3, CCL5, GM-CSF, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ compared to OVX animals that did not receive p62 (Figure 5). Meanwhile, enhanced secretion of anti-inflammatory molecules, such as IL-1α, IL-4, IL-6, IL-10, and IL-13, was observed in the OVX p62 plasmid-treated group (Figure 5). In contrast, p62 plasmid did not alter the levels of these molecules in SO rats. This data supports the notion of an osteoprotective effect of extramedullary-administered p62 vectors. Again, p62 plasmids exhibited significantly different effects in vivo under normal and stress / pathological conditions.

[0044] Finally, we tested whether IM-injected p62 plasmids could limit the increase in reactive oxygen species (ROS) levels induced by ovariectomy. As expected, ROS generation was increased in OVX rats (Zhang et al., 2007; Muthusami et al., 2005). However, p62 DNA treatment reduced the ROS increase and re-established ROS levels similar to those in the sham-operated group (Figure 6). MSCs represent an important therapeutic tool for various bone, bone marrow, and systemic inflammatory diseases due to their “hit-and-run” mechanism of action, which is based on the production of exosomes and microvesicles and the release of cytokines, chemokines, and other immunomodulatory factors, resulting in slow tissue “burn” and modulating inflammation (Wu et al., 2020).

[0045] Indeed, there are several animal disease models in which MSCs are thought to play a key role. The development of osteoporosis in ovariectomized animals is one of the most well-studied. Extensive evidence indicates that the prevalence of osteoporosis increases with age in both men and women. It has been hypothesized that sex hormone deficiency interacts with aging-related increases in oxidative stress and inflammation, thereby contributing to the development of degenerative osteoporosis (Almeida et al., 2017). Several factors contribute to the development of osteoporosis, including calcium and vitamin D deficiency, secondary hyperparathyroidism, reduced growth factors, nerve and muscle disorders, and medications.

[0046] Current insights have highlighted the unique immunological features of MSCs that allow their clinical adoption for a variety of diseases. Indeed, to our knowledge, 1,094 MSC-based clinical trials at various clinical stages have been registered, of which 149 trials focus on immune-related diseases, including certain forms of osteopenia ( Mancuso et al., 2019 ; Wu et al., 2020 ).

[0047] In parallel, preclinical studies based on MSC transplantation in OVX model animals such as mice ( Yang et al., 2013 ) and rats ( Yu et al., 2012 ) have revealed promising osteogenic results. Nevertheless, major challenges remain to manipulate and induce MSCs towards an osteogenic / anti-inflammatory bias and consequently enhance their immunomodulatory effects and involvement in bone and bone marrow regulation.

[0048] Therefore, the development of new MSC-modulating drugs is one of the promising directions in modern medicine. Interestingly, it has yet to be verified how many of the existing drug and / or therapeutic combinations that act through this mechanism. Here, we argue that a p62-encoding plasmid administered to animals or humans may bias MSCs toward an anti-inflammatory and / or differentiated phenotype through MSC modulation, thereby maintaining a younger / healthier state of the body. Furthermore, the results presented herein may suggest that administration of p62 plasmids may be an alternative to cell therapy for MSCs.

[0049] Recently, p62 plasmid has been reported to act as a promising anticancer agent in spontaneous cancers in laboratory models of rodents (Venanzi et al., 2013), dogs (Gabai et al., 2014), cats (unpublished data), and humans (Ponomarenko et al., 2017). This plasmid remodels the tumor microenvironment, making it more favorable for anticancer therapy (Venanzi et al., 2019). p62 plasmid can partially restore both metabolic and behavioral components of high-calorie diet-induced obesity (Halenova et al., 2017), partially prevent age-related macular degeneration (Kolosova et al., 2018), and induce bone and bone marrow "rejuvenation" (Sabbieti et al., 2015; Lacava et al., 2019; Agas et al., 2020; Agas et al., 2021). In particular, the p62 plasmid is thought to be a bone marrow niche orchestrator (Lacava et al., 2019; Agas et al., 2020; Agas et al., 2021). However, although the anti-inflammatory potential of the p62 plasmid is well documented, the mechanism of this effect remains largely unknown. In particular, the following two questions need to be addressed: (1) whether the plasmid acts directly or indirectly via messengers; and (2) whether MSCs are involved in the process.

[0050] We prepared four types of MSC cultures: (1) from sham-operated rats transfected with the backbone plasmid, (2) from ovariectomized rats transfected with the backbone plasmid, (3) from sham-operated rats transfected with a p62-encoding plasmid, and (4) from ovariectomized rats transfected with a p62-encoding plasmid. MSCs from ovariectomized rats transfected with the backbone plasmid were then grown in two types of media: fresh medium or medium collected from MSCs from ovariectomized rats transfected with the p62-encoding plasmid. Clearly, the medium collected from MSCs from ovariectomized rats transfected with the p62-encoding plasmid contained released soluble factors. Both MSCs from OVX rats transfected with the p62 plasmid directly or the backbone plasmid proliferated in medium collected from the p62-transfected cells and released "osteoinductive" anti-inflammatory cytokines / chemokines that compensated for the bone-destructive effects of estrogen deficiency.

[0051] The results we present here clearly demonstrate that p62-encoding plasmids act indirectly, allowing cells to internalize the p62 plasmid and secrete a factor that is sensed by distant MSCs. This factor is soluble and long-lived, and can confer anti-inflammatory / survival and lineage fate instructions to MSCs placed in conditioned medium collected from plasmid-transfected cultures. Demonstrating this fact raises many questions. For example, do all cells produce MSC-stimulating factors upon p62 plasmid internalization, or only some cell types? Also, do these cells secrete MSC-stimulating factors regardless of growth conditions, or are there other factors that regulate the ability of p62-plasmid-containing cells to secrete MSC-stimulating factors? Our in vivo experiments demonstrated that p62 plasmids elicit different MSC responses in ovariectomized and non-ovariectomized rats. However, this finding does not explain the specific factors that determine the differences in ovariectomized rats. It also remains to be explained whether ovariectomy affects factor-secreting cells, factor-cancelling bone mesenchymal stem cells, or both. Finally, our study warrants identifying the specific factors (e.g., exosomes, proteins, low-molecular-weight factors, etc.) secreted by p62-transfected cells that are censored by MSCs.

[0052] MSCs are known to be able to respond to cytokines, chemokines, growth factors, and other signals released in the microenvironment. The cytokine profile of cells directly transfected with p62 plasmid showed increased levels of anti-inflammatory cytokines and decreased levels of pro-inflammatory cytokines. A similar cytokine profile was observed in MSCs grown in conditioned medium collected from plasmid-transfected cells, but not directly transfected with p62 plasmid. Therefore, one possibility is that cells engulfing p62 plasmid secrete signals that act in an autocrine, paracrine, and / or endocrine manner. Furthermore, the fact that p62 plasmid upregulates IL-1ra suggests that p62 plasmid could be used to treat lameness in horses secondary to joint and soft tissue injuries.

[0053] To further test our in vitro results in a clinically relevant in vivo model, we administered p62 plasmid to OVX rats and assessed femoral BMD. Not surprisingly, p62 therapy reversed the osteoporotic phenotype in OVX rats and significantly reestablished bone and bone marrow physiology, as observed by whole-body imaging and immunohistochemical analysis. Within the bone marrow, IM-injected p62 plasmid was able to restrict adipocyte proliferation, exerting a "watchdog" role in adipogenesis. Imbalances in adipogenesis are often observed due to aging, comorbidities, and / or unhealthy lifestyles. Whether the downregulation of adipogenesis in bone we report here is related to the metabolic effects of p62 plasmid previously reported in animals receiving a high-fat, high-calorie diet remains to be examined (Halenova et al., 2017). Notably, excessive lipogenesis, which leads to the formation of unhealthy amounts of fat in various parts of the body, is an undesirable process, but excessive suppression of adipogenesis is undesirable because it is necessary for maintaining metabolic homeostasis, sequestering lipids, and avoiding lipotoxicity in the heart, liver, muscle, and other organs, as well as for the innate immune system. The fact that p62 plasmid selectively induces its effects in ovariectomized rats but not in sham-operated rats suggests that this product may be used to selectively balance adipogenesis in disease prevention and treatment, and this may not be limited to bone restriction, but could potentially be extended to the diabetic pancreas, liver, and diabetic fat.

