Compositions and methods for promoting ocular neovascularization

Abstract

To provide compositions and methods for promoting ocular angiogenesis.SOLUTION: Compositions and methods are provided for promoting ocular neovascularization comprising an IL-6 family protein, including leukemia inhibitory factor (LIF) or cardiotrophin-1 (CT-1).SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 788,174, filed January 4, 2019, which is incorporated herein by reference.

[0002] The present invention relates to promoting angiogenesis to alleviate ocular conditions. [Background technology]

[0003] Angiogenesis is a physiological process required for embryonic development, adult vascular homeostasis, and tissue repair (1). However, angiogenesis also contributes to various pathological conditions, such as tumors and several intraocular disorders, including wet age-related macular degeneration (AMD) (1). During tumor progression, new blood vessels provide nutrients and oxygen to tumor tissue and thus play a critical role. In intraocular disorders, abnormal, leaky vascular growth can destroy the retina and lead to blindness (1, 2). Extensive efforts to analyze the molecular basis of angiogenesis and identify therapeutic targets for neoplasia and other diseases have led to the discovery of key signaling pathways involved in vascular development and differentiation (1, 3). In particular, numerous studies have established the pivotal role of the VEGF pathway in physiological angiogenesis, and therapies targeting this pathway have been successful in treating ocular diseases such as cancer and wet AMD (4, 5). Conversely, stimulating angiogenesis has the potential to improve outcomes for patients with various ischemic diseases through improved perfusion (6). This hypothesis has led to a series of clinical trials over the past few decades in which angiogenic factors such as VEGF or bFGF delivered by gene therapy or as recombinant proteins were tested in patients with coronary artery or limb ischemia. Unfortunately, despite promising preclinical studies, none of these studies were successful. (7) Therefore, there is a need to identify novel strategies to improve angiogenic therapy.

[0004] Glioblastoma cells secrete a variety of angiogenic factors, contributing to the highly vascular phenotype of such tumors (8). Xenograft tumors derived from the LN-229 glioblastoma cell line are well vascularized despite very low expression of VEGF (9, 10). Therefore, the LN-229 secretome is of interest for characterizing putative endothelial mitogens.

[0005] The IL-6 superfamily of cytokines includes leukemia inhibitory factor (LIF), which has been widely used in experimental stem cell biology due to its ability to maintain the pluripotency of embryonic stem cells. Various roles for LIF have also been observed in different cell types and tissues, including embryo implantation, hematopoietic cell development, inflammatory responses, and tumor progression (67).

[0006] The role of LIF in angiogenesis remains controversial. It was initially characterized as an anti-angiogenic factor in bovine aortic endothelial cells and showed no effect on bovine adrenal cortical capillary endothelial cells (35), suggesting that LIF functions distinctly in different types of endothelial cells. Subsequent studies have shown considerable complexity. Transgenic mice overexpressing LIF exhibited reduced ocular vasculature and suppressed retinal vascular development (14), whereas mice carrying a homozygous LIF knockout allele had increased retinal vascular density (16). Early postnatal injection of recombinant LIF into rat pups resulted in a slight increase in the avascular area in the developing retina (22). Summary of the Invention

[0007] The present invention provides that members of the IL-6 superfamily, and functional fragments thereof, can be used to increase ocular angiogenesis in subjects in need of therapeutic treatment for conditions such as, but not limited to, age-related macular degeneration and retinopathy of prematurity (ROP). In an embodiment, the subject is a human.

[0008] In an embodiment, the present invention provides a method of treating a condition associated with insufficient angiogenesis in the eye of a subject, comprising administering an effective amount of an IL-6 family protein or a functional fragment thereof to a subject in need thereof to promote angiogenesis. In an embodiment, the present invention provides that the IL-6 family protein is leukemia inhibitory factor (LIF) or cardiotrophin-1 (CT-1).

[0009] In embodiments, the invention provides that administration increases retinal microvascular density. In embodiments, the invention provides that administration increases choroidal endothelial cell proliferation.

[0010] In an embodiment, the invention provides that the condition is age-related macular degeneration. In an embodiment, the invention provides that the condition is retinopathy of prematurity (ROP).

[0011] In embodiments, the invention provides that administration is via intravitreal injection. In embodiments, the invention provides that the effective amount does not induce vascular leakage. In embodiments, the invention provides that the effective amount does not induce edema.

[0012] In an embodiment, the present invention provides a method for inducing angiogenesis in the eye of a subject, comprising administering an effective amount of an IL-6 family protein or a functional fragment thereof to a subject in need thereof.

[0013] In embodiments, the invention provides that administration increases retinal neovascularization. In embodiments, the invention provides that administration increases choroidal endothelial cell proliferation.

[0014] In embodiments, the present invention provides that the subject has age-related macular degeneration. In embodiments, the present invention provides that the subject has retinopathy of prematurity (ROP).

[0015] In embodiments, the invention provides that administration is via intravitreal injection. In embodiments, the invention provides that the effective amount does not induce vascular leakage. In embodiments, the invention provides that the effective amount does not induce edema.

[0016] In an embodiment, the present invention provides that the IL-6 family protein is leukemia inhibitory factor (LIF). In an embodiment, the present invention provides that the IL-6 family protein is cardiotrophin-1 (CT-1). [Brief explanation of the drawings]

