KR20110117982A - Compositions comprising nfat5 inhibitor as an active ingredient for preventing or treating of angiogenesis-related diseases - Google Patents

Abstract

The present invention relates to a composition for preventing or treating angiogenesis-related diseases containing an NFAT5 inhibitor as an active ingredient, and more specifically, an antisense oligonucleotide and siRNA (small interference RNA) that complementarily bind to the nucleotide sequence of the NFAT5 gene. Composition for inhibiting angiogenesis comprising an antibody specific for an oligonucleotide or NFAT5 protein as an active ingredient, an agent for treating angiogenesis-related diseases comprising the composition, a method for screening for preventing or treating angiogenesis-related diseases, and the present invention It relates to a method for inhibiting angiogenesis using the composition of. Inhibitors that inhibit the expression or activity of NFAT5 according to the present invention inhibit the expression and activity of the NFAT5 gene or protein overexpressed in cells of diseases associated with angiogenesis, thereby proliferating, migrating and forming tube and endothelial cells that induce angiogenesis. It has the effect of inhibiting the production of cytokines, and has the effect of reducing the symptoms of diseases related to angiogenesis, and thus can be used as a pharmaceutical agent that can prevent or treat diseases caused by excessive angiogenesis. .

Description

Compositions comprising NFAT5 inhibitor as an active ingredient for preventing or treating of angiogenesis-related diseases}

The present invention relates to a composition for preventing or treating angiogenesis-related diseases containing an NFAT5 inhibitor as an active ingredient, and more specifically, angiogenesis containing an inhibitor that inhibits the expression of NFAT5 gene or NFAT5 protein as an active ingredient. A composition for inhibition, a therapeutic agent for angiogenesis-related diseases comprising the composition, a method for screening the therapeutic agent, and a method for inhibiting angiogenesis.

Angiogenesis is the process by which new blood vessels are formed from existing blood vessels and plays an important role in normal human defense and physiological phenomena and early development, such as wound healing and inflammatory reactions. The angiogenesis is a step in which new capillaries are generated by the reconstitution of blood vessels by the breakdown of the basal membrane by proteolytic enzymes, the movement of vascular endothelial cells forming the vascular wall, the proliferation, and the formation of tubes (tubes) by vascular endothelial cell differentiation. It takes place through a series of sequential steps, including.

Also the process of blood vessel is new is there strictly is regulated by a variety of negative and positive regulatory factor (Folkman and Cotran., Int. Rev. Exp. Patho., 16, 207 ~ 248, 1976), these angiogenesis is normally controlled Failure to do so can lead to a number of conditions, including cancer, rheumatoid arthritis, and diabetic retinopathy. In particular, abnormal angiogenesis is known to play a very important role in tumor growth and metastasis, firstly to provide nutrition and oxygen for tumor growth and proliferation, and secondly to neonatal capillary Blood vessels provide an opportunity for metastatic cancer cells to enter the blood circulation, allowing cancer cells to spread and spread throughout the body.

Therefore, the study of the mechanism of angiogenesis and the development of a material that can suppress it has been the focus of attention in the prevention and treatment of various diseases including cancer, and recently in animal cancer models and human clinical trials Investigation of the development of angiogenesis inhibitors is being actively conducted as it has been shown that inhibition of tumor angiogenesis can effectively inhibit tumor growth and development and prolong patient life. In addition, such angiogenesis inhibitors are particularly popular in anticancer therapies. For this reason, angiogenesis inhibitors have the potential to be commonly used in all solid tumors. Because of its rapid growth characteristics, it has a toxic effect on bone marrow cells and gastrointestinal cells that have a relatively rapid cell cycle, whereas angiogenesis inhibitors have relatively low side effects even after long-term administration. Since cells supply nutrients and oxygen to hundreds of cancer cells, they can suppress many cancer cells by suppressing one blood cell. Angiogenesis inhibitors act in direct contact with vascular endothelial cells As is because it has the advantage that the drug delivery can be facilitated.

On the other hand, about 200 types of angiogenesis inhibitors that have been developed so far have been reported. Such angiogenesis inhibitors include a mechanism for reducing the activity of certain angiogenesis-promoting factors, a mechanism for inhibiting the growth or death of vascular endothelial cells, There are four broad categories of mechanisms that inhibit the action of angiogenesis and indirect factors that control endothelial cell survival factors and increase the activity of angiogenesis inhibitors present in the body. Angiogenesis inhibitors such as angiostatin, endostatin, PK5, prothrombin kringle 2 are well known (O'Relly, MS et al. Cell ., 79, 315-328, 1994; Lee. TH, Biol . Che ., 273, 28805-28812, 1998).

However, most of the angiogenesis inhibitors developed to date are in the form of compounds, which are difficult to maintain good activity for a long time, are difficult to mass-produce, are expensive in the manufacturing process, and have low pharmaceutical properties and are easily There is a problem that can be denatured.

