KR20170119902A - Composition for treating diabetic retinopathy comprising inhibitors of Integrin αvβ5 and Screening method for the said inhibitors - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of diabetic retinopathy comprising an anti-integrin αvβ5 antibody, and a method for screening a substance for the treatment of diabetic retinopathy suppressing the interaction between angiopoietin-2 and integrin αvβ5 protein.

Description

 The present invention relates to a composition for treating diabetic retinopathy comprising an integrin < RTI ID = 0.0 > av5 < / RTI > inhibitor and a method of screening for the inhibitor of integrin &

This is a therapeutic field for diabetic retinopathy.

Diabetic Retinopathy (DR) is the most common microvascular complication and is the main cause of vision impairment in diabetic patients and producers. Although new blood vessels cause severe visual impairment in late DR as DR progresses, macular edema due to vascular leakage can occur at any stage of the DR and may impair vision.

Accordingly, various techniques for treating diabetic retinopathy have been disclosed.

U.S. Published Patent Application No. 2015-0045296 discloses a therapeutic method for diabetic retinopathy using Apolipoprotein E (apoE), a lipid protein.

While astrocytes play an important role in regulating brain-vascular barrier in the brain, their role in vascular-retinal barrier is not well known. In the retina, astrocytes are involved in vascular ensheathment and are affected by diabetes (hyperglycemia). However, the role of astrocytes in relation to blood vessel leakage in the diabetic retina is unknown. Angiopoietin 2 (Ang2) induces peripheral vascular cell death through integrin signaling (Park SW et al., Angiopoietin 2 induces pericyte apoptosis via alpha 3beta 1 integrin signaling in diabetic retinopathy. Diabetes 2014; 63: 3057- 3068), it is known that local angiopoiesis occurs early in diabetic mice before cell apoptosis occurs in peripheral blood cells, and that Ang2 is upregulated in hyperglycemia and diabetic retinas in endothelial cells.

However, it is not known whether astrocytes are involved in the regulation of Ang2 / integrin signaling, and whether angiogenesis can be prevented in early diabetic retinopathy through the modulation of signaling, and a more effective therapeutic agent and treatment method based on the new mechanism Development is needed.

The present invention provides a therapeutic agent for diabetic retinopathy by controlling Ang2 / integrin signaling in astrocytes.

In one embodiment, the present invention provides a method comprising: providing angiopoietin-2 (Ang2) and an integrin? V? 5 protein in a cell; Contacting said protein with a test substance which is expected to inhibit the interaction of said angiopoietin-2 and integrin? V? 5 protein; And detecting the interaction of the angiopoietin-2 and integrin [alpha] v [beta] 5 protein, comprising contacting the angiopoietin-2 and integrin [alpha] v [beta] 5 protein with the test substance in contact with the test substance, Screening a substance for the treatment of diabetic retinopathy, comprising the step of screening the test substance as a candidate substance inhibiting the interaction between angiopoietin-2 and integrin? V? 5 protein when the interaction is reduced, And a method for screening a cell death inhibitory substance of an astrocyte cell of a retinal tissue.

The method according to the present invention may be carried out in a cell or extracellularly, and in one embodiment said integrin? V? 5 protein is provided in the form of a cell expressing said integrin? V? 5 protein.

Inhibition of the interaction between angiopoietin-2 (Ang2) and the integrin? V? 5 protein in the method according to the present invention can be measured by whether the GSK-3? /? - catenin pathway is inhibited in cells expressing? V? 5.

In another aspect, the present invention also provides a pharmaceutical composition for the prevention or treatment of diabetic retinopathy comprising a polypeptide that specifically recognizes integrin [alpha] v [beta] 5.

The polypeptide specifically recognizing the integrin? V? 5 according to the present invention includes an antibody, an antigen-binding fragment of an antibody, an antibody mimetic, an aptamer, or a receptor, and in one embodiment, an antibody or a fragment thereof.

In another aspect, the invention also provides a method of inhibiting astrocytic cell death, comprising the step of culturing astrocytes in the presence of a substance that inhibits the interaction of integrin alpha v [beta] 5 and Ang2, thereby inhibiting the GSK-3 [beta] .

In another embodiment, the present invention also provides a method of inhibiting GSK-3? /? -Catenin pathway in astrocytes, comprising culturing astrocytes in the presence of a substance that inhibits the interaction of integrin? V? 5 with Ang2, Lt; RTI ID = 0.0 > GSK-3/3-catenin < / RTI >

In another embodiment, the present invention also provides a method of screening for a substance that modulates Ang2-regulated GSK (Glycogen synthase kinase) -3 beta / beta -catenin pathway in a cell, said method comprising administering to said cell Ang2,? V? 5, GSK (Glycogen synthase kinase) -3? and? -catenin; Contacting said cell with a test substance; And the phosphorylation of the β-catenin is increased as a result of the treatment of the test substance as compared to the control group in which the test substance is not treated in the cell, GSK (Glycogen synthase kinase) (Glycogen synthase kinase) -3β / β-catenin pathway; -3-beta is reduced and the phosphorylation of beta -catenin is reduced as a result of the treatment of the test substance as compared with the control group in which the test substance is not treated in the cell, the test substance is treated with GSK (Glycogen synthase kinase) (Glycogen synthase kinase) -3? /? - catenin pathway.

The composition for preventing and treating diabetic retinopathy comprising integrin αvβ5 inhibitor according to the present invention is useful for prevention and treatment of diabetic retinopathy by inhibiting intracellular astrocytic cell death through intracellular GSK-3β activation by Ang2 Can be usefully used.

In addition, the screening method of a substance for treating diabetic retinopathy which inhibits the interaction between Ang2 and Integrin? V? 5 protein according to the present invention is characterized in that the substance screened by the above method is activated by GSK-3? Can be effectively used for the prevention and treatment of diabetic retinopathy.