[0017] [Figure 1A] LIF is an endothelial cell mitogen derived from LN-229 conditioned medium. LN-229 conditioned medium stimulates the growth of bovine choroidal endothelial cells, n=3 (FIG. 1A). [Figure 1B] VEGF-neutralizing antibodies were unable to suppress BCE cell growth induced by LN-229 CM, n=3 (Fig. 1B). [Figure 1C] Reverse-phase chromatography fractions of LN-229 CM induce BCE cell growth. BCE cells were incubated with fractions (2 μl / well) as indicated, n=3 (FIG. 1C). [Figure 1D] Anti-LIF neutralizing antibodies abolished BCE cell growth induced by the reverse phase fraction, n=3 (Figure 1D). [Figure 1E] Recombinant human LIF protein stimulates the growth of BCE cells in a dose-dependent manner. BCE cells were cultured in the presence of vehicle, VEGF (10 ng / ml), and the indicated concentrations of recombinant human LIF (rhLIF), n=3 (Figure 1E). [Figure 1F] LIF and VEGF synergistically stimulate BCE cell growth. Cell proliferation was analyzed after 6 days using Alamar Blue, n = 3. Bars and error bars represent mean ± SD. *: p < 0.05, **: p < 0.01, #: p ≤ 0.0001, ns: not statistically significant (Figure 1F). [Figure 2A]We demonstrate that LIF promotes BCE cell growth via the JAK-STAT3 pathway. The JAK inhibitor baricitinib (Ba) blocks LIF-induced STAT3 activation. BCE cells were preincubated with DMSO, baricitinib (2 μM), cobimetinib (Co) (150 nM), or BEZ235 (BE) (5 nM) for 1 hour and then treated with vehicle or LIF (10 ng / ml) for 15 minutes. Control: no inhibitor preincubation (Figure 2A). [Figure 2B] Baricitinib inhibits LIF-induced BCE cell growth. BCE cells were preincubated with DMSO, baricitinib, cobimetinib, or BEZ235 for 1 hour and then treated with vehicle, LIF (10 ng / ml), or VEGF (10 ng / ml). Cell proliferation was analyzed 6 days later (n=3) (Figure 2B). [Figure 2C] Figures 2C and 2D show STAT3 knockdown in BCE cells. BCE cells were transfected with siNegative and siRNA targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 levels in the siNegative were set to 1. Data from three independent experiments were averaged and shown in Figure 2C. In Figure 2D, siRNA-transfected cells were treated with LIF (10 ng / ml) or vehicle for 15 minutes. Whole cell lysates were subjected to Western blotting using the indicated antibodies. [Figure 2D] Figures 2C and 2D show STAT3 knockdown in BCE cells. BCE cells were transfected with siNegative and siRNA targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 levels in the siNegative were set to 1. Data from three independent experiments were averaged and shown in Figure 2C. In Figure 2D, siRNA-transfected cells were treated with LIF (10 ng / ml) or vehicle for 15 minutes. Whole cell lysates were subjected to Western blotting using the indicated antibodies. [Figure 2E]LIF-induced BCE cell growth was abolished by STAT3 knockdown. BCE cells with STAT3 knockdown were cultured with LIF (10 ng / ml) or vehicle. Cell proliferation was analyzed after 3 days. The fluorescence reading at 590 nm for each vehicle group was set to 1, n=3. siNegative, negative control siRNA, does not target any known gene. **: p<0.01, ***: p<0.001, #: p≦0.0001, ns: not statistically significant (Figure 2E). [Figure 3A] This shows that LIF promotes angiogenesis in ex vivo and in vivo models. Figures 3A and 3B show the induction of choroidal sprouting in mice by LIF. The representative photograph in Figure 3A shows vascular growth from primary choroidal explants 6 days after seeding. Supplements were added to each sample as indicated. Quantification of vascular sprouting growth was performed using Axiovision software (n=5). [Figure 3B] This shows that LIF promotes angiogenesis in ex vivo and in vivo models. Figures 3A and 3B show the induction of choroidal sprouting in mice by LIF. The representative photograph in Figure 3A shows vascular growth from primary choroidal explants 6 days after seeding. Supplements were added to each sample as indicated. Quantification of vascular sprouting growth was performed using Axiovision software (n=5). [Figure 3C] Figures 3C and 3D show that intravitreal injection of LIF increases vascular density in mouse eyes. Adult mice were intravitreally injected with the indicated amounts of VEGF and LIF. Seven days after injection, PFA-fixed choroid-sclera complexes and retinas were subjected to CD31 IF. A representative image of CD31-positive blood vessels is shown in Figure 3C. Blood vessel density determined using ImageJ software is shown in Figure 3D, n = 5–8. [Figure 3D]Figures 3C and 3D show that intravitreal injection of LIF increases vascular density in mouse eyes. Adult mice were intravitreally injected with the indicated amounts of VEGF and LIF. Seven days after injection, PFA-fixed choroid-sclera complexes and retinas were subjected to CD31 IF. A representative image of CD31-positive blood vessels is shown in Figure 3C. Blood vessel density determined using ImageJ software is shown in Figure 3D, n = 5–8. [Figure 3E] Figures 3E and 3F show OCTA imaging of the retina of mice treated with LIF. Adult mice were intravitreally injected with 1 μl of LIF (50 ng) or vehicle solution (PBS). Retinal OCTA images were obtained 7 days after injection, and a representative image is shown in Figure 3E. Vascular density was determined as the percentage of vascularized area / total area surface using ImageJ software and is shown in Figure 3F, n = 7–8. [Figure 3F] Figures 3E and 3F show OCTA imaging of the retina of mice treated with LIF. Adult mice were intravitreally injected with 1 μl of LIF (50 ng) or vehicle solution (PBS). Retinal OCTA images were obtained 7 days after injection, and a representative image is shown in Figure 3E. Vascular density was determined as the percentage of vascularized area / total area surface using ImageJ software and is shown in Figure 3F, n = 7–8. [Figure 3G] Figures 3G and 3H show that LIF treatment increases retinal vascular density in mice. Adult mice were intravitreally injected with LIF (10 ng) or vehicle solution. Seven days after injection, frozen sections of mouse eyes were subjected to H&E staining and CD31 IF staining. A representative image is shown in Figure 3G. Quantification of CD31 positivity using ImageJ software is shown in Figure 3H, n = 4. [Figure 3H] Figures 3G and 3H show that LIF treatment increases retinal vascular density in mice. Adult mice were intravitreally injected with LIF (10 ng) or vehicle solution. Seven days after injection, frozen sections of mouse eyes were subjected to H&E staining and CD31 IF staining. A representative image is shown in Figure 3G. Quantification of CD31 positivity using ImageJ software is shown in Figure 3H, n = 4. [Figure 3I]In Figures 3I and 3J, 5-day-old neonatal mice were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days of treatment, the mouse retinas were subjected to IF staining using Dyight-488-labeled lectin. Representative images of similar ocular loci are shown in Figure 3I. Quantification of the lectin-labeled area using ImageJ software is shown in Figure 3J, n = 4. *: p < 0.05, **: p < 0.01. [Figure 3J] In Figures 3I and 3J, 5-day-old neonatal mice were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days of treatment, the mouse retinas were subjected to IF staining using Dyight-488-labeled lectin. Representative images of similar ocular loci are shown in Figure 3I. Quantification of the lectin-labeled area using ImageJ software is shown in Figure 3J, n = 4. *: p < 0.05, **: p < 0.01. [Figure 4A] This shows that LIF inhibits BAE cell growth via the JAK-STAT3 pathway. Recombinant human LIF inhibits BAE cell growth in a dose-dependent manner. BAE cells were cultured in the presence of vehicle and the indicated concentrations of recombinant human LIF (rhLIF). Cell proliferation was analyzed after 6 days, n = 3 (Figure 4A). [Figure 4B] The JAK inhibitor baricitinib blocks LIF-induced STAT3 activation. BAE cells preincubated with DMSO and inhibitor for 1 hour were treated with vehicle and LIF (10 ng / ml) for 15 minutes. Whole cell lysates were subjected to Western blotting using the indicated antibodies. Control: no inhibitor preincubation; Ba: baricitinib (2 μM); Co: cobimetinib (150 nM); BE: BEZ235 (5 nM) (Figure 4B). [Figure 4C] The JAK inhibitor baricitinib reverses LIF-induced BAE growth inhibition. BAE cells preincubated with the inhibitor for 1 hour were treated with vehicle, LIF (10 ng / ml), and VEGF (10 ng / ml). Cell proliferation was analyzed after 6 days using Alamar Blue (n=3) (Figure 4C). [Figure 4D]Figures 4D and 4E show STAT3 knockdown in BAE cells. BAE cells were transfected with siRNA targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 levels in the negative control were set to 1. Data from three independent experiments were averaged and shown in Figure 4D. In Figure 4E, siRNA-transfected cells were treated with LIF (10 ng / ml) and vehicle for 15 minutes. Total cell lysates were subjected to Western blotting using the indicated antibodies. [Figure 4E] Figures 4D and 4E show STAT3 knockdown in BAE cells. BAE cells were transfected with siRNA targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 levels in the negative control were set to 1. Data from three independent experiments were averaged and shown in Figure 4D. In Figure 4E, siRNA-transfected cells were treated with LIF (10 ng / ml) and vehicle for 15 minutes. Total cell lysates were subjected to Western blotting using the indicated antibodies. [Figure 4F] Figure 4F shows that STAT3 knockdown abolishes LIF-induced BAE cell growth inhibition. BAE cells with STAT3 knockdown were cultured with LIF (10 ng / ml) and vehicle. Cell proliferation was analyzed after 3 days. The fluorescence reading for each vehicle group was set to 1, n=3. Bars and error bars represent the mean ± SD. siNegative, a negative control siRNA, does not target any known gene. **: p<0.01, ***: p<0.001, #: p≦0.0001, ns: not statistically significant. [Figure 5A]We demonstrate that LIF does not induce vascular permeability in guinea pig skin and mouse retina. In Figure 5A, hairless male guinea pigs (control: HA-Hrhr / IAF, 450-500 g, Charles River Laboratories) were anesthetized with intraperitoneal (ip) administration of xylazine (5 mg / kg) and ketamine (75 mg / kg). Next, the animals received an intravenous injection (penile vein) of 1 ml of 1% Evans blue dye. Fifteen minutes later, intradermal injections (0.05 ml / site) of different doses of rhLIF in PBS (1, 5, 25, 100, 200 ng per injection site) were administered into the trunk area behind the shoulders. 0.05 ml of PBS and 25 ng of VEGF in 0.05 ml of PBS were injected as negative and positive controls. Thirty minutes after the intradermal injection, the animals were euthanized by intraperitoneal injection of pentobarbital (200 mg / kg). Skin tissue was dissected from connective tissue and photographed, n=2. [Figure 5B] In Figure 5B, vascular leakage is shown in the mouse retina. LIF (10 ng) or VEGF (100 ng) was injected into the vitreous cavity (0.1% BSA / PBS as a control). TRITC-dextran was used to demonstrate vascular leakage. Retinal vasculature was labeled with FITC-lectin (n=5). [Figure 6A] LIF induces cell death through upregulation of cathepsin L. Figures 6A and 6B show that LIF treatment induces cell death in BAE cells. After treatment with LIF (10 ng / ml) or vehicle for 24 hours, BAE cells were stained with Annexin V-Cy5. A representative image is shown in Figure 6A. [Figure 6B] The percentage of Annexin V-positive area versus total cell-covered area was calculated and shown in Figure 6B, n=3. [Figure 6C]Figures 6C and 6D show LIF-induced cathepsin L expression in BAE cells. After 24 hours of treatment with LIF (10 ng / ml) or vehicle, qRT-PCR was performed to examine cathepsin L (CTSL) mRNA levels in BAE cells. CTSL levels in the vehicle group were set to 1. CTSL mRNA levels in each sample were compared with the vehicle group and shown as fold changes in Figure 6C, n = 3. Total protein from LIF-treated BAE cells was used for bovine cathepsin L ELISA. Cathepsin L protein levels in the vehicle-treated group were set to 1. The fold change in cathepsin L protein induction (LIF-treated samples vs. vehicle group) was calculated, and the fold changes from three independent experiments are shown in Figure 6D. [Figure 6D] Figures 6C and 6D show LIF-induced cathepsin L expression in BAE cells. After 24 hours of treatment with LIF (10 ng / ml) or vehicle, qRT-PCR was performed to examine cathepsin L (CTSL) mRNA levels in BAE cells. CTSL levels in the vehicle group were set to 1. CTSL mRNA levels in each sample were compared with the vehicle group and shown as fold changes in Figure 6C, n = 3. Total protein from LIF-treated BAE cells was used for bovine cathepsin L ELISA. Cathepsin L protein levels in the vehicle-treated group were set to 1. The fold change in cathepsin L protein induction (LIF-treated samples vs. vehicle group) was calculated, and the fold changes from three independent experiments are shown in Figure 6D. [Figure 6E] Figures 6E and 6F show that cathepsin L inhibitors CA074me and CAA0225 attenuate LIF-induced BAE cell growth inhibition. BAE cells preincubated with the indicated concentrations of CA074me and CAA0225 for 1 hour were treated with vehicle, LIF (10 ng / ml), and VEGF (10 ng / ml). Cell growth was analyzed after 6 days (n=3). Bars and error bars represent the mean ± SD. *: p<0.05, **: p<0.01, ***: p<0.001, #: p≦0.0001, ns: not statistically significant. [Figure 6F]Figures 6E and 6F show that cathepsin L inhibitors CA074me and CAA0225 attenuate LIF-induced BAE cell growth inhibition. BAE cells preincubated with the indicated concentrations of CA074me and CAA0225 for 1 hour were treated with vehicle, LIF (10 ng / ml), and VEGF (10 ng / ml). Cell growth was analyzed after 6 days (n=3). Bars and error bars represent the mean ± SD. *: p<0.05, **: p<0.01, ***: p<0.001, #: p≦0.0001, ns: not statistically significant. [Figure 7A] Figures 7A and 7B show that LIF induces cell cycle arrest in BAE cells. Figures 7A and 7B show that LIF treatment reduces BrdU incorporation into BAE cells. After 48 hours of treatment with LIF (10 ng / ml) and vehicle, BAE cells were incubated with 10 μM BrdU for 4 hours. A representative image of BrdU incorporation detected with an Alexa Fluor-488-conjugated BrdU antibody is shown in Figure 7A. [Figure 7B] The percentage of BrdU-positive nuclei versus total DAPI-stained nuclei was calculated and shown in Figure 7B, n=3. [Figure 7C] Figure 7C shows the suppression of cyclin A and B expression by LIF in BAE. BAE and BCE cells were treated with LIF (10 ng / ml) and vehicle for 24 hours. qRT-PCR was performed to examine the mRNA levels of CTSL1, CCNA2, CCNB1, and MYC. For each gene probe, the vehicle-treated group level was set to 1. The mRNA levels in LIF-treated samples were normalized to the vehicle group, n = 3. Bars and error bars represent the mean ± SD. *: p < 0.05, ***: p < 0.001, #: p ≤ 0.0001, ns: not statistically significant. [Figure 8A] The effects of other IL-6 family proteins in a mouse eye model are shown. Recombinant LIF (50 ng) and different doses of CT-1 and PBS vehicle control in 1 μl were intravitreally injected into mouse eyes (FIG. 8A). The retinal vasculature was visualized by both OCT-A imaging and CD31 immunofluorescence staining in live mice, n=5 (FIG. 8A). [Figure 8B] Retinal flat-mount staining was imaged using a confocal microscope (FIG. 8B). Quantification of blood vessels was performed using Image J. [Figure 8C] Figures 8C and 8D show that sodium iodate was used to induce choriocapillaris injury in mice. After sodium iodate injection, the eyes were injected with the indicated amounts of LIF, CT-1, or OSM. Choriocapillaris was imaged with an OCT-A system (n=5). The avascular area of ​​the choroid was determined and quantified using Image J. [Figure 8D] Figures 8C and 8D show that sodium iodate was used to induce choriocapillaris injury in mice. After sodium iodate injection, the eyes were injected with the indicated amounts of LIF, CT-1, or OSM. Choriocapillaris was imaged with an OCT-A system (n=5). The avascular area of ​​the choroid was determined and quantified using Image J. DETAILED DESCRIPTION OF THE INVENTION