[0054] Previous studies have shown that ovariectomy induces enhanced ROS release (Zhang et al., 2007; Muthusami et al., 2005), whereas estrogen can reduce ROS in bone and bone marrow (Manolagas, 2010). The anti-ROS function of estrogen was demonstrated to be mediated not by binding to ERα DNA response elements but by activating various cytoplasmic molecules. Furthermore, the increase in mitochondrial ROS was found to be more pronounced in cells in which p62 was silenced (Alegre et al., 2017). Our observations show that increased ROS production in the bone marrow of OVX rats parallels increased obesity. However, intramuscular injection of p62 plasmid into OVX rats significantly reduced ROS levels in the bone marrow. This may indicate that administration of a p62-encoding vector can partially compensate for estrogen deficiency. Additionally, because increased ROS generation can upregulate inflammatory processes, the anti-inflammatory effects of p62 plasmids may involve downregulation of ROS. Considering the free radical theory of aging, which claims that ROS release causes the accumulation of oxidative cell damage, exacerbating cellular dysfunction and premature aging (Lopez et al., 2020), p62 vectors may be a potential therapeutic agent for combating cellular senescence and aging. Indeed, loss of proliferative cells can lead to osteoarthritis, atherosclerosis, diabetic pancreas, diabetic adipose tissue, and cancer (Childs et al., 2015). Furthermore, p62 plasmids may protect against drug-induced mitochondrial ROS generation and pro-inflammatory signaling, potentially reducing drug-induced toxicity. This may be particularly important in oncology, where treatment-related toxicity is a serious problem.

[0055] Overall, our results demonstrate that p62 plasmids are a potential preventative and therapeutic agent that can regulate distant MSCs, stimulating them to reduce inflammation and differentiate. This may be achieved without MSCs coming into physical contact with the injected p62 vector, because cells engulfing p62-encoding nucleic acids secrete signals sensed by MSCs. Because p62 plasmids have already demonstrated a favorable safety profile in humans, this may pave the way for the earliest clinical trials. Osteoporosis may be one of the first diseases targeted for this novel clinical trial and application. These data also support the concept of an indirect mechanism of action for p62 plasmids, which may elicit clinical benefits via messengers secreted by cells containing the p62 plasmid.

[0056] publications Agas D, Amaroli A, Lacava G, Yanagawa T, Sabbieti MG, Loss of p62 impairs bone turnover and inhibits PTH-induced bone formation, J Cell Physiol. 2020 Oct;235(10):7516-7529. Agas D, Sabbieti MG, Archetypal autophagy players through a novel lens for bone marrow stem / mature cell control, J Cell Physiol. 2021 Sep;236(9):6101-6114. Agas, D., Gusmao Silva, G., Laus, F., Marchegiani, A., Capitani, M., Vullo, C., Sabbieti, MG(2017), Administration of a plasmid encoding INF-γ causes bone loss and disrupts the bone marrow microenvironment, Journal of Endocrinology, 232,309-321.

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[0061] Manolagas SC From estrogen-centered to aging and oxidative stress: a revised perspective on the pathogenesis of osteoporosis, Endocr Rev. 2010 Jun;31(3):266-300. Zhang L, Fujii S, Kosaka H, Effect of estrogen on reactive oxygen species production in the aorta of ovariectomized Dahl salt-sensitive rats. J Hypertens. 2007;25(2):407-14. Numan, MS, Amiable, N., Brown, JP and Michou, L. (2015), Paget's disease of bone: an osteoimmune disease?, Drug Des. Dev. Ther.9,4695-4707. Song HB, Park SY, Ko JH, Park JW, Yoon CH, Kim DH, Kim JH, Kim MK, Lee RH, Prockop DJ, Oh JY, Mesenchymal stromal cells inhibit inflammatory lymphangiogenesis in the cornea by suppressing macrophages in a TSG-6-dependent manner, Mol Ther. 2018 Jan 3;26(1):162-172.

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[0064] Huang, Y., Simmons, C., Kaigler, D. et al., Bone regeneration in rat cranial defects by delivery of PEI-condensed plasmid DNA encoding bone morphogenetic protein-4 (BMP-4), Gene Ther 12, 418-426 (2005). Kanis JA, Borgstrom F, De Laet C, Johansson H, Johnell O, Jonsson B, Oden A, Zethraeus N, Pfleger B, Khaltaev N. 2005. Assessment of fracture risk. Osteoporos Int16:581-589. Jee WS, Yao W, Abstract: Animal models of osteopenia and osteoporosis, J Musculoskelet Neuronal Interact. 2001 Mar;1(3):193-207. Frost HM, Jee WS. Rat models of human osteopenia and osteoporosis. Bone Miner. 1992 Sep;18(3):227-36.

[0065] Bagi CM, Berryman E, Moalli MR, An anatomical comparison of bones in commonly used laboratory animals: implications for drug discovery, Comp Med. 2011 Feb;61(1):76-85. Muthusami S, Ramachandran I, Muthusamy B, Vasudevan G, Prabhu V, Subramaniam V, Jagadeesan A, Narasimhan S, Ovariectomy induces oxidative stress and impairs bone antioxidant system in adult rats, Clin Chim Acta. 2005;360(1-2):81-6. Ziada AS, Smith MSR and Cote HCF update free radical theory on the aging frontier, Cell Dev. Biol. September 16, 2020. Gabai V, Venanzi FM, Bagashova E, Rud O, Mariotti F, Vullo C, Catone G, Sherman MY, Concetti A, Chursov A, Latanova A, Shcherbinina V, Shifrin V, Shneider A, Pilot study of p62DNA vaccine in dogs with mammary tumors, Oncotarget.2014 Dec 30;5(24):12803-10. Kolosova NG, Kozhevnikova OS, Telegina DV, Fursova AZ, Stefanova NA, Muraleva NA, Venanzi F, Sherman MY, Kolesnikov SI, Sufianov AA, Gabai VL, Shneider AM, Prevention of age-related macular degeneration in a rat model by a p62 / SQSTM1-encoding plasmid. Aging (Albany NY). 2018 Aug 28;10(8):2136-2147.

[0066] Wu, X., Jiang, J., Gu, Z., et al., Mesenchymal stromal cell therapy: immunomodulatory properties and clinical advances, Stem Cell Res Ther 11,345 (2020). Almeida M., Laurent MR, Dubois V., Claessens F., O'Brien CA, Bouillon R., Vanderschueren D., Manolagas SC, Estrogens and androgens in skeletal physiology and pathophysiology, Physiol Rev. 2017 Jan;97(1):135-187. Mancuso P, Raman S, Glynn A, Barry F and Murphy JM (2019), Mesenchymal stem cell therapy for osteoarthritis: the key role of the cellular secretome, Biotechnol. 7:9. Yang Y, Bingyi S, Zhifei Z et al., The role of bone marrow-derived mesenchymal stem cells in the treatment of estrogen deficiency-induced osteoporosis, Chinese J Cellular Molecular Immunol. 2013;29(12):1267-1271. Yu Z, Zhu T, Li C, et al., Improvement of intertrochanteric bone quality in osteoporotic female rats after injection of polylactic-co-polyglycolic acid / collagen type I microspheres combined with bone mesenchymal stem cells, Int Orthop. 2012;36(10):2163-2171.