Figure 1 shows retinal vascular leakage and retinal astrocytic cell loss in early diabetic mice. Retinal vascular leakage and astrocytic cell distribution were evaluated in streptozotocin-induced diabetic mice (DM) and in the same-age control group (Con) for 3 weeks. (a) Representative sites of central cell loss in the medial peripheral retina in the diabetic retina (initial magnification x 400; scale bar, 100 탆). White arrowheads indicate astrocyte loss in diabetic retinal vessels. (b) Relative astrocytic cell coverage (co-localized area of stellate cells and blood vessels / area of blood vessel) was shown and normalized on the basis of the control group. (c) Retinal vascular leakage was measured in the presence of FITC-dextran (70 kD) and relative retinal vascular leakage (%) was normalized on the basis of that of control mice. (c) Sample size per group is shown in the bar graph. The bar graph represents the mean ± SEM; * P <0.01 by Student's t-test.
FIG. 2 shows that inhibition of Ang2 in early diabetic retinas decreased astrocyte cell loss and vascular leakage. (ac) 1, 3, 5 and 7 weeks after diabetes was induced by streptozotocin in the control (Con) and diabetic mouse (DM) retinas, retinal mRNA was measured by qPCR and normalized on the basis of Rn18s mRNA. (a) Ang2 mRNA. (b) Ang1 mRNA. (c) Vegfa mRNA. (d? f) Anti-Ang2-neutralizing antibody (Anti-Ang2 Ab, 1 μg) or PBS was injected into 2-week-old DM. One week after injection, retinal astrocytic cells and retinal vascular leakage were measured in 3-week-old DM. (d) The central astrocytic cell loss seen in diabetic retinas (PBS) was restored in diabetic mice injected with anti-Ang2 Ab (initial magnification x 400; scale bar, 100 μm). White arrowheads indicate astrocyte loss in diabetic retinal vessels. (e) Relative astrocytic cell coverage (%; Anti-Ang2 Ab) in IB4 + co-localized area per vascular area was calculated and normalized on the basis of the control group. (f) Relative retinal vascular leakage (%; Anti-Ang2 Ab) in the presence of FITC-dextran was expressed as normalized on the basis of that of control mice. (g) Weight and blood glucose levels of STZ-induced diabetic and non-diabetic control mice at 7 weeks. The sample size of each group is shown in the bar graph. The bar graph represents the mean ± SEM; * P < 0.01 and *** P < 0.001 by ANOVA and Student's t-test FIG. 3 shows that Ang2 induces synergistic cell apoptosis synergistically in the presence of high glucose (HG; 25 mM glucose). The effect of Ang2 on cell viability and stellate cell apoptosis under HG conditions was measured. Stromal cells were cultured with Ang2 (300 ng / ml) under HG for 48 hours and compared with the control (Con). (a) Cell viability was evaluated by MTT assay. Ang2 exacerbates HG-induced cell death in astrocytes. (b) Western blot analysis of Bax and cleaved PARP was performed in the fusants obtained from astrocytes under the same experimental conditions. beta -tubulin was used as the loading control. (c) stromal cells were stained with Annexin-V FITC and PI and analyzed by flow cytometry. Cell death was expressed as a percentage of apoptotic cells in the total cell population. HG induces astrocytic cell death and Ang2 aggravates the apoptosis. The bar graph represents the mean ± SEM of three independent experiments. HG, high mannitol (5 mM glucose + 20 mM mannitol); NG, normal blood glucose (5 mM glucose). * P < 0.05 by Student's t-test.
Figure 4 shows that Ang2 induces astrocytic cell death via GSK-3 beta / beta -catenin pathway in high glucose conditions (HG; 25 mM glucose). Although astrocytes were incubated for 48 h under HG conditions, Ang2 (300 ng / ml) was treated for the time indicated. (Ser9) and phospho-β-catenin (Thr41 / Ser45) for the fusants obtained from the stellate cells treated with Ang2 for 15, 30 and 60 min under HG (48h) Western blot analysis was performed. (b) Western blot analysis for beta -catenin was performed on the fusants obtained from astrocytes treated with Ang2 for 24 and 48 hours under HG conditions for a total of 48 hours. (c) 48 hours after induction, SB216763 (GSK-3 beta inhibitor, 10 μM) was pretreated for 1 hour. Thereafter, Ang2 was treated for 30 minutes. Western blot analysis was performed on phospho-GSK-3? (Ser9) and phospho-beta-catenin (Thr41 / Ser45). (d and e) in the presence of Ang2 or SB216764 for 48 hours under HG conditions. (d) Western blot analysis on? -catenin was performed. (e) Cell death cell count was evaluated by FACS analysis. The bar graph represents the mean ± SEM of three independent experiments. * P &lt; 0.05 by Student's t-test. The data represent three independent experiments. beta -tubulin was used as the loading control.
Figure 5 shows that in astrocytes increased integrin [alpha] v [beta] 5 as an Ang2 receptor in high glucose conditions. (a) Western blot analysis for Tie-2 and Tie-1 expression was performed on fusants from HRMEC and astrocytic cells. (b) TIA2 and TIE1 mRNA transcripts were evaluated by quantitative RT-PCR. TIE2 and TIE1 mRNA levels were normalized to [beta] -actin mRNA and reported as fold induction compared to HRMEC. * P < 0.001 by Student's t-test. (c) Phospho-β-catenin (Thr41) was incubated for 48 hours at 25 mM high glucose (HG) and then for 30 minutes with Ang2 (300 ng / ml) or GRGDS (0.5 mg / / Ser45) and [beta] -catenin were performed. (d) Western blot analysis for integrin [alpha] v, [beta] 3, and [beta] 5 was performed on the fusants obtained from astrocytes under HG conditions for 48 hours. beta -tubulin was used as the loading control. Data represent three independent experiments. The ITGαv (e), ITGβ3 (f), and ITGβ5 (g) mRNA transcripts were evaluated and normalized to β-actin mRNA by quantitative RT-PCR. All mRNA levels were induced several fold compared to normal glucose (NG, 5 mM). * P &lt; 0.05 by Student's t-test. (h) After incubation of Ang2 (500 ng), integrin alpha v beta 5 (500 ng) and integrin alpha v beta 5 antibody (2 μg), the immune complexes co-immunoprecipitated and showed that Ang2 binds directly to integrin αvβ5. They were then analyzed for Ang2 and integrin alpha v. The same amount of recombinant Ang2 and integrin [alpha] 3 [beta] l was used as input. Con, control.
Figure 6 shows that Ang2-induced astrocytic cell apoptosis is inhibited by? V? 5 integrin inhibition. (a) treated with Ang2 (300 ng / ml) or various integrin-blocking antibodies (10 [mu] g / ml, alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v) for 30 min in 25 mM high glucose conditions Western blot analysis of phospho-β-catenin (Thr41 / Ser45) and β-catenin was performed on the fused products obtained from the cells. (b and c) Ang2 and anti-integrin [alpha] 3 or [alpha] v antibodies for 48 hours. (b) Western blot for [beta] -catenin was performed (ImageJ quantification, N = 3, mean ± SEM). (c), the astrocytic cell death in the presence of the integrin [alpha] 3 antibody was analyzed by FACS and expressed as a percentage of apoptotic cells in the total cell group. (d and e) In the HG conditions, astrocytes were cultured with Ang2 and anti-integrin [beta] l antibody (10 [mu] g / ml). (d) Western blot analysis on? -catenin was performed. beta -tubulin was used as the loading control. Data represent three independent experiments. (e) The astrocytic cell death in the presence of integrin beta1 antibody was analyzed by FACS and expressed as a percentage of apoptotic cells in total cell group. (f and g) Astrocytes were cultured for 48 hours with Ang2 and anti-integrin αvβ3 or αvβ5 antibodies (10 μg / ml) under HG conditions. (f) Western blot analysis on? -catenin was performed. beta -tubulin was used as the loading control. Data represent three independent experiments. (g) The expression of astrocytic cell death in the presence of anti-integrin αvβ5 antibody was analyzed by FACS and expressed as a percentage of apoptotic cells in the total cell group. # P> 0.05, * P <0.05 compared with Ang2 treatment by Student's t-test.
Figure 7 shows that inhibition of [alpha] v [beta] 5 integrin reduces astrocyte loss in STZ-induced diabetic mice (DM). Anti-αvβ5-integrin-neutralizing antibody (Anti-αvβ5 Ab, 1 μg) or PBS was injected into the 2-week-old DM into the vitreous body. Retinal astrocytic cells were evaluated in 3-week-old DM that was one week after injection. (a) The central astrocytic cell loss seen in diabetic retinas (PBS) was recovered in anti-αvβ5 Ab-injected DM (original magnification × 400; scale bar, 100 μm). (b) Relative astrocytic cell coverage (%) was normalized to the numerical basis of control mice, calculated as the IB4 + co-location area of blood vessels and astrocyte cells per vessel area. The sample size for each group is shown in the bar graph. The bar graph is expressed as mean ± SEM: * P <0.001 by Student's t-test.