Therefore, in order to overcome these problems, researches have recently been conducted to develop an inhibitor of a nucleic acid form as an inhibitor of angiogenesis. The nucleic acid has a molecular characteristic of having higher bioavailability and affinity than a compound. There is an advantage that can be used as a better angiogenesis inhibitor.

However, there is still a lack of technology for an inhibitor capable of inhibiting angiogenesis by using a target gene having excellent angiogenesis inhibitory effect and a nucleic acid complementary to these genes.

Accordingly, it is an object of the present invention to provide a composition for inhibiting angiogenesis comprising an inhibitor of NFAT5 expression or activity as an active ingredient that can effectively prevent or treat diseases caused by excessive angiogenesis.

In addition, another object of the present invention to provide an agent for treating angiogenesis-related diseases comprising the composition.

Another object of the present invention is to provide a method for screening a substance for preventing or treating angiogenesis-related diseases.

Furthermore, another object of the present invention is to provide a method for inhibiting excessive angiogenesis comprising administering the composition according to the present invention to a mammal except for a person whose abnormal angiogenesis persists.

In order to achieve the object of the present invention as described above, the present invention is selected from the group consisting of an antisense oligonucleotide, siRNA (small interference RNA) oligonucleotide and NFAT5 protein specific antibody complementary to the base sequence of the NFAT5 gene. It provides a composition for inhibiting angiogenesis comprising an NFAT5 expression or activity inhibitor as an active ingredient.

In one embodiment of the present invention, the gene of NFAT5 may have a nucleotide sequence represented by SEQ ID NO: 1.

In one embodiment of the invention, the siRNA specific to the NFAT5 gene may have a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 to 7 and SEQ ID NO: 12 to 17.

The present invention also provides a therapeutic agent for angiogenesis-related diseases comprising the composition according to the present invention.

In one embodiment of the present invention, the disease is rheumatoid arthritis, osteoarthritis, sepsis arthritis, psoriasis, corneal ulcer, macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ophthalmic inflammation, cone cornea , Sjogren's syndrome, myopia ophthalmic tumor, corneal graft rejection, abnormal wound union, bone disease, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, myelin deficiency disease, cirrhosis, renal glomerular disease, inflammatory bowel disease It may be selected from the group consisting of periodontal disease, arteriosclerosis, restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging and cancer.

In addition, the present invention,

Contacting a candidate to be analyzed with a biological sample comprising the NFAT5 gene or NFAT5 protein;

Measuring the expression level of the NFAT5 gene, the amount of NFAT5 protein or the activity of NFAT5 protein; And

When the amount of expression of NFAT5 gene, the amount of NFAT5 protein or the activity of NFAT5 protein is reduced in comparison with the control group which did not contact the candidates, the sample is a substance capable of preventing or treating a disease caused by excessive angiogenesis. It provides a method for screening a material for preventing or treating angiogenesis-related diseases, comprising the step of determining.

In one embodiment of the invention, the measurement is reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, Western blot, Northern blot, ELISA (enzyme linked) immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assay.

Furthermore, the present invention provides a method for inhibiting excessive angiogenesis, comprising administering the composition according to the present invention to a mammal other than a person whose abnormal angiogenesis persists.

Inhibitors that inhibit the expression or activity of NFAT5 according to the present invention inhibit the expression and activity of the NFAT5 gene or protein overexpressed in disease cells associated with angiogenesis, thereby proliferating, migrating, and forming tube and endothelial cells that induce angiogenesis. Since there is an effect of inhibiting the production of caine, and reduces the symptoms of diseases associated with angiogenesis, there is an effect that can be used as a pharmaceutical agent that can prevent or treat diseases that can be caused by excessive angiogenesis.