[0067] Bone marrow mesenchymal / stromal stem cells (MSCs) have become a major research focus because their anti-inflammatory properties and involvement in bone formation can prevent inflammation-induced aging and various forms of osteopenia. We previously demonstrated that engineering and injecting p62 / SQSTM1 plasmids into adult mice can promote the release of anti-inflammatory cytokines / chemokines by MSCs. Furthermore, this can promote osteoblastogenesis at the expense of adipogenesis, improving bone mineral density and bone remodeling. On the other hand, the absence of a partially p62-depleted MSC pool led to the expansion of adipocytes and the accumulation of pro-inflammatory mediators in the bone marrow. Given the important function of p62 as a molecular hub for MSC dynamics, here we used MSCs from p62-knockout adult mice to investigate the effects of this protein on MSC survival and osteogenic molecular cascades. We found that in the absence of p62, key osteogenic pathways were impaired. P62 knockout suppresses Smad activation, Runx2, Osterix, and CREB expression, and reduces the nuclear colocalization of NFκB / Smad1 / 5 / 8 associated with NFκB activation. Considering the "teamwork" of TGFβ, PTH, and BMP2 in MSC homeostatic behavior, p62 plays an important role as a hub protein. Finally, pulsing p62-deficient MSCs ex vivo and administering them to patients as cell therapy may be considered as a treatment for bone and bone marrow diseases.

[0068] One of the criteria for individualizing MSCs is their ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages [1, 2]. The functional schedule of MSCs consists of self-renewal and differentiation, remaining under the control of intramural bone marrow cytokines / chemokines, minerals, growth factors, and systemic factors [2, 3]. Among these, parathyroid hormone (PTH) has been identified as a key regulator of calcium homeostasis and plays a fundamental role in the bone remodeling process by regulating the bone marrow microenvironment and enhancing osteogenic signaling pathways [4-7]. PTH induces transforming growth factor (TGF)-β, wingless MMTV integration site (Wnt), bone morphogenetic protein (BMP), insulin-like growth factor (IGF)-1, and other signaling cascades, modulating the bone marrow microenvironment and controlling the fate of MSCs [3]. For example, PTH induces MSC commitment to osteoblasts by activating BMP and Wnt signaling [8]. In particular, PTH enhances bone marrow CD8+ T cell production of Wnt10b, activating Wnt signaling in MSCs and osteogenic progenitor cells, leading to osteoblastogenesis [8].

[0069] TGF-β is considered one of the major mediators of bone remodeling. TGF-β1, released by osteoclasts, promotes the migration of MSCs to bone resorption sites [9]. TGF-β stimulates the production of FGF2 in osteoblasts and regulates osteoblast proliferation, differentiation, and deposition

[10] . Furthermore, TGF-β regulates MSC migration through SMAD family proteins [3]. Specifically, TGF-β1 induces MSC migration via the ALK5-SMAD2 / 3-SMAD4 cascade [9]. Importantly, TGF-β enhances autophagy through Smad activation, which is associated with the initiation of autophagy-related gene transcription

[11] . Experimental results support the notion that PTH specifically induces MSC differentiation toward bone formation, while TGF-β supports MSC spatial distribution and homing to specific bone marrow regions. Thus, the coordinated mode of action of these two mediators is essential for bone remodeling processes and MSC dynamics [3].

[0070] Along with PTH and TGF-β, BMPs play a fundamental role in skeletogenesis and the osteogenic commitment of MSCs. Indeed, early in vitro studies on rat MSCs demonstrated the efficacy of BMP-2 in enhancing MSC differentiation into osteoblasts through the expression of alkaline phosphatase and osteocalcin and N-cadherin regulation [12, 13]. Relatedly, the interconnection between BMP-2 and Wnt through β-catenin and N-cadherin signaling network interactions has also been reported as a clear link in MSC osteoinduction

[14] . Current research findings highlight the importance of crosstalk between the above factors and the autophagy machinery in facilitating MSC surgical tasks [15, 16]. Indeed, autophagy levels directly affect MSC behavior, and reduced autophagy activity in aged animals is associated with impaired MSC proliferation and lineage commitment (Ying et al., 2019). P62, Atg7, Atg5, and Beclin are representative examples of autophagy players involved in MSC survival and differentiation fate (Agas et al., 2021).

[0071] The adaptor protein p62 (also known as sequestosome 1 / SQSTM1) was initially considered a multifunctional hub involved in autophagy, cell death, inflammation, immune regulation, oxidative stress response, and the aging process (Seibenhener et al., 2004; Moscat et al., 2012; Lee et al., 2012; Kwon et al., 2012). Given the multitasking properties of p62, this hub protein has also been studied in bone and bone marrow homeostasis. Data regarding the association of p62 with bone remodeling remain contradictory in some cases. On the one hand, it has been proposed that p62 induced osteoclastogenesis via RANK-TRAF6-NFκB activation (Moscat et al., 2009). On the other hand, other findings attribute the anti-osteoclastogenic effect of p62 to the promotion of osteoclast differentiation in the absence of p62 (Zach et al., 2018).

[0072] We previously reported that exogenous p62 administration as a plasmid ameliorates inflammatory bone loss, suppresses proinflammatory cytokines / chemokines, and promotes bone deposition (Sabbieti et al., 2015). Furthermore, we demonstrated the importance of p62 in MSC differentiation and maintenance of the bone marrow MSC niche in aged mice. p62 knockout mice showed depletion of MSC subpopulations and reduced MSC differentiation toward adipogenesis. Lack of p62 accelerated the pathological scenario of inflammatory aging and increased bone loss (Lacava et al., 2019). Furthermore, p62 regulates osteoblast differentiation, concomitant with an increase in key osteogenic markers such as Runx2 and Osterix. Furthermore, PTH regulates p62 expression in adult mice, and the osteogenic effects of PTH were significantly abolished in p62- / - mice (Agas et al., 2020).

[0073] Motivated by these results, we questioned the functional relevance of p62 to MSC maintenance and differentiation pathways. Considering the "teamwork" of PTH, TGFβ, and BMPs in the osteogenic activity of MSCs within the bone marrow, we investigated the interplay between p62 and the aforementioned osteogenic signaling cascades using MSCs derived from p62- / - adult female mice. Furthermore, we addressed whether p62 DNA transfection could direct MSCs toward an anti-inflammatory / osteoinductive pathway.

[0074] Materials and Methods animal C57BL / 6J (Envigo, Udine, Italy) wild-type (WT) and p62 / SQSTM1 knockout (p62KO) mice were used. p62KO (p62- / -) mice were generated by conventional methods as previously described (Komatsu et al., 2007; Lacava et al., 2019). Prior to analysis, these mice were crossed with C57BL / 6J WT mice (Charles River) for over 10 generations to generate normally developing, fertile offspring. Mice were housed in laminar flow cages under standardized environmental conditions. Eight-month-old mice were used for the experiments. This age was chosen because adult mice typically reach peak bone mass at 6 months of age and gain an additional 5% to 10% between 6 and 12 months of age (Jilka, 2013). Mice were sacrificed by CO2 narcosis and cervical dislocation. The Italian Ethics Committee for Animal Experiments approved the procedures described in this study (approval number: 225 / 2018-PR).

[0075] DNA Plasmids and Therapeutics Human p62 (SQSTM, isoform 1) cloned into the pcDNA3.1 vector was provided by CureLab Oncology (USA) ( Sabbieti et al., 2015 ). Mouse p62 (SQSTM, isoform 1) was cloned into the pcDNA3.1 (Invitrogene) vector.

[0076] Immunohistochemical staining and immunofluorescence analysis Femurs from the p62+ / + and p62- / - mouse groups were dissected free of adherent tissue, fixed, decalcified, and embedded as previously described (Agas et al., 2017; Lacava et al., 2019). Sections (10–12 μm thick) were obtained using a Leica Reichert-Jung2040 microtome (Leica Microsystems Srl, All Microscopy and Histology Buccinasco [MI], Italy). For immunohistochemical staining, sections were incubated in 0.3% HO and then incubated with blocking buffer (PBS containing 0.3% Triton X-100 and 1% bovine serum albumin) containing 10% normal serum for 30–60 min at room temperature (RT) in a humidified chamber. Next, sections were incubated with the following primary antibodies diluted 1:80 in blocking buffer for 1 h at room temperature: rabbit anti-CD90, rabbit anti-Sca1 (Santa Cruz Biotechnology, DBA, Milan, Italy), rabbit anti-nestin, rabbit anti-LepR, and rabbit anti-osterix (Abcam, Prodotti Gianni, Milan, Italy). After washing with PBS, sections were incubated with biotinylated goat anti-rabbit IgG (Bether Laboratories, Aurogene srl, Rome, Italy) diluted 1:200 in blocking buffer for 30 min at room temperature. Control experiments were performed omitting the primary antibodies. Slides were imaged using a Leica DM 2500 microscope (Leica).