In early diabetic retinas, retinal cell loss occurs with retinal vascular leakage. In the present invention, retinal astrocytic cell loss and angiopoiesis are associated with Angiopoetin 2 (Ang2) in hyperglycosylated conditions, Ang2 induces retinal astrocytic cell apoptosis, and Ang2-induced astrocytic cell apoptosis is induced by GSK- β-catenin pathway, and that inhibition of Ang2 reduces astrocyte cell loss and vascular leakage, and is based on the finding that integrin αvβ5 is elevated as a receptor for Ang2 in high glucose conditions.

In one embodiment, the present invention relates to a composition for the prevention and treatment of diabetic retinopathy comprising a polypeptide specifically recognizing anti-integrin [alpha] v [beta] 5.

The integrin αVβ5 to which the polypeptide according to the present invention binds belongs to the 'integrin' family, and the integrin is a transmembrane glycoprotein composed of a hetero-bident of α and β subunits, and is classified by a combination of various kinds of α and β . Integrin is present on the cell surface and functions when cells adhere to extracellular matrix such as fibronectin and collagen, and sometimes it is involved in cell interaction, immunity, and hemostasis. Integrin αVβ5 is a form of integrin composed of two components: integrin alpha V and integrin beta 5.

Herein, integrin [alpha] v [beta] 5 acts as a receptor for Ang2 by directly binding to Ang2 under high glucose conditions, and thus a polypeptide specifically recognizing integrin [alpha] v [beta] 5 according to the present invention inhibits its interaction with Ang2, , Inhibits GSK-3? Activation, and inhibits cell death.

The polypeptide according to the present invention is a polypeptide capable of specifically binding to integrin [alpha] v [beta] 5 expressed in a target cell, and may contain various types of polypeptides that inhibit the interaction with Ang2 and exhibit such effects. For example, But are not limited to, antibodies, antigen-binding fragments of antibodies, antibody mimetics, aptamers, or receptors.

In one embodiment according to the present invention, an antibody is used, wherein the antibody comprises a polyclonal, monoclonal antibody, or chimeric or humanized antibody, a full length antibody or fragment thereof.

In another embodiment according to the present invention, an antigen-binding fragment is used, and the antigen-binding fragment includes all or a part of the antigen-binding site in the whole-length antibody. For example, scFv (see below), BITE (See for example U.S. Patent No. 7,235,641), TandAb (see, for example, U.S. Patent Publication No. 2005-089519), Immunobody (see, for example, U.S. Patent Publication No. 2004-146505), Flexibody (See, e. G., U.S. Patent No. 6,254,454), Nanobody (see, for example, U.S. Patent Publication No. 2003-088074), Triomab (See, for example, U.S. Patent Publication No. 2004-101905), Vaccibody (see, for example, U.S. Patent Publication No. 2004-253238), SMIP (see, for example, U.S. Publication No. 2008-227958) MAb2 (see, for example, (See, for example, U.S. Patent Publication No. 2009-298195), UniBody (see, for example, U.S. Patent No. 7235641), Fv (Fragment variable), dAB (see for example US Patent Publication No. 2006-280734), scFV-Fc , Diabodies (see, for example, U.S. Patent No. 7235641), Tetrabody (see, for example, U.S. Patent No. 7,235,641), Minibody (see, for example, U.S. Patent No. 7235641), scFab See, for example, U.S. Published Patent Application No. 2009-298195).

In another embodiment according to the present invention, an antibody mimetic is used, wherein such antibody mimetic is selected from the group consisting of DARPin (see, for example, US Pat. No. 7417130), Tetranectin (see, for example, US Patent Publication No. 2004-132094), Affibody (See, for example, U.S. Pat. No. 5,250,297), AdNectin (see, for example, U.S. Patent No. 6,818,418), Transbody ), Affilin (see, for example, U.S. Patent No. 7838629), Microbody (see, for example, U.S. Patent No. 7186524), Peptide aptamer (see, for example, U.S. Patent No. 6004746), Phylomer 6994982), Stradobody (see, for example, U.S. Patent Publication No. 2010-239633), Avimer (see, for example, U.S. Patent No. 7,803,907), Evibody (see, for example, U.S. Patent No. 7,166,697), or Fynomer See, for example, U.S. Published Patent Application No. 2010-119 446). &Lt; / RTI &gt;

The term &quot; prophylactic, &quot; as used herein, refers to any act that inhibits or delays the onset of diabetic retinopathy by administration of the composition herein. It will be apparent to those skilled in the art that the present compositions of the present invention, which have therapeutic effects on diabetic retinopathy, can prevent the early symptoms of diabetic retinopathy, or such diseases when taken before they appear.

As used herein, the term &quot; treatment &quot; refers to any action that improves or alleviates the symptoms of diabetic retinopathy by administration of the composition herein. Those skilled in the art will be able to ascertain, by reference to the data provided by the Korean Medical Association, the precise criteria of the disease for which the composition of the present invention is effective, .

As used herein, the term 'diabetic retinopathy' refers to a disorder of peripheral circulation caused by diabetes, which is a systemic disease, including eye complications resulting from impaired retinal microcirculation and decreased visual acuity. In early diabetic retinopathy, Red spots, microvessels, and retinal hemorrhage.