[0018] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0019] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, exemplary methods, devices, and materials are described herein.

[0020] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of those in the art. Such techniques may be found in, for example, Molecular Cloning: A Laboratory Manual, 2002. nd (Sambrook et al., 1989), Oligonucleotide Synthesis (MJGait, ed., 1984), Animal Cell Culture (RIFreshney, ed., 1987), Methods in Enzymology (Academic Press, Inc.), Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987, and periodic updates), PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994), Remington, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22 th This is fully described in references such as "Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012" and "Pharmaceuticals and Pharmacology: A Guide to Pharmacology and Pharmacy in the United States."

[0021] The present invention provides that members of the IL6 superfamily, and functional fragments thereof, can be used to increase ocular angiogenesis in subjects in need of therapeutic treatment for conditions such as, but not limited to, age-related macular degeneration and retinopathy of prematurity (ROP). In an embodiment, the subject is a human.

[0022] In an embodiment, the present invention provides a method of treating a condition associated with insufficient angiogenesis in the eye of a subject, comprising administering an effective amount of an IL-6 family protein or a functional fragment thereof to a subject in need thereof to promote angiogenesis. In an embodiment, the present invention provides that the IL-6 family protein is leukemia inhibitory factor (LIF) or cardiotrophin-1 (CT-1).

[0023] In embodiments, the present invention provides that administration increases retinal microvascular density. In embodiments, the present invention provides that administration increases choroidal endothelial cell proliferation. In embodiments, the present invention provides that administration stimulates angiogenesis.

[0024] In an embodiment, the invention provides that the condition is age-related macular degeneration. In an embodiment, the invention provides that the condition is retinopathy of prematurity (ROP).

[0025] In embodiments, the invention provides that administration is via intravitreal injection. In embodiments, the invention provides that the effective amount does not induce vascular leakage. In embodiments, the invention provides that the effective amount does not induce edema.

[0026] In an embodiment, the present invention provides a method for inducing angiogenesis in the eye of a subject, comprising administering an effective amount of an IL-6 family protein or a functional fragment thereof to a subject in need thereof.

[0027] In embodiments, the invention provides that administration increases retinal neovascularization. In embodiments, the invention provides that administration increases choroidal endothelial cell proliferation.

[0028] In embodiments, the present invention provides that the subject has age-related macular degeneration. In embodiments, the present invention provides that the subject has retinopathy of prematurity (ROP).

[0029] In embodiments, the invention provides that administration is via intravitreal injection. In embodiments, the invention provides that the effective amount does not induce vascular leakage. In embodiments, the invention provides that the effective amount does not induce edema.

[0030] In an embodiment, the present invention provides that the IL-6 family protein is leukemia inhibitory factor (LIF). In an embodiment, the present invention provides that the IL-6 family protein is cardiotrophin-1 (CT-1).

[0031] definition To facilitate understanding of the present invention, some terms and abbreviations used herein are defined below.

[0032] When introducing elements of the present invention or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0033] The use of the term "and / or" in the context of a list of two or more items means that any one of the listed items may be used alone or in combination with any one or more of the listed items. For example, the phrase "A and / or B" is intended to mean one or both of A and B, i.e., A only, B only, or a combination of A and B. The phrase "A, B, and / or C" is intended to mean A only, B only, C only, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0034] It will be understood that aspects and embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0035] It should be understood that descriptions in range format are for convenience and brevity only and should not be construed as inflexible limitations on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Also, herein, values ​​or ranges may be expressed as "about," "about" from one particular value, and / or "about" to another particular value. When such values ​​or ranges are expressed, other disclosed embodiments include the recited specific values ​​from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It is further understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. In embodiments, "about" can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0036] As used herein, "patient" or "subject" means a human or animal subject being treated.

[0037] As used herein, the term "pharmaceutical composition" refers to a pharmaceutically acceptable composition, which comprises a pharmaceutically active agent and, in some embodiments, further comprises a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition may be a combination of a pharmaceutically active agent and a carrier.

[0038] The term "combination" refers to either a fixed combination in a single dosage unit form or a kit of parts for combined administration, in which one or more active compounds and combination partners (e.g., another drug described below, also referred to as a "therapeutic agent" or "adjunct agent") can be administered simultaneously or separately within a time interval. In some situations, the combination partners exhibit a cooperative effect, e.g., a synergistic effect. As used herein, terms such as "co-administration" or "combined administration" are intended to encompass the administration of selected combination partners to a single subject (e.g., patient) in need thereof, and are intended to include therapeutic regimens in which the agents are not necessarily administered by the same route of administration or at the same time. As used herein, the term "pharmaceutical combination" refers to a product obtained by mixing or combining two or more active ingredients, including those in which the active ingredients are either fixed or loosely combined. The term "fixed combination" means that both active ingredients, e.g., a compound and a combination partner, are administered to a patient simultaneously in the form of a single entity or dosage. The term "unfixed combination" means that both active ingredients, e.g., a compound and a combination partner, are administered to a patient simultaneously, concurrently, or sequentially as separate entities without specific time constraints, whereby such administration provides therapeutically effective concentrations of the two compounds in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0039] As used herein, "effective" or "therapeutically effective" refers to the amount of a pharmaceutically active compound that is sufficient to treat or improve, or in some way reduce, the symptoms associated with disease and pathological conditions.When used in relation to a method, the method is effective enough to treat or improve, or in some way reduce, the symptoms associated with disease or condition.For example, with respect to age-related eye disease, an effective amount is an amount that is sufficient to prevent or prevent the onset of the disease, or, if the disease symptoms have begun, to alleviate, improve, stabilize, reverse, or delay the progression of the disease, or reduce the pathological consequences of the disease.In either case, the effective amount can be given in a single dose or in divided doses.

[0040] As used herein, the terms "treat," "treatment," or "treating" encompass at least ameliorating the symptoms associated with a disease in a patient, where amelioration is used broadly to refer to at least a decrease in the severity of a parameter, such as a symptom, associated with the disease or condition being treated. Thus, "treatment" includes situations in which a disease, disorder, or pathological condition, or at least the symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated), rendering the patient no longer susceptible to the condition, or at least the symptoms that characterize the condition.

[0041] As used herein, unless otherwise specified, the terms "prevent," "preventing," and "prevention" refer to the prevention of the onset, recurrence, or spread of a disease or disorder, or one or more symptoms thereof. In certain embodiments, these terms refer to treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agents, prior to the onset of symptoms, particularly to subjects at risk of a disease or disorder provided herein. These terms encompass the inhibition or reduction of symptoms of a particular disease. In certain embodiments, subjects with a family history of a disease are potential candidates for a preventative regimen. In certain embodiments, subjects with a medical history of recurring symptoms are also potential candidates for prevention. In this regard, the term "prevention" can be used interchangeably with the term "prophylactic treatment."

[0042] As used herein, unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder or prevent its recurrence. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent that, alone or in combination with one or more other agents, provides a prophylactic benefit in the prevention of a disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic effectiveness of another prophylactic agent.