[0077] For immunofluorescence analysis, other sections permeabilized with 0.3% Triton X-100 were incubated with the above antibodies diluted 1:80 in PBS. After washing, sections were incubated with Alexa Fluor 488 chicken anti-rabbit IgG secondary antibody (Euroclone, Milan, Italy) diluted 1:100 in PBS. Control experiments were performed by omitting the appropriate primary antibody or neutralizing the primary antibody with the corresponding blocking peptide.

[0078] Bone marrow mesenchymal stem cells (BM-MSCs) isolation, culture, and p62 DNA transfection Mesenchymal stem cells were isolated from the bone marrow of C57BL / 6J p62+ / + and p62− / − mice and cultured as described by Soleimani and Nadri (2009). Cultures were maintained in α-MEM containing 10% HIFCS, penicillin, and streptomycin until they reached 80% confluence. Some cultures were transiently transfected with 2.5 μg of human p62 DNA (hp62DNA) as previously described (Agas et al., 2020). Control cultures were transfected with pcDNA3.1 (vector control). Transfected and untransfected cultures were treated with different doses of TGFβ, PTH, and BMP2 for Western blotting and immunofluorescence staining.

[0079] Westerblotting For Western blotting, 5 × 10 MSCs from C57BL / 6J p62+ / + and p62− / − mice were plated in a 6-well plate. 6 Cells were seeded at a density of 100 cells / well. Cultures were maintained in α-MEM containing 10% heat-inactivated fetal bovine serum (HIFCS), penicillin, and streptomycin until they reached 80% confluence. Cultures were treated with different TGFβ, PTH, and BMP2 for different periods of time.

[0080] Proteins from BM-MSCs were extracted with cell lysis buffer (Cell Signaling, EuroClone, Milan, Italy), and the concentration was measured using BCA protein assay reagent (Pierce, EuroClone). Western blotting was performed as previously described in Sabbieti et al. (2010). Membranes were incubated with the following primary antibodies diluted in blocking buffer: rabbit anti-mouse p62 (1:800 dilution, Sigma-Aldrich, Milan, Italy); rabbit anti-phospho-Smad2 / 3 and rabbit anti-phospho-Smad4 (1:400 dilution, Abcam, Prodotti Gianni, Milan, Italy); rabbit anti-phospho-Smad1 / 5 / 8; rabbit anti-phospho-CREB (1:600 dilution, Abcam, Prodotti Gianni, Milan, Italy); and rabbit anti-NFκB (1:500 dilution, BioLegend, Microtech Srl, Naples, Italy). After washing with PBS-T, the blots were incubated with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (Abcam, Prodotti Gianni, Milan, Italy) diluted 1:100,000 in blocking solution for 1 hour at room temperature. Immunoreactive bands were visualized using LiteAblot Turbo Luminol Reagent and Hyperfilm-ECL film (Abcam, Prodotti Gianni, Milan, Italy) according to the manufacturer's instructions. To normalize bands, the filters were stripped and reprobed with monoclonal anti-α-tubulin (Sigma-Aldrich). Band density was quantified by densitometry using Image J software.

[0081] RNA interference MSCs from C57BL / 6J p62+ / + mice were cultured at 10 × 10 cells per well in 6-well plates in α-MEM containing 10% HIFCS, penicillin, and streptomycin until they reached 80% confluence. 6Cells were seeded at a density of 1000 cells / well. Cells were then transfected with p62 siRNA as previously described (Agas et al., 2020). p6 levels were analyzed by Western blotting using rabbit anti-mouse p62 antibody (Sigma-Aldrich, Milan, Italy), and the specificity of silencing was confirmed in three independent experiments.

[0082] Subcellular fractionation MSCs obtained from p62+ / + and p62- / - mice were plated as described above, grown to confluence, and stimulated with BMP-2 (10 ng / ml) for 24 hours. Control cultures were treated with the appropriate vehicle. Cell fractionation was performed using the Qproteome Cell Compartment Handbook (Qiagen SPA, Milan, Italy) according to the manufacturer's instructions. Fractions were then processed as described above in the Western blot section. Membranes were incubated with rabbit anti-phospho-Smad1 / 5 / 8 antibody (Abcam, Prodotti Gianni, Milan, Italy) or rabbit anti-NFκB (BioLegend, Microtech Srl, Naples, Italy), both diluted 1:500, for 2 hours at room temperature. The next step was performed as described above. To normalize bands, filters were stripped and reprobed with rabbit anti-B23 / nucleophosmin (Santa Cruz Biotechnology, Inc., tebu-bio, Milan, Italy) antibody. Band density was quantified by densitometry.

[0083] Immunofluorescence of Smads1 / 5 / 8 and NfκB Fixed and permeabilized MSCs (Marchetti et al., 2006) were incubated with the following primary antibodies: goat anti-NFκB (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:50 in PBS, and rabbit anti-phospho-SMAD1 / 5 / 8 (Abcam, Prodotti Gianni, Milan, Italy) diluted 1:80 in PBS. After washing with PBS, cells were incubated with Alexa Fluor 594 mouse anti-rabbit IgG (Texas Red; Molecular Probe, Invitrogen srl, Milan, Italy) diluted 1:100 and Alexa Fluor 488 chicken anti-IgG-rabbit IgG (FITC; Molecular Probe, Invitrogen srl, Milan, Italy) diluted 1:100 for 1 hour at room temperature. After washing, coverslips were mounted onto slides using PBS / glycerol (1:1). Slides were imaged using a fluorescent Zeiss Axioplan microscope. Fluorescence analysis was performed by a fluorometer Tecan Infinite with an excitation filter of 485 nm and an emission of 535 for Alexa Fluor 488 and 590 and 635 for Alexa Fluor 594. The amounts of Alexa Fluor 594-labeled anti-NFκB and Alexa Fluor 488-labeled anti-Smad1 / 5 / 8 were quantified with a Tecan Infinite fluorescence reader.

[0084] Quantitative immunohistochemistry analysis High-resolution, 36-megapixel digital photomicrographs (12 per experimental group) of the defined bone marrow were taken using a Leica DM 2500 light microscope. Pixel intensity was quantified using freely available ImageJ software (version 1.34j, National Institutes of Health, Bethesda, MD).

[0085] statistical analysis Statistical analysis was performed by Student's t-test. Two-way analysis of variance (ANOVA) was also used to compare variables between experimental groups. Tukey's test was used for multiple comparisons between all groups. All statistical analyses were performed using GraphPad Prism (v6.01). Data were presented as mean ± standard error. A value of P < 0.05 was considered significant.

[0086] P62 regulates MSC subpopulation pools Specific identification markers for individual MSC subtypes were used, including Osterix, Nestin, LepR, Sca1, CD90, CD146, and CD166 (Dominici et al., 2006; Morrison et al., 2014). Through immunofluorescence and immunohistochemistry, we demonstrated that the absence of p62 altered the pool and allocation of Sca1+ and CD90+ cells. Furthermore, we observed a decrease in the perivascular distribution of Nestin+ and LepR+ populations, as well as condensed endosteal and perivascular Osx+ cells (Figure 1). Because this is the first observation of a decrease in "typical" MSC populations, Sca1+ and CD90+, loss of p62 appears to affect all MSC subpopulations. The decrease in MSC populations seen in p62- / - cultures may reflect a dysregulation of MSC homeostasis. Interestingly, a pro-inflammatory environment caused by aging, infection, or aberrant gene expression in the bone marrow can alter the behavior of HSCs and MSCs by altering the stem cell niche (Schuettpelz et al., 2013; Josephson et al., 2019). Consequently, we analyzed the release of pro- and anti-inflammatory cytokines and chemokines from MSC supernatants of both genotypes ex vivo. Furthermore, parallel MSC p62+ / + and p62- / - cultures were treated with p62 DNA plasmid to examine the potential effects of exogenously administered p62 in both MSC microenvironments. Notably, pro-inflammatory cytokines / chemokines, such as INF-γ, IL-1α, IL-1β, IL-7, IL-17, IL-27, and IL-6, were significantly reduced after p62 DNA plasmid treatment (Figure 2). The effect of exogenous p62 was particularly pronounced in p62- / - mice, highlighting p62 action primarily within pathological / abnormal scenarios. In this context, we questioned the efficacy of key survival / differentiation MSC mediators such as TGFβ, PTH, and BMP2 in the absence of p62.