In addition, the pharmaceutical compositions of the present invention can be used alone or in combination with methods using surgery, drug therapy and biological response modifiers.

The pharmaceutical composition of the present invention may further comprise at least one pharmaceutically or physiologically acceptable carrier in addition to the above-mentioned effective ingredients.

The term carrier as used herein means a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cell or mammal exposed to the dose and concentration employed. Examples of such carriers include buffers such as saline, Ringer's solution, buffered saline, phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, Or immunoglobulins; Other hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, For example, mannitol or sorbitol, salt forming counter ions such as sodium, and / or nonionic surfactants such as tweens, polyethylene glycol (PEG) and PLURONICS.

If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Can further be suitably formulated according to the respective disease or ingredient according to the appropriate method in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have.

Solid formulations for oral administration include tablets, patients, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate Sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are included in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and parenteral administration is particularly preferred. The dose varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and time, but can be appropriately selected by those skilled in the art.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically or therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type, severity, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.01 μg to 100 mg, preferably 0.01 μg to 10 mg per 1 kg of body weight is administered daily or every other day Or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In another aspect, the present invention provides a method of inhibiting the interaction of angiopoietin-2 (Ang2) and integrin alpha v beta 5 protein and screening such substance with an astrocyte inhibitor or a therapeutic agent for diabetic retinopathy.

In one embodiment, the method comprises providing angiopoietin-2 (Ang2) and integrin alpha v beta 5 protein; Contacting said protein with a test substance which is expected to inhibit the interaction of said angiopoietin-2 and integrin? V? 5 protein; And detecting the interaction of the angiopoietin-2 and integrin [alpha] v [beta] 5 protein, comprising contacting the angiopoietin-2 and integrin [alpha] v [beta] 5 protein with the test substance in contact with the test substance, When the interaction is reduced, the test substance is selected as a candidate substance inhibiting the interaction between angiopoietin-2 and integrin [alpha] v [beta] 5 protein.

The test substance used in the method of the present invention is a substance that is expected to inhibit the interaction of integrin alpha v [beta] 5 with Ang2. For example, for screening purpose of a drug, a compound having a therapeutic effect of a low molecular weight may be used. For example, compounds having a weight of about 1000 Da such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may constitute a part of a library of compounds, and the number of compounds constituting the library may vary from several tens to several millions. Such a library of compounds can be prepared by reacting peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular weight compounds based on a template, such as benzodiazepines, hydantoins, biaryls, carbocycles, and polycycle compounds such as naphthalene, Carbodihydrate and amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely exemplary It is not limited thereto. In one embodiment, it may be an anti-integrin [alpha] v [beta] 5 antibody.

The angiopoietin-2 (Ang2) and integrin [alpha] v [beta] 5 proteins that can be used in the screening method of the present application may be those described above or those described in the embodiments of the present application. According to a specific method of screening, -2 (Ang2) and integrin [alpha] v [beta] 5 protein and / or cells expressing it can be used in the present method. In one embodiment, animal cells, retinal cells, or astrocytes may be used, but the present invention is not limited thereto as long as the above effects can be measured.

Angiopoietin-2 (Ang2) belongs to the angiopoietin family and is known as a growth factor for blood vessels. Its sequence is known, and the protein sequence is known, for example, as the human protein sequence, NM_001147.

Various methods for qualitatively or quantitatively detecting a known protein may be used for the analysis of inhibition of the interaction of angiopoietin-2 (Ang2) and integrin? V? 5 protein according to the present method.

The Ang2 and integrin [alpha] v [beta] 5 proteins used herein may be prepared using methods known in the art. In particular, a screening protein is produced using a gene recombination technique. For example, a plasmid containing the corresponding gene encoding the protein may be transferred to prokaryotic or eukaryotic cells such as insect cells and mammalian cells for overexpression, followed by purification. The plasmid can be used, for example, by transfection into an animal cell line such as that used in the exemplary embodiments herein, followed by purification of the expressed protein, but is not limited thereto.

Alternatively, a DNA or RNA sequence encoding the screening protein is expressed in an appropriate host cell to produce a cell lysate thereof, or the mRNA of the screening protein is translated in vitro, followed by purification of the screening protein by a protein separation method known in the art . Usually, the cell lysate or the translation product in vitro is centrifuged to remove cell debris and the like, and then precipitation, dialysis, various column chromatography and the like are applied. Ion exchange chromatography, gel-permeation chromatography, HPLC, reversed phase-HPLC, SDS-PAGE for affinity column, and affinity column are examples of column chromatography. Affinity columns can be made, for example, using anti-screening protein antibodies.

In one embodiment, angiopoietin-2 (Ang2) and integrin [alpha] v [beta] 5 protein may be provided in the form of cells expressing it. In particular, the [alpha] v [beta] 5 protein can be provided in the form of a cell expressing it as a monoprotein present in the membrane. For example, mammalian cells, retinal cells or astrocytes can be used. After culturing the cells on a cell culture plate, the test substance is added to the cells, the total protein is extracted from the cells after a certain period of time, , It is possible to confirm whether HBx and APC interact with each other through the co-immunoprecipitation method and the immunofluorescence method. Compared with the control group (when the test substance is not treated), inhibition of protein interactions can be selected as candidates for diabetic retinopathy or astrocytic death inhibitor.

The contact of the purified protein can be directly confirmed in the absence or presence of the test substance by directly using them in vitro. In this case, the protein may be labeled using various known markers such as biotin, fluorescent material, acetylation, radioisotope or the like using a known protein labeling kit or a commercially available labeled substance Lt; RTI ID = 0.0 &gt; detectors &lt; / RTI &gt; Protein-protein interactions can be measured using a variety of methods known in the art. For example, the yeast two-hybrid method, the confocal microscope method, which confirms the binding / interaction of intracellular proteins. (SPR) and spectroscopy methods, and further references to comparative and detailed experimental methods for such methods are provided in Berggard et al., (2007) "Methods for protein-protein interactions &quot;, PROTEOMICS Vol 7: pp 2833-2842.

In another aspect, the inhibition measure of the interaction between angiopoietin-2 (Ang2) and the integrin? V? 5 protein in the method according to the invention is determined by whether the GSK-3? /? - catenin pathway is modulated in cells expressing said? V? And this method can be carried out with reference to what is described in the embodiments of the present application. The GSK-3 [beta] / [beta] -catenin pathway is GSK-3 [beta] dephosphorylated by the integrin [alpha] v [beta] 5 protein as described in this Example, and this dephosphorylated GSK-3 [beta] phosphorylates the beta -catenin to induce its degradation.