[0043] As used herein, the term "pharmaceutically active" refers to the beneficial biological activity of a substance on the cells and tissues of an organism, particularly the human body. A "pharmaceutically active agent" or "drug" is a substance that is pharmaceutically active, and a "pharmaceutically active ingredient" (API) is the pharmaceutically active substance in a drug.

[0044] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias, in addition to other formulations that are safe for use in animals, more specifically, humans and / or non-human mammals. The present invention contemplates compositions for the treatment of the eye formulated for ophthalmic delivery, including intravitreal injection.

[0045] As used herein, the term "pharmaceutically acceptable carrier" refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle administered with the demethylenated compound. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity, such as sodium chloride or dextrose, may also be carriers. Methods for combining carriers to prepare compositions are known to those skilled in the art. In some embodiments, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.See, for example, Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003).Except insofar as any conventional media or agent is incompatible with the active compound, its use in the present compositions is contemplated.

[0046] As used herein, the term "pharmaceutically acceptable salt" refers to an acid or base addition salt of a compound, such as a multi-drug conjugate, disclosed herein. A pharmaceutically acceptable salt is any salt that retains the activity of the parent drug or compound and does not impart any harmful or undesirable effects to the subject to which it is administered and in the context in which it is administered. Pharmaceutically acceptable salts can be derived from amino acids, including, but not limited to, cysteine. Methods for producing compounds as salts are known to those skilled in the art (e.g., Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J. Pharm. Sci. 66:1, 1977). In some embodiments, "pharmaceutically acceptable salt" is intended to mean a free acid or base salt of a drug or compound described herein that is non-toxic, biologically acceptable, or otherwise biologically suitable for administration to a subject. See generally Berge, et al., J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without undue toxicity, irritation, or allergic response. The agents or compounds described herein may have sufficiently acidic groups, sufficiently basic groups, both types of functional groups, or more than one of each type, and therefore may react with several inorganic or organic bases, and inorganic and organic acids, to form pharmaceutically acceptable salts.

[0047] Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dione, hexyne-1,6-dione. Acid salts include benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, [gamma]-hydroxybutyrates, glycolates, tartrates, and mandelates.

[0048] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, α-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid but function similarly to a naturally occurring amino acid.

[0049] The IL-6 family of proteins for use in the present invention includes leukemia inhibitory factor (LIF) or cardiotrophin-1 (CT-1). The IL-6 family of proteins for use in the present invention can also include other IL-6 cytokines that promote angiogenesis, such as interleukin-11 (IL-11), ciliary neurotrophic factor (CNTF), cardiotrophin-like cytokine (CLC), and interleukin-27 (IL-27), a heterodimeric cytokine that can also be classified as part of the IL-12 family. However, oncostatin M (OSM) has the opposite effect. Using the knowledge of the invention described herein, one skilled in the art can routinely screen additional IL-6 family members for angiogenesis-promoting activity for use in the present invention. IL-6 family proteins can be isolated or partially purified naturally occurring proteins or recombinantly produced proteins.

[0050] The amino acid sequences of such naturally occurring IL-6 family members are well known in the art. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," resulting in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present invention.

[0051] In embodiments, the present invention is directed to promoting angiogenesis for the prevention or treatment of diseases or conditions characterized by insufficient or insufficient angiogenesis, including, but not limited to, retinopathy of prematurity (ROP), age-related macular degeneration, diabetic retinopathy, glaucoma, diabetic foot ulcers, pulmonary hypertension, ischemia, chronic ulcers, alopecia or graying of hair, skin flap regeneration, wound and burn healing, artificial skin implants, embryonic development, and preparation of blood vessels for transplantation.

[0052] The present invention identifies LIF as a mitogen for primary choroidal endothelial cells. Prior to the present invention, LIF had long been characterized as a negative regulator of endothelial cell growth / angiogenesis, although the precise mechanism was largely unknown. In 1992, LIF was first reported to be an inhibitor of BAE cell growth (35). Subsequent studies have also described LIF as an inhibitor of bFGF- and VEGF-induced endothelial cell proliferation (15, 41). The only exception was a study demonstrating some mitogenic effects of LIF in immortalized endothelial cell lines generated via SV40 large T antigen (42).

[0053] This study demonstrates for the first time that LIF can stimulate primary endothelial cell growth in vitro. Furthermore, the present study demonstrates that the LIF-JAK-STAT3 signaling axis is responsible for the mitogenic effects in endothelial cells. Intravitreal injection of recombinant LIF significantly increases retinal vascular density in adult mice, confirming the angiogenic role of LIF. Interestingly, CT-1 also induces retinal angiogenesis and is protective in the NaIO3 model.

[0054] In genetically engineered mouse models (GEMMs), LIF expression levels are negatively correlated with the development of the retinal vasculature (14, 16). Nevertheless, it has previously been reported that LIF affects multiple cell types (16, 43) and even completely disrupts retinal development in GEMMs (44). In particular, LIF negatively affects the maturation of retinal astrocytes, which in turn promotes VEGF expression by immature astrocytes, which may contribute to increased vascular density (16, 31, 32, 45). Therefore, the alterations in the retinal vasculature in GEMMs may not be a direct effect of LIF on endothelial cells. Another study showed that both intraperitoneal and intravitreal LIF injections moderately reduced vascular density in neonatal rat eyes (22), and such an inhibitory role of LIF could also be explained by its effect on retinal development. Furthermore, the dose of LIF injected intravitreally was not clearly defined in that study (22). Given the tight, bell-shaped dose-response profile disclosed in this study, it is difficult to compare this study with previous studies. Indeed, at least some of the discrepancies in the literature may be explained by the widely varying doses of LIF used in different studies, ranging from a few nanograms to hundreds of nanograms ( 16 , 22 ).

[0055] Retinopathy of prematurity (ROP), a common blinding disease in premature infants, is characterized by delayed vasculature development and regression of existing blood vessels, followed by hypoxia-induced retinal neovascularization (46). Dramatic downregulation of VEGF expression in the eye is associated with the onset and progression of ROP (47), and administration of exogenous VEGF attenuates the severity of ROP in mice (47). However, concerns remain about using VEGF as a therapeutic agent because VEGF contributes to pathological neovascularization accompanied by increased vascular permeability (48). In this study, LIF, unlike VEGF, does not induce vascular permeability in guinea pig skin (Figure 5A). Furthermore, retinal microvascular leakage in mice was determined using TRITC-labeled dextran. LIF (10 ng) or VEGF (100 ng) was injected intravitreally 15 min before TRITC-dextran injection. The results show that, unlike VEGF, LIF does not induce retinal microvascular leakage (Figure 5B). Thus, LIF may be used at some stages of ROP to prevent vascular regression.

[0056] Consistent with previous reports (35), we demonstrate that LIF inhibits BAE cell growth. This is due, at least in part, to cell death, as evidenced by increased Annexin V staining upon LIF treatment. Interestingly, two inhibitors of the lysosomal cysteine ​​protease cathepsin L (i.e., CA-074me and CAA0225), but not caspase inhibitors, reversed LIF-induced cell death, suggesting the involvement of caspase-independent cell death. Furthermore, the cathepsin B-specific inhibitor CA074 failed to rescue BAE cell death, and cathepsin L, but not cathepsin B, was upregulated in LIF-treated BAE cells, indicating that cathepsin L is the executor of LIF-induced lysosomal cell death.

[0057] Induction of cathepsins B and L is involved in autophagy and cell death (49, 50). This study is the first to implicate the LIF-cathepsin L pathway in the induction of endothelial cell death. This raises the question of whether such a signaling pathway is involved in specific physiological or pathological processes. Interestingly, both LIF and cathepsin L are involved in the development and progression of vascular diseases, such as abdominal aortic aneurysms and atherosclerosis (51-53). These data collectively suggest a role for the LIF-cathepsin L pathway in regulating the vasculature in pathological settings.

[0058] In the present study, LIF also reduced BrdU incorporation, accompanied by a decrease in cyclin A / B expression in BAE cells, suggesting that LIF-induced cell cycle arrest plays a role in BAE growth inhibition. It has previously been reported that cyclin A1 and cyclin B1 are direct STAT3 targets (54). STAT3 has also been implicated in both the up- and down-regulation of cyclin A / B depending on the specific context (55-58), and STAT3-mediated suppression of cyclin A expression was mediated by its direct target, PIM1 (58). This explains why LIF suppresses cyclin A / B expression in BAE cells but not in BCE cells, since PIM1 induction by LIF is only observed in BAE cells.

[0059] The present invention discloses opposing responses (proliferation vs. growth inhibition) elicited by the same signaling pathway in two types of endothelial cells. Activated STAT3 transactivates unique sets of genes in these two cell types. Indeed, upon LIF treatment in BCE and BAE cells, several genes are differentially expressed, including downregulation of the S-phase and G2 / M cyclin genes CCNA2 and CCNB1, and upregulation of the lysosomal cysteine ​​protease CTSL in BAE cells but only the proliferation gene MYC in BCE cells. Different types of endothelial cells have unique gene expression patterns / epigenetic profiles that determine their distinct responses to the same stimuli (59-61). Our disclosure of the opposing effects of LIF in different endothelial cells illustrates a novel aspect of such diversity: the same signaling pathway mediates divergent effects depending on endothelial cell type-specific transcriptional programs. The present invention reports for the first time that LIF-induced lysosomal protease cathepsin L leads to endothelial cell death.