[0087] P62 as a hub protein in TGFβ signaling To determine whether p62 can affect TGFβ osteogenic signaling, we performed Western blotting analysis on p62-deficient mice and their WT counterparts. Our findings revealed that stimulating MSCs with TGFβ at doses of 1.25, 2.5, and 5 ng / ml increased p62 protein expression in MSCs, with a maximal response at 5 ng / ml for 6 hours (Figure 3A, B). Interestingly, 5 ng / ml of TGFβ was able to stimulate p.Smad2 / 3 (6 hours) and p.Smad4 (6 and 24 hours) in p62+ / + MSC cultures. The above-mentioned effects were abolished in TGFβ-treated p62- / - MSCs (Figure 3C, D). Subsequently, p62- / - MSCs were transfected with hp62 DNA for 24 hours and then stimulated with 5 ng / ml of TGFβ for 6 hours. The TGF-β response to p.Smad2 / 3 and p.Smad4 proteins was only potent in p62- / - cultures transfected with hp62 DNA compared with cells transfected with the empty vector (control) (Figure 3E). Furthermore, specific siRNA against p62 was applied to abolish synthesis in WT BMSCs. siRNA efficiently and specifically suppressed p62 synthesis (more than 65%). The fact that TGF-β (5 ng / ml, 6 h) failed to induce an increase in p.Smad2 / 3 and p.Smad4 proteins in p62-silenced MSCs (compared to scrambled siRNA "control" cultures) is considered further evidence of the importance of p62 in TGF-β signaling in MSCs (Figure 3F). Indeed, even partial (approximately 65%) p62 ablation can prevent the activation of Smad proteins.

[0088] PTH enhances osteogenic signaling only in p62+ / + MSCs Because PTH can promote the TGF-β response of MSCs, questions remain as to whether p62 can influence PTH-induced osteoblastogenesis and whether co-treatment with PTH and TGF-β can enhance MSC osseointegration. Western blotting analysis showed that MSCs stimulated with 10-9M PTH strongly increased p.CREB expression after 6 hours of treatment in p62+ / + MSCs, but only very weakly upregulated it in p62- / - MSC cultures (Figure 4A). PTH-induced expression of p62, as well as Runx2 and Osx, in WT MSCs can be considered a predictable result, as we have previously described the stimulatory effect of PTH on p62 levels in MSCs and preosteoblasts from 8-week-old mice (Agas et al., 2020). Based on age-related p62 expression, our findings were expected but not conclusive. Furthermore, simultaneous treatment with PTH and TGFβ failed to increase p62, likely due to attenuated cellular responses to these dose-dependent bone anabolic effectors ( Fig. 4B ).

[0089] p62 signaling enhances the BMP2 downstream pathway Interestingly, we observed that BMP2 (at concentrations up to 10 ng / ml) could increase p62 expression (Figure 5A), with a maximum response observed at 24 hours (Figure 5B). Consequently, dose-response and time-course experiments were performed to clarify the BMP2 intramural signaling cascade in the presence and absence of p62. As expected, active downstream Smad1 / 5 / 8 signaling was reduced in p62KO MSCs, even after 24 hours of BMP2 treatment (Figure 5C, D). The involvement of p62 in the BMP2 osteoinductive cascade was further confirmed in p62-knockdown MSCs. Partial (>65%) p62 ablation was sufficient to reduce Smad1 / 5 / 8 expression in response to BMP2 (Figure 5E). Interestingly, the absence of p62 increased NFκB expression in MSCs, enhancing the nuclear accumulation of NFκB-p.Smads1 / 5 / 8. Similarly, the effect of BMP2 on Smads and their nuclear translocation in p62- / - cultures was impaired (Figure 5F). To further analyze the spatial distribution of NFκB and p.Smad1 / 5 / 8 in the absence of p62 after BMP2 treatment, we applied immunofluorescence techniques. Quantitative immunofluorescence analysis revealed that nuclear colocalization of NFκB and p.Smad1 / 5 / 8 was evident in p62- / - MSCs (Figure 6A). The spatial overlap of NF-κB and Smads1 / 5 / 8 in the nuclei of p62- / - MSCs predicts reduced Smad transcriptional activity, reflecting reduced osteoclastogenic mediator release. Transfecting p62- / - MSCs with hp62 DNA for 24 hours and subsequently stimulating them with BMP2 for 24 hours partially, but not completely, reversed the above phenotypes (Figure 6B).

[0090] Contemporary research on bone marrow mesenchymal / stromal stem cells asserts that the autophagy machinery plays a prominent role in maintaining stemness (Sbrana et al., 2016) and differentiation (Nuschke et al., 2014). Autophagy can (re)program MSC osteogenic commitment, revealing that hallmarks of osteoblastogenesis and osteocyte homeostasis require the monitoring and control of the autophagy agenda (Ying et al., 2019). Indeed, typical autophagy players, within autophagy-related or -independent mechanisms of action, exert anti-aging effects on mesenchymal stem cells and protect bone integrity from age-related diseases (Rubinsztein et al., 2011; Sabbieti et al., 2015; Agas et al., 2021).

[0091] In this study, we addressed the involvement of p62 in MSC signaling dynamics. First, we focused on the behavior of various MSC subpopulations in the absence of p62. Stem cell antigen 1 (Sca1) was developed as a selectable marker for mouse MSC populations (Houlihan et al., 2012). Interestingly, ablation of Sca1+ progenitor cells promotes the depletion of all precursor MSC populations, including hematopoietic stem / progenitor cells in the bone marrow niche (Hu et al., 2016). Based on immunofluorescence and immunohistochemistry assays that better delineate the topography and distribution of various bone marrow residents, we demonstrated that the Sca1+ cell pool is significantly altered in the absence of p62. In parallel, we found that nestin+, Osx+, and LepR+ subgroups were reduced and scattered within the p62- / - bone marrow. Our results predict impaired expansion of MSC subpopulations, likely due to impaired proliferation / survival signaling in p62- / - MSCs.

[0092] Deviations from the survival / operation schedule of MSCs have been observed primarily during predominant abnormalities in the bone marrow caused by inflammatory diseases. The release of proinflammatory cytokines and chemokines may strengthen the inflammatory network, shorten MSC lifespan, and affect the operation schedule of MSCs (Lepperdinger, 2011; Deshpande et al., 2013; Agas et al., 2015). In this perspective, we examined the pro- and anti-inflammatory mediators secreted by MSCs from both genotypes and found that p62 knockout increased inflammatory cytokines.