The amount of the integrin [alpha] v [beta] 5 protein used in the present method, the type of the cell, and the amount and kind of the test substance will vary depending on the specific experimental method used and the kind of the test substance. Those skilled in the art will be able to select an appropriate amount. As a result of the test, a substance which inhibits integrin [alpha] v [beta] 5 is selected as a candidate substance in the presence of the test substance as compared with a control substance not in contact with the test substance. About 95% more, about 90% more, about 85% more, about 80% more, about 75% more, more than 70% more, more than 65% more than the control , About 60% increase, about 55% increase, about 50% increase, about 45% increase, about 40% increase, about 30% increase, about 20% increase, It is not excluded.

As a method for screening such therapeutic agents, a number of known methods can be used, and those skilled in the art can select appropriate methods according to specific embodiments. Samples containing test substances for use in screening include, but are not limited to, tissue extracts, expression products of gene libraries, synthetic compounds, synthetic peptides, and natural compounds.

In another aspect, the invention provides a method for inhibiting astrocyte death comprising culturing astrocytes in the presence of a substance that inhibits the interaction of integrin [alpha] v [beta] 5 and Ang2, and inhibiting the GSK-3 [ . In one embodiment, the method is performed in Invitro, and the substances and cells that inhibit the interaction may refer to those mentioned above and those described in the Examples herein.

In another aspect, the invention also provides a method of inhibiting GSK-3? /? -Catenin pathway in astrocytes, comprising culturing astrocytes in the presence of a substance that inhibits the interaction of integrin? V? 5 with Ang2, Lt; RTI ID = 0.0 &gt; GSK-3/3-catenin &lt; / RTI &gt; pathway in a cell. In one embodiment, the method is performed in Invitro, and the substances and cells that inhibit the interaction may refer to those mentioned above and those described in the Examples herein.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Materials and Methods

Cell culture. Human retinal microvascular endothelial cells (HRMEC) (Applied Cell Biology Research Institute, Kirkland, WA, USA) and human astrocyte (Applied Cell Biology Research Institute) Were maintained in DMEM supplemented with 20% FBS (Hyclone, Logan, UT, USA). All cells were incubated in an incubator at 5% CO ₂ and 37 ° C.

Reagents and antibodies. Mouse Angl2 ELISA kit, mouse VEGF ELISA kit, human phospho-kinase assay kit, mouse anti-angiogenesis kit, recombinant human Ang2, human integrin alpha v beta 5, PE (phycoerythrin) -binding anti-endotypic receptor tyrosine kinase 2 (Tie- Anti-integrin αvβ3, and anti-integrin αvβ5 (clone P5H9, MAB2528) neutralizing antibodies were purchased from R & D systems (Minneapolis, MN, USA). Mouse Ang1 ELISA kit was purchased from MyBioSource (San Diego, CA, USA). Anti-Bax antibodies were purchased from Epitomics (Burlingame, CA, USA). Anti-phospho GSK-3? (Ser9), anti-phospho? -Catenin, anti-beta -catenin, and anti-PARP antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-Tie-1 and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-Tie-2 antibody, GRGDS peptide, and SB216763 were purchased from Millipore (Billerica, MA, USA). MTT ((3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolinium bromide), STZ and FITC-dextran (70 kD) were purchased from Sigma-Aldrich FITC-conjugated annexin-V / PI assay kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-GFP, anti-integrin αv, β1, β3, Antigen-Ang2 antibody was purchased from Abcam (Cambridge, Mass., USA). Anti-mouse Ang2-block antibody (Angy-2-1) was purchased from Adipogen (San Diego, CA, USA) α3 (clone P1B5, MAB1952), anti-integrin β1 (clone 6S6, MAB2253), and α-integrin-blocking and IHC kit (α1-6, v) were purchased from Millipore and used for functional blockocking.

Animal experiments. All animal experiments in this specification were conducted in accordance with the Animal Use Regulations for Ophthalmological Research and the guidelines of the Animal Care Use Committee of Seoul National University. Six-week-old aseptic male C57BL / 6J mice were obtained from Central Lab. Animal Inc. (Seoul, Korea). STZ was dissolved in a citrate buffer (pH 4.5) of 10 mM at a concentration of 180 mg / kg of body weight and was injected intraperitoneally to induce diabetes. In the control group of the same age, only the citrate buffer was injected. After 4 days of STZ injection, mice with a blood glucose level of 4300 mg / dl were treated with diabetes induction.

Diabetic and control mice were euthanized 3 weeks after diabetes induction. The mouse eyes were anesthetized and tissues used for ELISA were immediately frozen at -80 ° C. The tissues used for retinal flat mount And fixed in 4% paraformaldehyde. Blood sugar levels (Accu-Chek, Roche, Indianapolis, IN, USA) and body weight were monitored continuously.

ELISA. Two retinas of the mouse were collected (N = 6 per group). Samples containing the retina, vitreous, and retina were homogenized and briefly sonicated and fused in 500 [mu] l RIPA buffer with complete protease inhibitor cocktail (Roche). After centrifugation at 12,000 rpm for 20 minutes, the supernatant was collected. Angl, Ang2, and VEGF levels were measured using an ELISA kit according to the manufacturer's manual. The protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, Ill., USA). Ang1 (ng), Ang2 (pg), and VEGF levels (pg) were normalized to retinal protein (mg).

Quantitative and qualitative evaluation of retinal vascular leakage. Three-week-old STZ-induced diabetic mice were used to measure vascular leakage. Deeply anesthetized mice were intravenously injected with FITC-dextran dissolved in PBS. One hour after injection, eyes were removed and fixed in 4% paraformaldehyde for 1 hour. The retina was dissected in 2 x PBS, flat-mounted, and observed with a fluorescence microscope (Eclipse 90i, Nikon, Tokyo, Japan) at x40 and x100 magnification.

To quantitate vascular leakage, four representative leak sites in the mid-peripheral retina (0.5 μm x 0.5 μm) of each mouse were selected (N = 6-7). The mid-peripheral retina was defined as the middle third of the retina from the optic nerve head to the ciliary body. The images were sent to ImageJ (1.47v, NIH, Bethesda, MD, USA). The image was adjusted for the color threshold based on the automatic iso data algorithm, and the area of interest with FITC was displayed in red. The red area was then measured. The data was expressed as vessel leakage (%), which normalized on the basis of the control mice.