[0060] In embodiments, the present invention discloses an unexpected mitogenic role for LIF in choroidal and retinal endothelial cells, demonstrating that both LIF and CT-1 increase retinal microvascular density in vivo. Indeed, protecting ocular blood vessels, such as the choriocapillaris layer, in patients with wet or dry AMD is beneficial because it may prevent atrophy (62). Both LIF and CT-1 have protective effects in the NaIO3 model, suggesting that these agents protect the retinal pigment epithelium and choriocapillaris and therefore have therapeutic value in preventing atrophy in AMD. The lack of a direct permeabilizing effect of LIF and possibly CT-1 is particularly useful in this regard. Notably, OSM has the opposite effect, demonstrating the specificity of the effects of LIF and CT-1. [Example]

[0061] material and method reagent Antibodies: human PDGF-AA antibody (R&D Systems, CAT# AF-221-NA), human CCL2 / MCP-1 (R&D Systems, CAT# AF-279-NA), human LIF antibody (Sigma, CAT# L9277), regular goat IgG isotype control (R&D Systems, CAT# AB-108-C), and Alexa Fluor-488 conjugated BrdU antibody 3D4 (Biolegend, CAT# 364106)

[0062] Small molecule inhibitors: baricitinib (Apexbio Technology, CAT# A414150), cobimetinib (MedChemExpress, CAT# HY-13064), BEZ235 (Selleckchem, CAT# S1409), Z-VAD-FMK (R&D Systems, CAT# FMK001), Z-DEVD-FMK (R&D Systems, CAT# FMK004), Q-VD(OMe)-OPh (Apexbio Technology, CAT# A8165), 5-AIQ hydrochloride (Sigma, CAT# A7479), CA-074me (Calbiochem, CAT# 205531), CA-074 (Tocris, CAT# 4863), and CAA0225 (Calbiochem, CAT# 219502)

[0063] Recombinant proteins: human LIF (Sigma, CAT#SRP9001), human LIF (Biolegend, CAT#593902), human PDGF-AA (Peprotech, CAT#100-13A), human peroxiredoxin 1 (Abcam, CAT#ab74172), human IL-8 (Biolegend, CAT#574202), and human VEGF165 (R&D Systems, CAT#293-VE)

[0064] cell culture LN-229 human glioblastoma cells were maintained in high-glucose DMEM supplemented with 5% FBS. Bovine choroidal endothelial (BCE) (P5-P9) and bovine retinal endothelial (BRE) (P5-P9) cells were maintained on fibronectin-coated culture plates in DMEM-low glucose supplemented with 10% fetal calf serum (BCS), 2 mM glutamine, 5 ng / ml bFGF, and 10 ng / ml VEGF. Bovine aortic endothelial (BAE) cells (P5-P10) were maintained in DMEM-low glucose supplemented with 10% BCS. Human retinal microvascular endothelial (HRME) cells (P4-P9) were maintained on gelatin-coated culture plates in EGM2 medium with antibiotics. All cells were maintained at 37°C in a humidified atmosphere of 5% CO2.

[0065] Endothelial cell proliferation assay Bovine endothelial proliferation assays were performed essentially as previously described (63, 64). BCE (1 × 10 3 cells / well) or BRE (5 × 10 2 BAE cells were seeded at 2 x 10 cells / well in a 96-well plate in culture medium (DMEM-low glucose supplemented with 10% BCS, 2 mM glutamine, and antibiotics) and test material in a total volume of 200 μl per well. BAE cells were seeded at 2 x 10 cells / well in culture medium (DMEM-low glucose supplemented with 1% BCS and antibiotics) and test material in a total volume of 200 μl per well. 3 HRME cells were plated in 96-well plates at a density of 1 × 10 cells per well in gelatin-coated 96-well plates in assay medium (DMEM-low glucose supplemented with 20% FBS and antibiotics) and test material making a total volume of 200 μl per well. 3 Cells were seeded at a density of 1000 cells / well. For assays involving antibody or small molecule inhibitors, the inhibitor or vehicle control was added first, followed by the test material 1 hour later. After 6 days (unless otherwise specified), cells were incubated with Alamar Blue for 4 hours. Fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Each experiment was performed in duplicate / triplicate and repeated at least three times.

[0066] LN-229 cell conditioned medium 5×10 6 LN-229 cells were seeded into 15 cm culture dishes with 35 ml of medium (DMEM-high glucose with 0.5% FBS and 1% antibiotics) and incubated at 37 °C for 72 h. LN-229 CM was collected by centrifugation, filtered through a 0.22 μm filter, and stored at -80 °C for later use.

[0067] Chromatographic enrichment of endothelial mitogens in LN-229 CM Approximately 400 ml of LN-229 CM was subjected to enrichment of endothelial mitogens by sequential chromatographic purification. The CM was buffer-exchanged into 20 mM Tris, pH 8.0, filtered (0.2 μm), and loaded onto a 5 ml HiTrap Q™ HP column (GE Healthcare, Pittsburgh, PA) using a GE AKTA Explorer System (GE Healthcare). After stepwise elution with 0.2 M, 0.5 M, 1 M, and 2 M NaCl in Tris buffer, aliquots of the eluted fractions were tested in the BCE cell growth assay as described above. The mitogen fractions were then pooled, diluted with 0.1% trifluoroacetic acid / HO (TFA, ThermoFisher), and applied to a SynChropak RP C4 reversed-phase column (4.6 x 100 mm, Eichrom Technologies, Darien, IL). Fractions were eluted with a linear gradient of acetonitrile / 0.1% TFA. Elution fractions were evaporated using a MiVac DUO concentrator (Genevac, Ipswich, UK), washed, resuspended in PBS, and tested as above. Mitogenic fractions and adjacent negatives were subjected to mass spectrometry.

[0068] ELISA VEGF and LIF levels in LN-229 CM samples were determined by a human VEGF ELISA kit (R&D Systems, CAT# DVE00) and a human LIF ELISA kit (Biolegend, CAT# 443507), respectively, according to the manufacturer's instructions. Cathepsin L levels in BAE cells were measured using a bovine cathepsin L ELISA kit (MyBioSource, Inc, CAT# MBS2887609), according to the manufacturer's instructions.

[0069] STAT3 knockdown by siRNA BCE and BAE cells were cultured at 1.5 × 10 5BCE cells were plated onto 6-well culture plates at a density of 100 cells / well. BCE cells were incubated overnight in 2 ml of DMEM-low glucose supplemented with 10% BCS, 2 mM, 5 ng / ml bFGF, 10 ng / ml VEGF, and antibiotics. BAE cells were cultured overnight in 2 ml of DMEM-low glucose supplemented with 10% BCS and antibiotics. 2 ml of antibiotic-free medium was used instead of the old medium. siRNAs, including siNegative (Ambion, CAT#AM4611), siSTAT3-915 (Invitrogen, CAT#361146C04), siSTAT3-1492 (Invitrogen, CAT#361146C05), and siSTAT3-454 (Invitrogen, CAT#384235A10), were mixed with Lipofectamine RNAiMAX Reagent (ThermoFisher Scientific, CAT#13778150) in Opti-MEM™ I Reduced Serum Medium (Gibco, CAT#31985062) according to the manufacturer's instructions. Briefly, cells in each well were transfected using a mixture containing 25 pmol of siRNA, 7.5 ul of RNAiMAX Reagent, and 125 ul of Opti-MEM medium, resulting in a final siRNA concentration of 12.5 nM. A mixture of RNAiMAX and Opti-MEM was used as a non-siRNA control. Cells were incubated with siRNA for 8 hours, and then fresh regular culture medium was used to replace the siRNA-containing medium. 24 hours after transfection with siRNA, cells were used for endothelial proliferation assays and RNA / protein extraction.

[0070] Western blotting BCE and BAE cells were cultured overnight in growth medium. The growth medium was removed, and then the cells were washed twice with PBS. Recombinant human LIF was added to the cells for 15 minutes, followed by 3-hour incubation in the following media: DMEM-low glucose supplemented with 10% BCS, 2 mM glutamine, and antibiotics for BCE cells, and DMEM-low glucose supplemented with 1% BCS and antibiotics for BAE cells. Where applicable, small molecule inhibitors (i.e., baricitinib, cobimetinib, BEZ235, and vehicle control DMSO) were added to the cells 1 hour prior to LIF treatment. Next, the cells were lysed with RIPA lysis buffer (Life Technologies, CAT#89901) and a protease and phosphatase inhibitor cocktail (ThermoFisher Scientific CAT#78440). Protein concentration in the cell lysates was measured by BCA assay (ThermoFisher Scientific CAT#23227). Equal amounts of protein were electrophoresed on NuPAGE 4-12% Bis-Tris gels (ThermoFisher Scientific, CAT# NW04125BOX) and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in TBST for 1 hour at room temperature and incubated with the following primary antibodies in TBST containing 0.5% nonfat milk overnight at 4°C, followed by secondary HRP-conjugated antibodies (1:2000, GE Healthcare) for 1 hour at room temperature. Signals were developed using SuperSignal™ West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific).Primary antibodies used were: anti-phospho-STAT3 (Cell Signaling, CAT#9131, 1:3000), anti-STAT3 (Cell Signaling, CAT#4904, 1:3000), anti-phospho-ERK (Cell Signaling, CAT#4376, 1:5000), anti-ERK (Cell Signaling, CAT#4695, 1:5000), anti-phospho-AKT Ser473 (Cell Signaling, CAT#4060, 1:2000), anti-AKT (Cell Signaling, CAT#4691, 1:2000), and HRP-conjugated anti-beta-actin (Sigma, CAT#AC-15, 1:10000).