[0093] Because the role of cytokines and chemokines in MSC commitment to osteogenesis has been clearly defined, our findings encourage further investigation of the effects of p62 knockout on MSC differentiation. Because the initiation of autophagy attenuates the production of inflammatory cytokines (Tilija Pun et al., 2017), it is possible that p62 functions as part of the autophagy machinery and / or as a hub protein in the anti-inflammatory cascade. Furthermore, the autophagy machinery is strictly linked to the differentiation and mineralization agenda of progenitor and osteocyte cells (Ying et al., 2019). Similarly, key osteogenic induction factors are being investigated. Data on the role of TGF-β in autophagy revealed that this effector enhances the mRNA expression levels of various autophagy-related genes, such as BECN1, ATG5, ATG7, and DAPK (Kiyono et al., 2009). Furthermore, TGF-β-Smad signaling stimulates the proliferation, chemotaxis, and early differentiation of committed MSCs (Wu et al., 2016). Downstream events of activated TGF-β receptors culminate in the phosphorylation and nuclear translocation of R-Smads (Smad2 / 3) (Massague, 2012; Nallet-Staub et al., 2015). R-Smads can recruit other transcription factors, such as CREB-binding protein and p300, to the nucleus to induce gene transcription (Wu et al., 2016). Notably, TGF-β anabolism was impaired in p62- / - MSC cultures, indicating that the presence of p62 plays a direct role in the signaling cascade that culminates in Smad activation.

[0094] PTH regulates the activity of several factors, such as TGF-β and BMP2, released into the bone matrix by MSCs and bone-lineage cells (Crane et al., 2014). In this context, PTH stimulates the internalization of the PTH1R-TGFβR2 complex, resulting in the expression of osteogenic genes (Atfi et al., 2010; Chen et al., 2012). It is also well known that the transcription factor cAMP response element-binding protein (CREB) mediates PTH responses in differentiating osteoblasts, and PTH-CREB signaling enhances BMP2 expression (Zhang et al., 2011). Accordingly, we demonstrated that PTH stimulation of MSCs induces the expression of Runx2, Osterix, and CREB in the presence of p62. Our results therefore support the fact that p62 protein functions as an important intermediate signaling molecule for PTH / TGF-β-related osteogenic MSC commitment.

[0095] To obtain further information regarding the involvement of p62 in bone marrow dynamics, we treated p62+ / + and p62- / - MSCs with BMP2. The requirement for BMP and Wnt signaling in MSC osteoinduction is well established, as treatment with specific BMP / Wnt antagonists abolished osteoblast formation (Eyckmans et al., 2010). The downstream mediators Smad1 / 5 / 8 are directly linked to MSC differentiation and osteogenic programming (Chen et al., 2004; Miyazono et al., 2005). Impairment of BMP signaling by overexpression of specific BMP inhibitors, such as Noggin and Gremlin, results in reduced bone deposition (Zhu et al., 2006; Davis et al., 2007). Furthermore, the interaction between TGF-β1 and BMP signaling on the osteogenic commitment of MSCs was also specified, as TGFβ can induce Smad1 / 5 / 8 signaling in MSCs via the ALK1 pathway (Chen et al., 2012; Wu et al., 2016). Previous findings also claimed that the osteoinductive BMP2 action is mediated by the autophagy mediator Atg7, which is involved in the activation of Wnt16 and metalloproteinase-13 and osteoblast differentiation (Ozeki et al., 2016).

[0096] Here, we found evidence for the first time that BMP2 increases p62 expression 4 to 24 hours after treatment (Figure 5). Interestingly, in p62KO mice, active Smad1 / 5 / 8 were unaffected after both PTH and BMP2 treatment. Because Atg7 itself, or within the autophagy scenario, was not sufficient to trigger an osteoinductive response, we speculate that p62 functions upstream, possibly signaling through PTH. Indeed, our findings, consistent with previous data, strongly suggest that the PTH anabolic cascade intersects with BMP2 and Wnt signaling and thus can amplify their osteoinductive responses. Therefore, suppression of PTH signaling in p62KO mice may attenuate the BMP2 / Smads pathway. It has been demonstrated that BMP2 induces osteoblast differentiation when NF-κB is blocked (Eliseev et al., 2006). Furthermore, the interaction of nuclear NF-κB with Smads1 / 5 / 8 inhibits Smads binding to target promoters (Yamazaki et al., 2009). In this context, we observed increased nuclear expression and colocalization of NF-κB / Smads1 / 5 in p62- / - MSCs. The above data predict an indirect involvement of p62 in NF-κB expression and nuclear localization. Consequently, the lack of p62 may regulate the differentiation schedule of MSCs and shift their commitment toward adipogenesis by impairing PTH / BMP2 signaling.

[0097] Despite its promising mechanism of action, previous attempts to utilize BMP2-encoding plasmids as therapeutic agents were unsuccessful due to safety concerns. In contrast, preclinical toxicology and Phase I / IIa clinical trials using p62-encoding plasmids demonstrated a desirable level of safety. Therefore, our results suggest that p62 plasmids can be used as a safer alternative to BMP2 plasmids. Furthermore, ex vivo MSC cell therapy does not work if MSCs isolated from patients encode mutated p62 or are deficient in p62 expression. Our data suggest that pulsing these cells with a plasmid encoding wild-type p62, expanding these cells, and then administering them to patients may be a solution to this problem. Overall, our findings reveal that the operational challenges of bone marrow MSCs are at least partially dependent on p62 function. P62 can be designed as a critical hub protein, the absence of which may impede typical survival / osteoinductive PTH / TGF-β / BMP-related signaling in MSCs. The intracellular spatiotemporal interactions of p62 and its placement within the complex MSC signaling cascade remain important questions that require further investigation.

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[0109] Text description of the illustration image022.gif. Figure 1. Key anti- and pro-inflammatory cytokines and chemokines were analyzed in medium from bone marrow mesenchymal stem cell cultures obtained from experimental groups. Two-way ANOVA revealed a statistically significant effect of in vitro administration of p62 plasmid and the ability of conditioned medium to stimulate anti-inflammatory factors in parallel MSC cultures: (A) MSCs transfected with pcDNA3.1, (B) MSCs transfected with p62 plasmid, (C) untransfected MSCs treated with conditioned medium from "(A)," and (D) untransfected MSCs treated with conditioned medium from "(B)." Lowercase letters indicate homogenous subsets (p<0.05).

[0110] Figure 2. X-ray images of the pelvis and hind limbs of sham-operated and OVX rats. The area of the ROI is indicated (small green oval). Histograms of bone mineral density measured at the levels of the distal epiphysis, diaphysis, and proximal epiphysis from the original 16-bit DICOM radiograph (A). Pseudocolor images of the pelvis and hind limbs from the same radiograph, obtained by converting the original 16-bit DICOM radiograph to 8-bit TIF format, were pseudocolored using a 16-color lookup table. In the pseudocolor image, low bone mineral content is represented by green and yellow, while high bone mineral content is represented by red and purple. The histograms represent BMD levels within the subchondral bone ROI (B). Note the partial BMD recovery after p62 plasmid treatment in OVX rats (p<0.05).

[0111] Figure 3. Representative reconstructions of toluidine blue (A), hematoxylin / eosin (B), and Masson's trichrome staining (C) of the metaphyseal region of the distal femur from sham-operated and p62 plasmid-treated OVX rats. Note the expansion of bone marrow adipocytes, particularly in OVX littermates, and the significant decrease in adipocytes after p62 treatment. Oil red staining (D) further supports the hypothesis that p62 plasmid promoted MSC differentiation into osteocytes at the expense of adipocytes. Quantification of adipocytes (E) revealed the ability of p62 plasmid to inhibit the OVX adipose inflammatory bone marrow microenvironment. Lowercase letters indicate homogenous subsets (p<0.05).

[0112] Figure 4. Immunostaining of CD90 (A), Sca1 (B), nestin (C), LepR (D), osterix (E), and osteocalcin (F) positive MSC subpopulations in the medullary submetaphyseal region of rat femurs. Note the ability of the p62 plasmid to completely (LepR) or partially (CD90, Sca1, nestin) reconstitute MSC subpopulations and osteoprogenitor cells (Osx, OC). Two-way ANOVA indicates a statistically significant effect of the p62 plasmid, particularly in the OVX-treated group, with no statistically significant interaction between the variables. Lowercase letters indicate homogenous subsets (p<0.05).