Quantification of retinal vascular coverage by astrocytes . To determine retinal astrocytic morphology and quantitative vascular coverage, stromal cells were immunostained for GFAP and retina flat mounts were contrasted with IB4 against blood vessels. The retina was observed with a confocal microscope (Leica TCS STED, Leica Microsystems, Wetzlar, Germany). Briefly, eyes were harvested and fixed with 4% formaldehyde for 1 hour. The retina was dissected in 2xPBS and incubated overnight in 100% methanol at -20 &lt; 0 &gt; C. The retina was then blocked with Perm / Block solution (0.3% Triton-X and 0.2% BSA in PBS) for 1 hour at room temperature. Thereafter, the cells were cultured in a Perm / Block solution overnight at 4 DEG C in the presence of an anti-GAPAP antibody (1: 100) and an Alexa Fluor 594-conjugated anti-IB4 antibody (1: 100). The retina was then repeatedly washed with PBS and incubated for 1 hour at room temperature in the presence of secondary antibody (1: 200). After washing with PBS, the retina was mounted with Fluoromount liquid mounting medium (Sigma-Aldrich). Representative sites where astrocytes disappeared in the mid-peripheral retina of each mouse were selected (N = 6-8). Stellate cell morphology was analyzed with a maximum projection image of z-stack (magnification x 400). To analyze blood vessel coverage by astrocytes, the astrocytes and the retinal vessels were divided into areas of retinal vessels (astrocytic coverage = joint site / vessel site). Co-location sites were calculated using Leica Application Suite Advanced Fluorescence (LAS AF) software (Leica Microsystems, threshold and background values of 30 and 20%, respectively). The image was sent to Imaris software (Ver.7.6.5, Bitplane, Belfast, UK) to analyze the blood vessel area. Thereafter, vascular coverage by astrocytes was normalized on the basis of the control group (relative stellate cell coverage%; Fig. 3).

Intravitreal injection of anti- Ang2 and anti- integrin -neutralizing antibodies . Two weeks after the induction of diabetes with STZ, one microliter containing 1 μg of anti-Ang 2-neutralizing antibody and 1 μg of anti-integrin αvβ5 antibody (clone P5H9) was injected into the vitreous under deep anesthesia. PBS was injected as a control. Seven days after injection, retinal flat mounts were performed on the retina for vascular leakage and / or astrocytic analysis.

Cell viability analysis. 2.5x10 &lt; ⁴ &gt; cells were seeded in 96-well plates in all experiments. After 24 hours, Ang2 (300 ng / ml) was treated with high glucose (5 mM glucose), high glucose (25 mM glucose) and high mannitol (5 mM glucose plus 20 mM mannitol) as an osmotic control for 48 hours. Cell viability was measured by MTT assay according to the manufacturer &apos; s manual. Three independent experiments were performed for each experimental condition.

FACS analysis. To assess apoptosis, ⁵ x 10 ⁵ cells in normal glucose, high mannitol, and high glucose conditions were treated with Ang2 (300 ng / ml) at 37 ° C for 48 hours. To determine the effect of integrin blocking, anti-integrin-blocking antibody (10 [mu] g / ml) was treated for 1 hour before adding Ang2. The cells were collected and washed twice with PBS. Cells were stained with FITC Annexin-V and PI for 15 minutes, and the cells were analyzed by flow cytometry. Annexin V-positive / PI-negative cells were determined to be apoptotic.

Quantitative RT- PCR . Total RNA was collected from the cells using RNeasy Plus Mini kit (Qiagen, Valencia, CA, USA). CDNA was prepared using 2.5 [mu] M oligo-dT primer, 1 mM dNTPs, and MuLV reverse transcriptase. Quantitative real-time PCR (qPCR) was performed on a qPCR master mix for SYBR Green PCR master mix (Applied Biosystems, Life Technologies, Gaithersburg, MD, USA) using 7900HT real-time PCR (Applied Biosystems). The qPCR conditions were set as follows: (i) 95 ° C for 10 min and (ii) 50 cycles of 5 s at 95 ° C and 20 s at 60 ° C. The average amount was calculated by qPCR three times per sample and normalized on the basis of the control gene.

Total RNA was isolated from the retina using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's manual. CDNA was prepared with High Capacity RNA-to-cDNA kit (Life Technologies). Real-time PCR was performed using a StepOnePlus Real-Time PCR System (Life Technologies) with TaqMan Fast Advanced Master Mix (Life Technologies) and specific Gene Expression Assays (Cat. No. 4331182; Life Technologies). The product ID of the gene expression analysis is as follows: Angpt1: Mm00456503_m1; Angpt2: Mm00545822_m1; Vegfa: Mm00437306_m1; And Rn18s: Mm03928990_g1. All procedures were performed according to the guidelines of MIQE (Bustin et al., Clin Chem. 2009 Apr; 55 (4): 611-22), Minimum Information for Publication of Quantitative Real-Time PCE Experiments.

Immunoprecipitation and immunoblotting . For immunoprecipitation, Ang2 (500 ng) and integrin [alpha] v [beta] 5 (500 ng) were preclear with G-Sepharose beads (Millipore) at 4 DEG C for 1 hour. The complex and anti-integrin [alpha] v [beta] 5 antibody (2 [mu] g) were then incubated overnight at 4 [deg.] C in the presence of G-Sepharose beads. The immunocomplex was collected by centrifugation and washed three times with buffer. For immunoblotting, cells were pooled and lysed in RIPA buffer with protease inhibitor cocktail. The protein fructose was analyzed by SDS polyacrylamide gel and transferred to nitrocellulose membrane. The membrane was incubated with primary antibody (1: 1000) overnight at 4 ° C and incubated with secondary antibody for 1 hour at room temperature. The membrane was incubated with an enhanced chemiluminescent substrate (Pierce) and exposed to film or LAS 4000 (GE Healthcare, Piscataway, NJ, USA).

Statistical analysis. Statistical analysis was performed using standard two-tailed Student's t-test for the two groups, and post hoc tests with one-way ANOVA and Bonferroni's multiple comparisons were performed for multiple groups (SPSS Statistics 20.0, IBM , Armonk, NY, USA). * P &lt; 0.05 is considered to be statistically significant. Quantitative data and figures are presented as means ± SEM.

Example  1. Retinal blood vessels in early diabetic mice Leakage  Together with the retina Astrocytes  Confirm loss occurrence

To determine whether retinal astrocytic cell coverage versus retinal blood vessels is attenuated in the diabetic mouse retina, immunohistochemistry was performed on retinal flat mounts of diabetic mice. Glial fibrillary acidic protein (GFAP) -labeled astrocytes were evaluated in STZ-induced 3-week old diabetic mice and non-diabetic control mice of the same age. Interestingly, it was observed that the center of astrocytic coverage for retinal vessels was lost in the diabetic retina (Fig. 1a). Relative astrocyte cell coverage (42.0 ± 6.4%) to retinal vessels was significantly reduced (100.0 ± 5.4%, P <0.001; Fig. 1b) in the diabetic retina compared to that of the control retina. Retinal vascular leakage was also assessed in the presence of FITC (fluorescein isothiocyanate) -dextran. Retinal vascular leakage was significantly increased in diabetic retinas (196.5 ± 7.1%) compared with control (100.0 ± 16.0%) (P <0.001; Fig. 1c). These results indicate that retinal astrocytic cell loss is accompanied by an increase in retinal vascularity in early diabetic mice.