[0071] RNA extraction and qRT-PCR After the indicated treatments, BCE and BAE cells were lysed with Trizol reagent (Invitrogen, CAT# 15596026) and subjected to RNA extraction according to the manufacturer's instructions. RNA concentration was determined using a Nanodrop2000 (ThermoFisher Scientific), and 1 μg of total RNA was reverse-transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CAT# 4368814). Equal amounts (typically 10 ng / reaction) of cDNA were subjected to qRT-PCR analysis using TaqMan Fast Advanced Master Mix (Applied Biosystems, CAT# 4444557) and a ViiA7 Real-Time PCR System. Relative mRNA levels of the examined genes were determined by normalizing to the internal control RPLP0 (ribosomal protein lateral stalk subunit P0) and comparing with the control sample group and reported as fold changes. TaqMan gene expression assay probes were used: bovine RPLP0 (Bt03218086_m1), bovine STAT3 (Bt03259865_m1), bovine CTSL1 (Bt03257307_m1 and Bt03257309_m1), bovine CTSB (Bt03259161_m1), bovine MYC (Bt03260377_m1), bovine JunB (Bt03246919_s1), bovine CCNA2 (Bt03240503_g1), bovine CCNB1 (Bt03237853_g1), and bovine PIM1 (Bt03212957_m1). Experiments were performed in triplicate and repeated three times.

[0072] Annexin V staining for cell death BAE cells were plated in 12-well plates at 2 × 10 with 1 ml of culture medium (DMEM-low glucose and 10% BCS). 4Cells were plated at a density of 1000 cells / well and then incubated overnight at 37°C. After removing the culture medium, the cells were incubated with 0.5 ml of DMEM-low glucose and 1% BCS. LIF (10 ng / ml) and vehicle control (0.1% BSA in PBS) were added to the cells. After 24 hours of LIF treatment, the cells were examined for the cell death marker annexin V using the Annexin V-Cy5 Apoptosis Staining Detection Kit (Abcam, CAT# ab14150) according to the manufacturer's instructions. Briefly, the cell culture medium was removed, and 0.5 ml of annexin V-binding solution was placed on the cells. 5 μl of annexin V-Cy5 was added, followed by incubation of the cells at room temperature for 5 minutes. The staining solution was then discarded and replaced with 0.5 ml of annexin V-binding solution. Imaging of annexin V staining was performed using a Keyence BZ-X710 microscope (Keyence Corporation, Osaka, Japan). Four random fields were selected, and the percentage of Annexin V-stained area in the total cell coverage area was determined using ImageJ software as an indicator of cell death. Imaging of Annexin V staining was performed using a Keyence Microscope BZ-X710 (Keyence Corporation, Osaka, Japan). Experiments were performed in triplicate and repeated three times.

[0073] BrdU incorporation assay BAE cells were cultured at 2 × 10 with 1 ml of culture medium (DMEM-low glucose and 10% BCS) in 12-well plates using 18 mm poly-D-lysine-treated coverslips in each well. 4Cells were plated at a density of 1000 cells / well and incubated overnight at 37°C. After removing the culture medium, the cells were incubated with 0.5 ml of DMEM-low glucose and 1% BCS. LIF (10 ng / ml) and vehicle control (0.1% BSA in PBS) were added to the cells. After 48 hours of LIF treatment, 2.5 μl of 2 mM BrdU in DMSO was added to each well to a final concentration of 10 μM and incubated for 4 hours to subject the cells to BrdU incorporation. Next, the cells were subjected to BrdU immunofluorescence staining using an antibody against BrdU conjugated with fluoroAlexa-488 (Biolegend, CAT#364106, 1:400). Briefly, the BrdU-labeling medium was removed from the culture plate, and the cells were fixed with 3.7% formaldehyde in PBS for 15 minutes at room temperature. Cellular DNA was denatured with 1N HCl on ice for 10 minutes and 2N HCl at room temperature for 10 minutes, followed by permeabilization of cells with 0.1% Triton X-100 in PBS (PBST). Cell coverslips were incubated with FluoroAlexa-488-conjugated BrdU antibody in 5% goat serum-PBST overnight at 4°C. Coverslips were then mounted on glass slides using Fluoroshield Mounting Medium with DAPI (Abcam, CAT# ab104139). Imaging of BrdU staining was performed using a Keyence BZ-X710 microscope (Keyence Corporation, Osaka, Japan). Four fields were randomly selected for each sample, and BrdU-positive nuclei, as well as total nuclei (DAPI-positive), were manually counted. The percentage of BrdU-positive cells was determined by dividing the number of BrdU-positive nuclei by the total number of nuclei. Experiments were performed in duplicate and repeated three times.

[0074] Mouse choroid explant assay In a 48-well plate, 60 μL of growth factor-reduced basement membrane extract (GFR-BME) (Corning, CAT#354230) was added to each well and allowed to solidify at 37°C for 20 minutes. A cubic peripheral choroid-sclera complex (approximately 1 mm x 1 mm) dissected from a male C57BL / 6J mouse (20 years old) was added to the center of each well as previously described (23). A top layer of 60 μL of GFR-BME was added to each well and incubated at 37°C for 30 minutes. Endogenous VEGF activity in choroidal explants was blunted by 5 μg / ml of anti-VEGF MAb B20-4.1.1 upon addition of 500 μL of endothelial cell growth basal medium EBM-2 (Lonza, CAT#CC3156) supplemented with 2% FBS and antibiotics. After 90 minutes of incubation with the antibody, 10 ng / ml LIF or PBS control was added to the test wells. The tissues were incubated in standard cell culture conditions with 5% CO2, and fresh medium was replaced every 48 hours. Phase-contrast Z-stack images of each explant were taken on day 5 using a Keyence microscope. The area of ​​vascular sprouting was quantified using ImageJ software. Experiments were repeated three times, and data were obtained by analyzing five replicates per condition.

[0075] Intravitreal injection of recombinant proteins in mouse eyes Male C57BL / 6J mice (6–8 weeks and P5) were anesthetized with a ketamine / xylazine cocktail. The indicated amount of recombinant LIF (Sigma, CAT# SRP9001) in 1 μl of PBS or PBS vehicle control was injected intravitreally with a 33-gauge Hamilton syringe. Seven days (for adult mice) or three days (for neonatal mice) after injection, the animals were euthanized, and the eyes were then enucleated and fixed in 4% paraformaldehyde (PFA) for 15 minutes. The choroid-sclera complex and retina were isolated, and anti-CD31 immunofluorescence (IF) or lectin labeling was performed to demonstrate the vasculature by whole-mount staining of both retinal and choroidal tissues or retinal flat-mounts. For CD31 IF, rat anti-mouse antibody (BD Biosciences, CAT# 550274) was diluted 1:100 and incubated overnight at 4°C. After 4 hours of incubation with Alexa Fluor-488-conjugated anti-rat antibody (Life Technologies, Cat. #A11006), whole mounts were imaged via the 488 nm channel using a Keyence Microscope BZ-X710 (Keyence Corporation, Osaka, Japan) or an A1R Confocal STORM Super Resolution System (Nikon). For lectin staining, Dylight-488-labeled lectin (Vector Laboratories, Cat. #DL-1174) was diluted 1:200 and images were acquired using an A1R Confocal STORM Super Resolution System (Nikon). Quantification of choroidal and retinal vascular density was performed using Image J. Statistical analysis was performed using Student's t-test. Each experiment was repeated three times with similar results, and each treatment group consisted of four or five individual samples in all experiments. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego, and were performed in accordance with the Animal Care Program (ACP) guidelines.

[0076] Sodium iodate model Eight-week-old C57BL / 6J mice were anesthetized with a ketamine / xylazine cocktail. Sterile NaIO3 was administered as a single intravenous injection (20 mg / kg body weight) (28) (29). Control mice were injected with PBS. PBS, LIF (50 ng), CT-1 (various doses), or OSM (10 ng) were intravitreally injected into groups of five mice. Five, seven, and nine days after injection, the choriocapillaris was monitored using an OCT-A system. Nine days after injection, the mice were sacrificed, and the eyes were harvested for H&E and immunofluorescence staining. The avascular area of ​​the choriocapillaris was analyzed using ImageJ.

[0077] Measurement of retinal vascular leakage Recombinant human VEGF (100 ng) or LIF (10 ng) was injected into the vitreous (0.1% BSA in PBS as a vehicle control). TRITC-dextran (50 mg / ml, 100 μl) was then injected into the tail vein. After 10 min, the animals were sacrificed and the eyes were enucleated. Retinal flatmounts were imaged under a microscope (65).

[0078] Optical Coherence Tomography Angiography (OCTA) Imaging Optical coherence tomography angiography (OCTA) imaging of the adult mouse retina was performed 7 days after LIF injection using a 1300 nm optical coherence tomography (OCT) system developed by Dr. R.K. Wang's group at the University of Washington, Seattle, consistent with previously described methodology. (66) Briefly, a swept laser operating in single longitudinal mode with a 90 nm bandwidth centered at 1300 nm and a 200 kHz A-line rate was used to scan the mouse retina, capturing a 1.5 × 1.5 mm 2Images of the vasculature in the visual field were generated. 2,500 B-frames were captured at 500 cross sections, with five B-frames repeated at each cross section. To quantify retinal vascular density, the retinal and choroidal layers in the 3D structural OCT scans were separated by the hyperreflective retinal pigment epithelium (RPE). Next, a maximum intensity projection of the emmetropic view was generated. Vascular density was then determined by calculating the percentage of vascular coverage in the total visual field using ImageJ software.