[0113] Figure 5. Cytokines and chemokines were examined in media obtained from bone marrow mesenchymal stem cell cultures from all experimental groups of rats. Two-way ANOVA revealed a statistically significant characteristic of the p62 plasmid in osteoporosis scenarios. Note the reduction in pro-inflammatory cytokines / chemokines after p62 treatment in the OVX group. Lowercase letters indicate homogenous subsets (p<0.05). Figure 6. Intracellular ROS production in MSCs in different experimental groups of rats. Note the statistically significant reduction in ROS in the OVXp62-treated group. Lowercase letters indicate homogenous subsets (p<0.05).

[0114] Figure 1. (A) Immunofluorescence analysis of CD90-, Sca1-, nestin-, LepR-, and osterix-positive MSC subpopulations in the submetaphyseal region of the femur of p62+ / + and p62- / - mice. Twelve sections from each sample were analyzed by fluorescence microscopy (green: FITC staining). A light microscopy tissue section from the same region is shown in the inset (magnification: 20x). (B) Immunostaining of CD90-, Sca1-, nestin-, LepR-, and osterix-positive MSC subpopulations in the femoral bone marrow (submetaphyseal region) of p62+ / + and p62- / - mice. Decreased perivascular and periosteal distribution was observed in p62- / - mice. Values are presented as mean ± SD and statistically analyzed by t-test (p<0.05).

[0115] Figure 2. Cytokine and chemokine release was analyzed in the medium from bone marrow mesenchymal stem cell cultures obtained from various experimental groups. Two-way ANOVA shows statistically significant effects of p62+ / + and p62- / - mice, and p62 DNA plasmid treatment or untreated, but no statistically significant interactions between variables. Lowercase letters indicate homogenous subsets (p<0.05).

[0116] Figure 3. (A) Dose-response and (B) time-course effects of TGFβ on p62 synthesis. MSCs were treated with 1.25 ng / ml to 5 ng / ml TGFβ or vehicle for 4 to 24 hours. Representative Western blot analysis showed that TGFβ could increase p62 protein levels, primarily at 5 ng / ml after 6 hours of stimulation. The graph represents the results of three independent experiments. Values are expressed as mean ± SD and statistically analyzed by t-test (p<0.05). (C) Dose-response and (D) time-course effects of TGFβ on p.Smad2 / 3 and p.Smad4 expression from p62+ / + and p62- / - MSCs. Representative Western blot analysis highlighted the decrease in p.Smad2 / 3 and p.Smad4 expression after 6 hours in p62 knockout MSCs treated with TGFβ (5 ng / ml). The graph represents the results of three independent experiments. Values are presented as mean ± SD and statistically analyzed by t-test (p<0.05). (E) Smad2 / 3 and Smad4 levels on MSCs from p62- / - mice after transfection with p62 DNA and stimulation with TGFβ. Representative Western blot analysis of Smad2 / 3 and Smad4 in MSCs transfected with hp62 DNA or pcDNA3.1 and treated with TGFβ. Graph represents the results of three independent experiments. Values are presented as mean ± SD and statistically analyzed by t-test (p<0.05). (F) TGFβ-stimulated Smad2 / 3 and Smad4 levels in BMSCs after p62 knockdown. Representative Western blot analysis of Smad2 / 3 and Smad4 in BMSCs transfected with p62 siRNA or control siRNA and treated with TGFβ (5 ng / ml, 6 hours). Graph represents the results of three independent experiments. Values were expressed as mean ± SD and statistically analyzed by t test (p < 0.05).

[0117] Figure 4. (A) Time-dependent effect of PTH on phospho-CREB expression from p62+ / + and p62- / - MSCs. Representative Western blot analysis highlighted impaired responses of the above proteins in p62 knockout MSCs treated with PTH (10-9 M) for 24 hours. The graph represents the results of three independent experiments. Values are expressed as mean ± SD and statistically analyzed by t-test (*p<0.05). (C) Co-stimulation of p62+ / + MSCs with PTH (10-9 M) and TGFβ (5 ng / ml for 24 hours). It was noted that p62 expression was significantly reduced under the influence of these two effectors. The graph represents the results of three independent experiments. Values are expressed as mean ± SD and statistically analyzed by t-test (*p<0.05).

[0118] Figure 5. (A) Dose-response and (B) time-course effects of BMP2 on p62 synthesis. MSCs were treated with BMP2 at doses ranging from 1 ng / ml to 50 ng / ml. Representative Western blot analysis showed that this effector was able to increase p62 protein levels, primarily at 10 ng / ml, after 24 hours of stimulation. The graph represents the results of three independent experiments. Values are presented as mean ± SD and statistically analyzed by t-test (p<0.05). (C) Dose-response and (D) time-course effects of BMP2 on Smad2 / 3 and Smad4 expression from p62+ / + and p62- / - MSCs. Representative Western blot analysis highlighted a decrease in Smad1 / 5 / 8 synthesis in p62- / - MSCs treated with BMP2 (10 ng / ml, 24 hours). The graph represents the results of three independent experiments. Two-way ANOVA shows statistically significant effects between exp groups, but no statistically significant interaction between variables (p<0.05). (E) Smad1 / 5 / 8 levels in p62+ / + BMSCs with p62 knockdown after BMP2 treatment. Representative Western blot analysis of Smad1 / 5 / 8 in BMSCs transfected with p62 siRNA or control siRNA and treated with BMP2 (10 ng / ml, 24 h). The graph represents the results of three independent experiments. Two-way ANOVA shows statistically significant effects between exp groups, but no statistically significant interaction between variables (p<0.05). (F) Cell fractionation was performed using the Qproteome Cell Compartment Handbook. Western blotting analysis was performed, and membranes were probed with rabbit anti-Smad1 / 5 / 8 or rabbit anti-NFkB antibodies. Filters were then stripped and reprobed with rabbit anti-B23 to demonstrate equal loading. Quantitative analysis was performed as previously described in the "Materials and Methods" section. The graph represents the results of three independent experiments. Two-way ANOVA shows a statistically significant effect between the experimental groups, but no statistically significant interaction between the variables (p<0.05).

[0119] Figure 6. (A) Effect of BMP-2 on Smad1 / 5 / 8 and NFκB labeling patterns in p62+ / + and p62- / - MSCs. MSCs of both genotypes were treated with BMP2 (10 ng / ml) for 24 hours. Smad1 / 5 / 8 and NFκB localization was analyzed by fluorescence microscopy using goat anti-NFκB antibodies and rabbit anti-phospho-Smad1 / 5 / 8 (green: Alexa Fluor 488 staining; red: Alexa Fluor 594) as described in the "Materials and Methods" section. Note that p62- / - BMP2-treated and untreated cultures capture the extended nuclear colocalization of Smad1 / 5 / 8-NFκB, as evidenced by merged images. Fluorescence analysis from a pool of four independent experiments was quantified using a Tecan Infinity fluorescence reader, and values were analyzed using Magellan v4.0 software. The bar represents 50 μm. (B) Effects on Smad1 / 5 / 8 and NFκB labeling patterns in p62- / - MSCs transfected with p62 DNA and treated with BMP2 (10 ng / ml) for 24 hours. Note the partial "reverse" effect of exogenous p62 on the nuclear colocalization of Smad1 / 5 / 8 and NFκB (merged image). Fluorescence analysis from a pool of four independent experiments was quantified using a Tecan Infinity Fluorescence Reader, and values were analyzed using Magellan v4.0 software. Bars represent 50 μm. Two-way ANOVA shows statistically significant results between experimental groups, but no statistically significant interactions between variables. Lowercase letters indicate homogeneous subsets (p<0.05).

Claims

1. 1. A method for preventing, treating, alleviating, ameliorating, reducing, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms of a disease associated with dysfunction of mesenchymal stem cells (MSCs) and a disease associated with elevated levels of reactive oxygen species (ROS) in a subject, comprising: The method includes administering to the subject an agent; The drug is p62 / SQSTM1 (p62) polypeptide, or A method characterized by comprising a nucleic acid encoding p62 / SQSTM1 (p62).

2. The drug is Contains deletions of one or more domains, the p62 polypeptide is at least 95% identical to SEQ ID NO:2; or The method of claim 1, wherein the nucleic acid encoding p62 encodes a p62 polypeptide that is at least 95% identical to SEQ ID NO:

2.