Example  2. Early diabetic retinas Of Ang2  Due to inhibition Astrocytes  Confirmed loss and reduced blood vessel leakage

ELISA was performed on retinal vascular endothelial growth factor (VEGF) and Ang1 / 2 to determine if growth factors were associated with increased angioreactivity and astrocytic cell loss in early diabetic retinas. At 1 week and 3 weeks of diabetic induction, retinal Ang2 levels were increased (from 88.9 ± 1.3 pg / mg to 98.11 ± 2.2 and 102.7 ± 4.6 pg / mg, respectively, P = 0.018 by one-way ANOVA). In the post-test, Ang2 at 3 weeks increased significantly compared to the control (P = 0.017). However, the level of retinal VEGF and Ang1 did not change significantly at 3 weeks after diabetes induction. In qPCR analysis of diabetic retinas (7 weeks after diabetic induction), Ang2 and Ang1 mRNA significantly increased after 3 weeks (Fig. 2a and b). However, Vegfa mRNA significantly increased after 5 weeks (Fig. 2C). The physical metabolism parameters for mice used in ELISA and qPCR are shown in Figure 2g. Ang2-block antibody was injected into diabetic mice at 2 weeks after STZ injection in order to investigate whether the increase in blood vessel leakage was due to Ang2-induced astrocytic cell loss. In contrast to astrocyte coverage loss (100.0 ± 15.2%, P <0.05; FIGS. 2d and e) in PBS-injected diabetic mice as control, astrocytic coverage in the retina of the anti-Ang2- ± 49.8%), respectively. In addition, anti-Ang 2 -blocking Ang2 attenuated vascular leakage (72.4 ± 6.3%) compared to vascular leakage in PBS-injected diabetic retinas (100.0 ± 5.3%, P <0.05; These results indicate that Ang2-induced astrocytic cell loss is associated with retinal vascular leakage, which results in retinal vascular leakage in STZ-diabetic mice.

Example  3. In high glucose On Ang2  by Astrocytes  Confirmation of induction of apoptosis

(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolinium bromide} assay and FACS (fluorescence in situ hybridization) assay to examine the effect of Ang2 on the cell viability of astrocytes in vitro (fluorescence-activated cell sorting) analysis. In high glucose, Ang2 led to pericyte death. Similarly, this enhanced astrocyte apoptosis in high glucose (76.8 ± 2.2% from 85.5 ± 3.3%, P <0.05; FIG. 3a). Next, we examined whether astrocyte apoptosis is mediated by apoptosis pathway. As a result, Ang2 induces apoptosis pathway with Bax increase in high glucose and cleaves poly (ADP-ribose) polymerase (PARP) (Fig. 3b). Thereafter, astrocytic cell death was evaluated by annexin-V / propidium iodide (PI) FACS analysis. Ang2 increased apoptotic cell populations regardless of glucose concentration. Importantly, high glucose-induced astrocytic cell death was significantly enhanced by Ang2 (32.6 ± 4.4% from 18.0 ± 2.6%, P <0.05; FIG. 3c). These data indicate that Ang2 induces astrocytic cell death, especially in high glucose conditions.

Example  4. High glucose  In condition On Ang2  Induction mechanism of apoptosis induced by: GSK - 3 β / β-catenin pathway

We investigated the mechanism by which Ang2 mediates astrocytic cell death in high glucose. The astrocytes were cultured in high glucose conditions for 48 h and treated with Ang2 (300 ng / ml) for a period of time. Ang2 induces GSK-3? Activation (Fig. 4a) by dephosphorylating GSK-3? (Ser9) and phosphorylating? -Catenin, which leads to? -Catenin degradation under high glucose conditions (Fig. 4b) Respectively. The astrocytes were then treated with the GSK-3? Inhibitor (SB216763, 10 μM, 1 h pretreatment) to determine the role of the GSK-3? /? -Catenin pathway to Ang2-mediated astrocytic cell apoptosis. As a result, GSK-3? Phosphorylated? -Catenin by induction of Ang2 at 30 minutes (Fig. 4c) and decreased? -Catenin degradation (Fig. In other words, inhibition of GSK-3? Effectively weakened Angk2 induction of astrocytic cell death under high glucose (13.4 ± 0.8% from 27.8 ± 3.2%, P <0.05; FIG. 4e). These results indicate that Ang2 induces astrocytic cell death through GSK-3β and its β-catenin pathway in high glucose conditions.

Example  5. In high glucose  due to In astrocytes Ang2  Receptor Integrin αvβ 5 increase

And to identify which receptors mediate Ang2-induced cell death in astrocytes. In order to investigate whether the well known Ang2 receptor, Tie-2 receptor, is associated with Ang2-induced astrocytic cell apoptosis, Tie-2 and Tie-1 were assayed for HRMEC (human retinal microvascular endothelial cell) Western blot analysis (Figure 5a) and real time PCR (Figure 5b) were performed. As shown in previous studies (Park SW, et al., Diabetes 2014; 63; 3057-3068; Lee J, et al., Sci Transl Med 2013; -1 but HRMEC expressed Tie-2 and Tie-1. Compared with HRMEC expressing Tie-2, astrocytes did not express Tie-2 in FACS analysis (data not shown). To investigate whether integrin acts as a receptor for Ang2 in astrocytic cell death in hyperglycosylated conditions, it was tested with Ang2 and H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) peptides at high glucose conditions The astrocytes were cultured. GRGDS (0.5 mg / ml) attenuated Ang2-induced? -Catenin phosphorylation (Fig. 5c). These results indicate that integrin signaling is involved in Ang2-induced? -Catenin phosphorylation.

Next, quantitative RT-PCR and Western blot studies were performed on αv, β3, and β5, which are known to be expressed in astrocytes, in order to examine whether high glucose changes the expression pattern of integrin. The astrocytes mainly expressed? V and? 5 integrin, and interestingly, hyperglycosylation increased? 5-integrin expression (Fig. 5d). Although high glucose did not increase ITGαv (FIG. 5e) or ITGβ3 (FIG. 5f) mRNA levels for 48 hours, it significantly increased ITGβ5 mRNA levels (P = 0.002 by one-way ANOVA, 24 h: 1.24 ± 0.04-fold ; P <0.05 and 48 h: 1.41 + 0.00-fold, P = 0.002 by post hoc test, respectively;

On the other hand, high glucose decreased ITGβ8 mRNA (0.72 ± 0.01-fold for 48 h, P <0.01). We also wanted to clarify that Ang2 binds directly to integrin αvβ5 since αv is an exclusive partner for β5. As a result, in the co-immunoprecipitation assay, Ang2 directly binds to integrin [alpha] v [beta] 5 (Fig. 5h). These results indicate that integrin αvβ5 as a receptor for Ang2 in astrocytes is increased by high glucose conditions.