[0079] statistical analysis Experiments were repeated at least three times with similar results, except for mass spectrometry. Bar graphs represent the mean ± standard deviation (sd). For comparisons of only two groups in studies, a two-tailed Student's t-test was performed. For comparisons between groups in studies with three or more data groups, a one-way analysis of variance with multiple comparisons was performed. For comparisons between groups in studies with two or more variables, a two-way analysis of variance with multiple comparisons was performed. p<0.05 was considered statistically significant. All statistical analyses were performed using the Graphpad Prism software package.

[0080] result Identification of LIF as a mitogen for choroidal endothelial cells LN-229 cell-conditioned medium (LN-229 CM) can stimulate the growth of bovine choroidal endothelial (BCE) cells (Figure 1A). However, consistent with previous studies (9, 10), LN-229 cells secrete little VEGF into the culture medium. The anti-VEGF antibody B20-4.1 (11) did not suppress the mitogenic effect of LN-229 CM (Figure 1B), suggesting the involvement of a VEGF-independent pathway. The angiogenic factor profile of LN-229 CM was examined using a specific antibody array. This analysis revealed that most known angiogenic factors were undetectable, with the exception of PDGF-AA, CCL2 (also known as MCP-1), and interleukin-8 (IL-8), which were abundant in CM. However, antibodies neutralizing PDGF-AA or CCL2 were unable to suppress BCE cell growth induced by LN-229 CM. Furthermore, recombinant PDGF-AA and IL-8 were unable to stimulate BCE cell growth (Table 1). [Table 1]

[0081] To identify mitogenic factors in LN-229 CM, we employed a proteomics approach. BCE mitogenic activity was enriched through two sequential chromatographic steps: anion exchange and reverse-phase chromatography. At each step, only one peak of absorbance, consisting of 4–5 consecutive fractions, exhibited mitogenic activity. After the reverse-phase column step, the peak mitogenic fractions (R26 and R27), the minimal mitogenic fractions (R25 and R28), and the adjacent negative (R24 and R29) fractions (Figure 1C) were subjected to mass spectrometry analysis. By screening intracellular proteins, we generated a short list of five candidate proteins (Table 2). [Table 2]

[0082] Of the five proteins listed, four are serum components and function as redox enzymes, including peroxiredoxins (PRDX)-1, -2, and -6, and alpha-2-macroglobulin. LIF, a cytokine, stood out. LIF, a member of the interleukin-6 (IL-6) family of proteins, is widely expressed, exerts effects in multiple cell types and tissues, and is involved in various critical physiological processes, including embryonic stem cell self-renewal, blastocyst implantation, and astrocyte differentiation (12, 13). The presence of LIF here was unexpected, as this cytokine has previously been characterized as an endothelial cell growth inhibitor and antiangiogenic agent (14-16). However, antibodies against LIF completely suppressed BCE cell growth induced by the reverse-phase fraction (Figure 1D). The LIF levels in each fraction correlated strongly with mitogenic activity: the most bioactive fractions, R26 and R27, exhibited the highest LIF concentrations, R25 and R28 had trace amounts of LIF, whereas the inactive fractions R24 and R29 were devoid of LIF (Table 3). [Table 3]

[0083] These observations suggested that LIF might be responsible for the mitogenic effect. Indeed, recombinant LIF stimulated the growth of BCE cells (Figure 1E), whereas another candidate, PRDX1, had no effect (Table 1), further confirming that LIF is a mitogenic factor. When tested on bovine retinal endothelial (BRE) cells, LIF also exerted mitogenic activity. Interestingly, VEGF and LIF together had a greater than additive mitogenic effect in both BCE (Figure 1F) and BRE cells, suggesting a synergistic relationship between LIF and VEGF. Indeed, although LIF did not induce a strong mitogenic response in human retinal microvascular endothelial cells, its addition significantly enhanced VEGF-stimulated growth.

[0084] The effect of LIF on endothelial cell growth is mediated by the JAK-STAT3 pathway Although all members of the IL-6 family share the receptor component gp130, LIF signaling is transmitted through the gp130:LIFR receptor dimer, and IL-6 activates its downstream signaling via the IL6Rα:gp130:gp130:IL6Rα tetramer (12). Among the four gp130-associated Janus kinases (JAK1, JAK2, JAK3, and TYK2), LIF signaling selectively activates JAK1 through transphosphorylation (12, 17, 18). Upon activation by LIF, JAKs induce three distinct signaling cascades: JAK-STAT, PI3K-AKT-mTOR, and RAS-MAPK, which contribute to distinct functions in specific cell types (12, 19). Regarding the JAK-STAT pathway, LIF signaling preferentially activates STAT3, whereas STAT1 and STAT5 can be phosphorylated by JAK1 (19, 20). To investigate which pathways are responsible for LIF-induced growth stimulation of BCE cells, we used a set of small molecule inhibitors: baricitinib, cobimetinib, and BEZ235, which specifically inhibit JAK1 / 2, MEK1 / 2 (MAPK pathway), and PI3K / mTOR, respectively. In BCE cells, 15 min of LIF treatment induced phosphorylation of STAT3 and ERK, but had little effect on AKT phosphorylation (Figure 2A). Preincubation with the JAK1 / 2 inhibitor baricitinib almost completely suppressed LIF-induced phosphorylation of STAT3 and ERK MAPK (Figure 2A). Cobimetinib pretreatment blocked ERK phosphorylation but had no effect on STAT3 or AKT phosphorylation (Figure 2A). BEZ235 had only a modest effect on AKT phosphorylation, regardless of LIF treatment (Figure 2A). Furthermore, baricitinib completely blocked LIF-induced cell growth, whereas cobimetinib showed minimal effects, and the PI3K / mTOR inhibitor BEZ235 had no effect on LIF-stimulated cell growth (Figure 2B). These observations suggest that the MAPK and PI3K pathways may not be the primary drivers of LIF stimulation in BCE cells, and thus JAK-STAT pathways are involved.Because STAT3 is the preferred mediator of the LIF-induced JAK-STAT signaling cascade (19, 20) and is involved in the proliferation and survival of a wide variety of cell types (21), we further investigated the role of STAT3 in BCE by siRNA knockdown. siRNA successfully suppressed STAT3 levels at both the RNA and protein levels in BCE cells (Figures 2C and 2D). Downregulation of STAT3 blocked LIF-induced BCE cell growth in vitro (Figure 2E). These observations indicated that the JAK-STAT3 signaling axis mediates the mitogenic effects of LIF in BCE cells.

[0085] LIF promoted endothelial cell growth ex vivo and in vivo LIF can induce the proliferation of choroidal and retinal endothelial cells in vitro. However, previous reports have suggested that LIF may adversely affect vascular function in the developing eye (14, 16, 22). To resolve these apparent discrepancies, we investigated whether LIF functions differently ex vivo and in vivo, particularly in ocular endothelial cells. We first examined the effects of LIF on choroidal endothelial cells in an ex vivo choroidal explant model modified from a previous report (23). In response to LIF, microvessel growth from the explant into Matrigel was significantly enhanced compared to that in controls (Figures 3A and 3B). Next, we examined the effects of LIF in vivo by intravitreal injection into 6- to 8-week-old mice. As assessed by immunohistochemistry (IHC) using an antibody against the endothelial cell surface marker CD31, administration of LIF at a dose of 10 ng per eye significantly increased retinal microvascular density, whereas a dose of 100 ng was less effective (Figures 3C and 3D), consistent with the bell-shaped response observed with many cytokines (24). Optical coherence tomography angiography (OCTA) also documented a significant increase in retinal vascular density after LIF injection (Figures 3E and 3F). Immunofluorescent staining for CD31 in cross-sections of mouse eyes also demonstrated that LIF injection increased vascular density in the adult mouse retina (Figures 3G and 3H).

[0086] To confirm that such angiogenesis-stimulating effects were truly induced by LIF and not by trace contaminants such as endotoxin or nonspecific events associated with injection, recombinant LIF was heat-inactivated by exposure to 95°C for 2 hours, which does not affect endotoxin stability (30). Such treatment abolished LIF's ability to promote mitogenesis in vitro and angiogenesis in vivo. However, previous studies using LIF knockout mice suggested that LIF expression was negatively associated with retinal vascular density (16). The discrepancy between such observations and our data raises the possibility that LIF plays a unique role in regulating retinal angiogenesis at different developmental stages. Importantly, LIF also plays a critical role in the maturation of retinal astrocytes, which may secondarily influence the development of the retinal vasculature (31, 32). To examine the effects of LIF on the developing retinal vasculature and minimize its effects on astrocyte development, we intravitreally injected LIF into postnatal day 5 (P5) mice, when the retinal vasculature is developing but the astrocyte network is already established and maturing (33, 34). LIF treatment in such neonatal mice also resulted in a significant increase in vascular density, assessed 3 days after injection (Figures 3I and 3J), confirming the angiogenic stimulatory effect of LIF on the retinal vasculature.

[0087] Because LIF is a member of the interleukin-6 (IL-6) family (25), we tested the effects of two other family members, cardiotrophin-1 (CT-1) (26) and oncostatin M (OSM) (27), on retinal neovascularization. Compared to 50 ng of LIF, 20 ng and 100 ng of CT-1 resulted in approximately 30% and 50% increases in retinal density, respectively. However, instead of promotion, vascular density was reduced in the retinas of OSM-treated mice. The distinct effects of OSM on the retinal vasculature from those of LIF and CT-1 suggest that OSM may not activate the same signaling pathways activated by LIF and CT-1. OSM can bind to both the gp130::LIFR and gp130::OSMR receptor complexes, whereas LIF and CT-1 utilize only the gp130::LIFR complex.