3. 2. The method of claim 1, wherein the nucleic acid is a plasmid, RNA, cosmid, virus, virus particle, or bacterium containing a p62 plasmid or p62 RNA.

4. 2. The method of claim 1, wherein the disease associated with dysfunction of mesenchymal stem cells is any of acute respiratory disease syndrome (ARDS), novel coronavirus-associated pneumonia, viral-associated pneumonia, graft-versus-host disease, spinal cord injury, liver fibrosis / cirrhosis, bronchopulmonary dysplasia, critical limb ischemia, solid organ transplant conditions, diabetic foot conditions, stroke, fractures, perianal leakage fistula, rectovaginal leakage fistula, surgical leakage fistula, and erectile dysfunction.

5. 2. The method of claim 1, wherein the disease associated with elevated reactive oxygen species (ROS) levels is any one of periodontitis, tendinopathy, neurodegeneration, adipogenesis disorders, Down's syndrome, Parkinson's disease, mental retardation, dementia, Alzheimer's disease, cardiovascular disease, myocardial ischemia, cardiac hypertrophy, heart failure, ischemia-reperfusion injury, atherosclerosis, organ failure, kidney stones, muscular dystrophy, spermatogenesis disorders, sarcopenia of aging, hair loss, premature aging, osteoporosis, asthma, chronic obstructive pulmonary disease, chronic inflammation, insulin resistance, decreased glucose uptake in skeletal muscle and liver, ectopic fat lipotoxicity, pancreatic fat / metabolism disorders, hepatic lipid accumulation, hepatic lipid inflammation, age-related increase in bone marrow adipose tissue, age-related decrease in trabecular bone volume, telomere shortening, mitochondrial dysfunction, and cellular senescence.

6. 10. The method of claim 1, wherein the administration is by injection, oral, subcutaneous, rectal, intranasal, via a microbiome carrier, intraocular, subcutaneous, or inhalation.

7. The method of claim 1, wherein the administration comprises using a pharmaceutical carrier that facilitates delivery of p62 into the body, delivery to a specific organ or tissue, penetration of p62 into cells, or stability of p62.

8. isolating cells from the subject; transfecting the cells with a vector encoding p62 or a pharmaceutical formulation comprising a p62 polypeptide; Expanding the cells in vitro; and returning the cells to the subject.

9. 10. The method of claim 1, wherein the method is carried out by administering to the subject a drug, treatment, or application of a combination of methods to prevent, treat, alleviate, ameliorate, relieve, delay the onset of, inhibit the progression of, reduce the severity of, and / or reduce the incidence of one or more symptoms of a disease associated with dysfunction of mesenchymal stem cells (MSCs) and a disease associated with elevated levels of reactive oxygen species (ROS) in the subject.

10. The method of claim 1 , wherein the subject is not receiving MSC cell therapy at the time of said administering.

11. 10. The method of claim 1, wherein the subject is a human, dog, cat, horse, cow, or bird.

12. A method for selecting a subject to which the method of claim 1 is applied, comprising: obtaining MSCs from the subject; and selecting the subject if the MSCs exhibit a low or insufficient ability to differentiate or secrete signaling molecules, or if the subject exhibits a gene expression profile indicative of a disease associated with mesenchymal stem cell (MSC) dysfunction.

13. A method for selecting a subject to which the method of claim 1 is applied, comprising: A method for selecting a subject, comprising selecting the subject if the subject exhibits high levels of ROS, is diagnosed with a disease associated with elevated ROS levels, or is at high risk for a disease associated with elevated ROS levels.

14. 1. A drug that prevents, treats, alleviates, improves, relieves, delays the onset of, inhibits the progression of, reduces the severity of, and / or reduces the incidence of one or more symptoms of a disease associated with dysfunction of mesenchymal stem cells (MSCs) and a disease associated with elevated levels of reactive oxygen species (ROS) in a subject, p62 polypeptide, or A drug characterized by containing a nucleic acid encoding p62.

15. The drug is Contains deletions of one or more domains, the p62 polypeptide is at least 95% identical to SEQ ID NO:2; or The agent of claim 14, wherein the nucleic acid encoding the p62 encodes a p62 polypeptide that is at least 95% identical to SEQ ID NO:

2.

16. The method of claim 14, wherein the nucleic acid encoding p62 is a p62 plasmid or a plasmid containing p62 RNA, RNA, a cosmid, a virus, a virus particle, or a bacterium.

17. The drug of claim 14, wherein the disease associated with dysfunction of mesenchymal stem cells is any of acute respiratory disease syndrome (ARDS), novel coronavirus-associated pneumonia, viral-associated pneumonia, graft-versus-host disease, spinal cord injury, liver fibrosis / cirrhosis, bronchopulmonary dysplasia, critical limb ischemia, solid organ transplant conditions, diabetic foot conditions, stroke, fractures, perianal leakage fistula, rectovaginal leakage fistula, surgical leakage fistula, and erectile dysfunction.

18. The drug of claim 14, wherein the disease associated with elevated reactive oxygen species (ROS) levels is any one of periodontitis, tendinopathy, neurodegeneration, adipogenesis disorders, Down's syndrome, Parkinson's disease, mental retardation, dementia, Alzheimer's disease, cardiovascular disease, myocardial ischemia, cardiac hypertrophy, heart failure, ischemia-reperfusion injury, atherosclerosis, organ failure, kidney stones, muscular dystrophy, spermatogenesis disorders, sarcopenia of aging, hair loss, premature aging, osteoporosis, asthma, chronic obstructive pulmonary disease, chronic inflammation, insulin resistance, decreased glucose uptake in skeletal muscle and liver, ectopic fat lipotoxicity, pancreatic fat / metabolism disorders, hepatic lipid accumulation, hepatic lipid inflammation, age-related increase in bone marrow adipose tissue, age-related decrease in trabecular bone volume, telomere shortening, mitochondrial dysfunction, and cellular senescence.

19. The agent of claim 14, further comprising a pharmaceutical carrier that facilitates delivery of p62 to the body, delivery to a specific organ or tissue, penetration of p62 into cells, and / or stability of p62.

20. 15. The method of claim 14, wherein the subject is a human, dog, cat, horse, cow, or bird.

21. 15. The agent of claim 14, formulated to be administered by injection, orally, subcutaneously, rectally, intranasally, via a microbiome carrier, intraocularly, subcutaneously, or via inhalation.

22. 15. The pharmaceutical agent of claim 14, formulated for use in combination with a method for administering a drug to a subject, treating, or preventing, treating, alleviating, ameliorating, reducing, delaying the onset of, inhibiting progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms of a disease associated with dysfunction of mesenchymal stem cells (MSCs) and a disease associated with elevated levels of reactive oxygen species (ROS) in said subject.

23. 15. A method of using the medicament of claim 14, comprising:

15. The method of claim 14, further comprising administering the medicament The method of using the agent, wherein the subject is not receiving MSC cell therapy during the administration.

24. A method for selecting a subject to administer the drug according to claim 14, comprising: obtaining MSCs from the subject; and selecting the subject if the MSCs exhibit a low or insufficient ability to differentiate or secrete signaling molecules, or if the subject exhibits a gene expression profile indicative of a disease associated with mesenchymal stem cell (MSC) dysfunction.

25. A method for selecting a subject to administer the drug according to claim 14, comprising: A method for selecting a subject, comprising selecting the subject if the subject exhibits high levels of ROS, is diagnosed with a disease associated with elevated ROS levels, or is at high risk for a disease associated with elevated ROS levels.

26. A method for selecting a subject to administer the drug according to claim 14, comprising: obtaining a liquid biopsy; (i) selecting the subject if the kid biopsy shows low or insufficient efficacy in mesenchymal stem cell (MSC) differentiation or secretion of signaling molecules, or if a gene expression profile indicative of a disease associated with mesenchymal stem cell (MSC) dysfunction is shown.

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