Example  6. αvβ5 Integrin  By inhibition On Ang2  Induced by Astrocytes  Inhibition of cell death

To determine whether Ang2-induced astrocytic cell death was caused by the integrin subunit, the integrin alpha subunits (alpha 1-6 and alpha v) were screened. Integrin a3- and av-blocking antibodies attenuated Ang2-induced beta -catenin phosphorylation (Fig. 6A). To further investigate the integrin complexes, integrin [alpha] 3-, [alpha] v-, and [beta] l- blocking antibodies were tested. Only integrin [alpha] v-blocker antibody significantly inhibited Ang2-induced [beta] -catenin degradation (Figures 6b and d). In fact, integrin [alpha] 3- and [beta] l- blocking antibodies did not affect Ang2-induced cell death (Fig. 6c and e). Thus narrowing it to? V and its corresponding subunits? 3 and? 5. The same experimental procedure was also performed for the [alpha] v [beta] 3- and [alpha] v [beta] 5-integrin-blocking antibodies and Ang2-induced beta-catenin degradation was inhibited only when blocking the [alpha] v [beta] 5 integrin (Fig. Inhibition of [alpha] v [beta] 5 integrin effectively reduced angi-induced astrocytic apoptosis (19.3 ± 1.4% from 25.2 ± 1.2%, P <0.05; These results indicate that inhibition of [alpha] v [beta] 5 integrin can reduce astrocytic cell apoptosis.

Example  7. αvβ5 Integrin's  Inhibition of diabetic retinopathy Astrocytes  Loss reduction effect

Based on the in vitro experiment, 1 μg of anti-αvβ5-integrin antibody was injected into the vitreous of diabetic mice at 2 weeks after STZ induction. One week after injection into the vitreous body, the retina was separated and stained with GFAP to measure astrocytes (Fig. 7A). In vivo, when the anti-αvβ5-integrin antibody was injected into the vitreous body as compared with the astrocytic loss (53.1 ± 8.6%, P = 0.003; FIG. 7b) in diabetic mice injected with PBS as a control group, Cell loss was effectively reduced (128.3 ± 16.4%). These in vivo data show that astrocyte loss occurs via the αvβ5 integrin in the diabetic retina.

Taken together, it has been demonstrated that Ang2 induces astrocytic cell death through αvβ5 integrin in diabetic retinas and that GSK-3β-specific inhibitors prevent said apoptosis. In other words. It has been disclosed herein that inhibiting GSK-3? Is a therapeutic target of diabetic retinopathy, in particular, integrin? V? 5 is a therapeutic target of Ang2-induced astrocytic cell death. That is, inhibition of Ang2 / integrin signaling can prevent stellate cell loss and vascular leakage in diabetic retinopathy.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (10)

Providing angiopoietin-2 (Ang2) and integrin [alpha] v [beta] 5 protein;
Contacting said protein with a test substance which is expected to inhibit the interaction of said angiopoietin-2 and integrin? V? 5 protein; And
2 and the integrin [alpha] v [beta] 5 protein, wherein the test substance is contacted with the test substance in comparison with a control group not in contact with the test substance, and the interaction of the angiopoietin-2 and integrin [ Screening a substance for treating diabetic retinopathy, wherein the test substance is selected as a candidate substance inhibiting the interaction between angiopoietin-2 and integrin? V? 5 protein when the action is reduced.
2. The method of claim 1, wherein the integrin [alpha] v [beta] 5 protein is provided in the form of a cell expressing it.
Providing angiopoietin-2 (Ang2) and integrin [alpha] v [beta] 5 protein;
Contacting said protein with a test substance which is expected to inhibit the interaction of said angiopoietin-2 and integrin? V? 5 protein; And
2 and the integrin [alpha] v [beta] 5 protein, wherein the test substance is contacted with the test substance in comparison with a control group not in contact with the test substance, and the interaction of the angiopoietin-2 and integrin [ Wherein the test substance is selected as a candidate substance that inhibits the interaction between angiopoietin-2 and integrin &lt; RTI ID = 0.0 &gt; avb5 &lt; / RTI &gt; protein when the action is reduced, Lt; / RTI &gt;
4. The method according to claim 3, wherein the integrin [alpha] v [beta] 5 protein is provided in the form of a cell expressing it.
5. The method according to any one of claims 2 to 4,
Inhibition of the interaction between angiopoietin-2 (Ang2) and integrin? V? 5 protein is measured by whether the GSK (Glycogen synthase kinase) -3? -? - catenin pathway is inhibited in cells expressing? V? 5 , Way.
A pharmaceutical composition for preventing or treating diabetic retinopathy comprising a polypeptide specifically recognizing integrin? V? 5.
The pharmaceutical composition for preventing or treating diabetic retinopathy according to claim 6, wherein the polypeptide specifically recognizing the integrin? V? 5 is an antibody, an antigen-binding fragment of an antibody, an antibody mimetic, an aptamer, or a receptor.
A method for inhibiting astrocyte death comprising culturing astrocytes in the presence of a substance that inhibits the interaction of integrin alpha v [beta] 5 and Ang2, thereby inhibiting the GSK-3 [beta] -catenin pathway in the cell.
3β-β-catenin pathway in astrocytic astrocytes, comprising culturing astrocytes in the presence of a substance that inhibits the interaction of integrin αvβ5 and Ang2, and inhibiting the GSK-3β-β- - a method for inhibiting the catenin pathway.
A method of screening for a substance that modulates Ang2-regulated GSK (Glycogen synthase kinase) -3 beta / beta -catenin pathway in a cell,
Expressing Ang2,? V? 5, GSK (Glycogen synthase kinase) -3? And? -Catenin in the cell;
Contacting said cell with a test substance; And
When GSK (Glycogen synthase kinase) -3β dephosphorylation is increased and the β-catenin phosphorylation is increased as a result of the treatment of the test substance as compared with the control group in which the test substance is not treated in the cell, Glycogen synthase kinase) -3β / β-catenin pathway; -3-beta is reduced and the phosphorylation of beta -catenin is reduced as a result of the treatment of the test substance as compared with the control group in which the test substance is not treated in the cell, the test substance is treated with GSK (Glycogen synthase kinase) Lt; RTI ID = 0.0 &gt; (Glycogen &lt; / RTI &gt; synthase kinase) -3 beta / beta -catenin pathway.

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