[0088] The NaIO3 mouse model is widely used as a preclinical model of atrophic AMD (28). In this model, both the RPE layer and choriocapillaris are severely damaged (29). Therefore, LIF, CT-1, and OSM were tested for their ability to promote choriocapillaris recovery in this model. After intravenous injection of NaIO3, LIF, CT-1, or OSM was intravitreally injected. Consistent with their effects on the retinal vasculature, LIF and CT-1 reduced the avascular area compared with the PBS group. In contrast, the avascular area of ​​the OSM-treated choroid was greater than that of the PBS group (Figures 8C and 8D). The protective effects of LIF and CT-1 on the retinal vasculature in response to NaIO3 treatment may be due to both their direct mitogenic activity on retinal endothelial cells and their ability to protect retinal RPE cells from oxidative stress-induced damage, which in turn supports the maintenance of the retinal vasculature through the secretion of angiogenic stimulators such as VEGF.

[0089] LIF caused growth inhibition via the JAK-STAT3 pathway Consistent with previous studies (35), LIF inhibited BAE cell growth (Figure 4A), suggesting a complex role for LIF in regulating endothelial function. To investigate the LIF-induced signaling cascade in BAE cells, we used baricitinib, cobimetinib, and BEZ235 to inhibit JAK1 / 2, MEK1 / 2, and PI3K / mTOR, downstream components of LIF-gp130:LIFR. In BAE cells, 15 min of LIF treatment induced phosphorylation of STAT3, ERK (MAPK), and AKT (Figure 4B). Baricitinib pretreatment significantly suppressed LIF-induced phosphorylation of STAT3, ERK, and AKT, and cobimetinib and BEZ235 pretreatment also effectively blocked ERK and AKT phosphorylation, respectively (Figure 4B). Interestingly, baricitinib was the only inhibitor that reversed LIF-induced growth inhibition in BAE cells (Figure 4C), suggesting that the JAK-STAT pathway mediated the effect of LIF in BAE cells. To further investigate whether LIF-induced inhibition of BAE cells was due to the JAK-STAT3 cascade, STAT3 was knocked down by approximately 80% with three different siRNAs in BAE cells (Figure 4D and 4E). Interestingly, knockdown of STAT3 in BAE cells ameliorated LIF-induced growth inhibition (Figure 4F). These observations demonstrate that the LIF-JAK-STAT3 signaling pathway may play opposing roles in regulating endothelial cell growth, which was dependent on the endothelial cell type.

[0090] LIF inhibited BAE cell growth via cathepsin L-dependent cell death and cell cycle arrest Next, we investigated which growth inhibitory effects (e.g., cell cycle arrest, cellular senescence, or programmed cell death) were induced by LIF in BAE cells. Because IL-6-STAT3 signaling has been closely associated with cellular senescence (36-38), it was initially hypothesized that the LIF-STAT3 axis also induces senescence in BAE cells. However, in a senescence-associated β-galactosidase assay, we did not observe an increase in the number of senescent cells in BAE cells treated with LIF for 48 h, suggesting that senescence was not the primary effect induced by LIF in BAE cells. Interestingly, staining for the cell death marker, Annexin V, showed an increase in the proportion of Annexin V-positive cells in BAE cells treated with LIF for 24 h (Figures 6A and 6B), indicating that LIF treatment induced cell death. Surprisingly, co-incubation with caspase inhibitors (Q-VD-OPH, Z-VAD-fmk, and Z-DEVD-fmk) or a poly(adenosine 5'-diphosphate ribose) polymerase (PARP) inhibitor (5-AIQ) failed to rescue the LIF-induced cell death phenotype. These data suggest that a caspase-independent pathway may be involved in LIF-mediated cell death of BAE cells. To investigate the molecular basis of the differentiated roles of LIF in BAE and BCE cells, genes induced / repressed by LIF were analyzed by RNA-seq in BAE and BCE cells incubated with LIF for 6 hours. Notably, LIF treatment resulted in unique gene expression patterns in these two cell types. Notably, IGFBP3, a secreted protein previously shown to be an angiogenesis inhibitor (39), at least in some circumstances, was upregulated approximately eightfold in BAE but not BCE cells, a finding later confirmed by qRT-PCR. However, recombinant IGFBP3 had no effect on BAE cell growth. Furthermore, conditioned medium from BAE cells treated with LIF for 72 h did not inhibit BAE cell growth in the presence of LIF-neutralizing antibodies, contradicting the hypothesis that LIF-induced BAE growth inhibition is mediated by a secreted factor.It has previously been reported that STAT3 can induce caspase-independent cell death through upregulation of the lysosomal proteases cathepsin B and L (40). Therefore, we investigated whether LIF could induce such a signaling cascade in BAE cells. Interestingly, CTSL, but not CTSB, was dramatically upregulated by LIF at both the mRNA and protein levels in BAE cells after 24 h of treatment (Figures 6C and 6D). Co-incubation with CA074me, an inhibitor that antagonizes both cathepsin B and L, attenuated LIF-induced growth inhibition of BAE cells in a CA074me dose-dependent manner (Figure 6E). Furthermore, CAA0225, another cathepsin L-specific inhibitor, also abrogated LIF-induced growth inhibition in BAE cells, albeit to a lesser extent (Figure 6F). In contrast, CA074, a cathepsin B-selective inhibitor, failed to suppress LIF-induced effects in BAE cells, even at 50 μM, the highest dose tested. Interestingly, cathepsin L mRNA (CTSL) levels in BCE cells were undetectable by qRT-PCR, regardless of whether the cells were incubated with vehicle or LIF. These data collectively demonstrated that LIF induced cathepsin L upregulation in BAE cells, which in turn led to caspase-independent cell death. Furthermore, after 48 h of incubation with LIF, BAE cells showed a significant reduction in BrdU incorporation compared to vehicle controls (Figures 7A and 7B), suggesting that cell cycle arrest was induced by LIF. This notion was supported by the downregulation of cyclin A / B in LIF-treated BAE but not BCE cells (Figure 7C).

[0091] In embodiments, the present invention provides novel and unexpected activities of IL-6 cytokines such as LIF and CT-1 to induce blood vessel growth, ie, angiogenesis.

[0092] For example, the present invention provides that LIF, a molecule previously characterized as an inhibitor of endothelial cell growth, has unexpected angiogenic stimulatory properties in the eye, as assessed by in vitro, ex vivo, and in vivo studies.

[0093] The present study demonstrates that LIF can directly stimulate choroidal endothelial cell proliferation while inhibiting the growth of aortic endothelial cells, highlighting the specificity and uniqueness of its effects on endothelial cells. Furthermore, when injected into the vitreous of mice, LIF promoted endothelial sprouting and angiogenesis from choroidal explants.

[0094] LIF is a well-characterized cytokine and member of the IL6 family that interacts with the LIF receptor and then heterodimerizes with GP130, resulting in, among other effects, activation of Stat3.

[0095] The present invention provides evidence that LIF can promote the growth of subsets of endothelial cells, providing opportunities for therapeutic intervention in a variety of conditions, including hypoperfusion in retinal / choroidal, coronary, and myocardial diseases (Reboucas et al., 2016; Simon-Yarza et al., 2012; Wang et al., 2013). The observation that LIF does not induce vascular permeability suggests that administration of this factor avoids the undesirable vascular leakage associated with VEGF (Niu et al., 2016).

[0096] The present invention suggests that IL-6 family members, such as LIF and CT-1, can protect the RPE from damage, including that caused by oxidative stress, which may represent a novel therapeutic strategy for treating retinal conditions associated with RPE damage or degeneration. 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Claims

1. A method of treatment for a condition associated with insufficient angiogenesis in the eye of a subject, comprising administering to a subject in need thereof an effective amount of an IL-6 family protein or a functional fragment thereof to promote angiogenesis.

2. The method of claim 1 , wherein the administration increases retinal microvascular density.

3. The method of claim 1 , wherein the administration increases proliferation of choroidal endothelial cells.

4. The method of claim 1 , wherein the condition is age-related macular degeneration.

5. 10. The method of claim 1, wherein the condition is retinopathy of prematurity (ROP).

6. The method of claim 1 , wherein the administration is via intravitreal injection.

7. The method of claim 1 , wherein the effective amount does not induce vascular leakage.

8. The method of claim 1 , wherein the effective amount does not induce edema.

9. The method of claim 1, wherein the IL-6 family protein is leukemia inhibitory factor (LIF).

10. The method of claim 1, wherein the IL-6 family protein is cardiotrophin-1 (CT-1).

11. A method for inducing angiogenesis in the eye of a subject, the method comprising administering to a subject in need thereof an effective amount of an IL-6 family protein or a functional fragment thereof.

12. The method of claim 11 , wherein the administration increases retinal neovascularization.

13. The method of claim 10, wherein the administration increases proliferation of choroidal endothelial cells.

14. The method of claim 10 , wherein the subject has age-related macular degeneration.

15. 11. The method of claim 10, wherein the subject has retinopathy of prematurity (ROP).

16. The method of claim 10 , wherein the administration is via intravitreal injection.

17. The method of claim 10, wherein the effective amount does not induce vascular leakage.

18. 11. The method of claim 10, wherein the effective amount does not induce edema.

19. The method of claim 10, wherein the IL-6 family protein is leukemia inhibitory factor (LIF).

20. The method of claim 10, wherein the IL-6 family protein is cardiotrophin-1 (CT-1).

Images