1 is a photograph showing the expression level of the NFAT5 expressed in the cells of the synovial cells of rheumatoid arthritis and osteoarthritis of the angiogenesis-related diseases through immunochemical staining, Figures 1a to 1e is the synovial membrane of rheumatoid arthritis 1F to 1H are observed in synovial cells of osteoarthritis, and FIG. 1I is a diagram illustrating the expression of NFAT5 over time after inducing osmotic stress by treating 100 mM NaCl in synovial cells. 1J and 1K show that the expression level of NFAT5 and the movement in the cells according to the cytokine treatment were confirmed.
Figure 2 shows the results of examining the effect on the symptoms of angiogenesis-related diseases when inhibiting the expression of NFAT5, Figure 2a is a collagen in the control mice and the mice lacking the expression of the NFAT5 gene NFAT5 gene expression After inducing arthritis as a graph showing the degree of pain over time, Figure 2b is a graph showing the measure of the swelling of the mouse foot, Figure 2c is a portion of the joint tissue of the mouse hematoxylene and eosin It is a photograph observing the degree of inflammation, the proliferation of synovial cells and the degree of joint destruction by staining with, and FIG. One average value is plotted.
Figure 3 shows the results of using NFAT5 siRNA to investigate the effect of NFAT5 on the survival and proliferation of synovial cells, Figure 3a is a CCK cell survival rate and morphology of the scrambled siRNA (control) and the introduction of NFAT5 siRNA CCK -8 shows the results confirmed by microscopic observation, 3b shows the result confirmed by the TUNEL analysis of the degree of apoptosis, 3c is treated with scrambled siRNA (control) and NFAT5 siRNA to the cells and induces cell proliferation, respectively After treatment with cytokines TNF-a and TGF-β, respectively, a graph showing the degree of cell proliferation, 3d is a graph showing the change of cell number by NFAT5 siRNA, 3e is a cell growth factor by NFAT5 siRNA The change in expression of incyclin D and E was confirmed by Western blot.
Figure 4 shows the results of the changes in the proliferation and migration of vascular endothelial cells according to the inhibition of NFAT5 expression, Figure 4a after treatment with NFAT5 siRNA in vascular endothelial cells, the cell survival rate over time analyzed by MTT assay One result. Figure 4b shows the result of examining the effect of the endothelial growth factor VEGF on the degree of proliferation of vascular endothelial cells treated with NFAT5 siRNA, Figure 4c shows the degree of inhibition of tube formation by endothelial cells according to NFAT5 siRNA under a microscope 4D and 4E show that NFAT5 siRNA inhibits the migration and chemotaxis of vascular endothelial cells.
FIG. 5 shows the results of investigating the effects of NFAT5 on macrophage migration and cytokine production. FIG. 5A shows 3% thioglycol in control mice expressing NFAT5 gene and mice lacking expression of NFAT5 gene. After intraperitoneal injection of rate, macrophages isolated from the mice were treated with LPS and IL-1β, and then the degree of migration of macrophages was shown in a graph. FIG. 5B shows control mice and NFAT5 genes expressing NFAT5 gene The graph shows the result of confirming the expression level of TNF-a in macrophages of mice lacking expression by flow cytometry. FIGS. 5C and 5D show control mice expressing NFAT5 gene and mice lacking expression of NFAT5 gene. After stimulating macrophages of LPS or IL-1β, the amount of TNF-a and IL-12 produced was measured by ELISA. Shows.

The present invention relates to an inhibitor of NFAT5 having an activity of inhibiting angiogenesis, and more specifically, to an antisense oligonucleotide, siRNA (small interference RNA) oligonucleotide, and NFAT5 protein, which complementarily bind to the nucleotide sequence of the NFAT5 gene. It is characterized by providing a composition for inhibiting angiogenesis comprising an NFAT5 expression or activity inhibitor selected from the group consisting of antibodies as an active ingredient.

In the present invention, the focus of the NFAT5 gene as a target of a novel inhibitor that inhibits angiogenesis, the transcription factor NFAT5 (nuclear factor of activated T cells 5) is a type of NFAT family, NFAT protein is IL-2 promoter It has been shown to act to promote transcription by binding to, and five types have been identified to date. In other words, the NFAT family consists of NFAT1 (NFATp or NFATc2), NFAT2 (NFATc or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx or NFATc3), and NFAT5. It is known to modulate the immune response. In addition, NFAT protein in T cells is involved in the development and activity of thymocytes, differentiation of T cells, self-tolerance, etc., and by the interaction of NFAT family calcium signal with other signals NFAT5 was initially identified as a tonity enhancer binding protein (TonEBP) in the NFAT family, and binds to tonicity response elements present in the upregulatory region of genes induced by osmotic stress. Recently, NFAT5 plays an important role in the proliferation and survival of lymphocytes, and CYR61-dependent pathways have been reported to mediate myoblast migration during muscle differentiation.

On the other hand, in the present invention, in the case of a disease caused by excessive angiogenesis, it was confirmed that the NFAT5 gene is overexpressed in the cells of the disease, and furthermore, when the expression of the NFAT5 gene was suppressed, the severity of the disease was confirmed to be reduced. Therefore, it was first identified that an inhibitor of NFAT5 can be used as a therapeutic agent for preventing and treating angiogenesis-related diseases.

That is, according to one embodiment of the present invention, NFAT5 protein expressed in the cells of the synovial cells obtained from patients with rheumatoid arthritis and osteoarthritis, which are diseases caused by excessive angiogenesis, through immunochemical staining, It was confirmed that NFAT5 was overexpressed in synovial cells of rheumatoid arthritis and osteoarthritis patients compared to normal synovial cells (see Example 1).

Therefore, the present inventors found that NFAT5 is overexpressed when angiogenesis is not normally regulated.

In general, angiogenesis, or neovascularization, refers to the process by which new capillaries are formed from existing vasculature in tissues, and pathological angiogenesis is almost consistently associated with some degree of inflammation, and thus the substances that cause inflammation. Blood vessels can be formed or neoplastic.

The present inventors confirmed that the expression of NFAT5 gene overexpressed in angiogenic diseases is regulated by inflammatory cytokines and osmotic stress. According to one embodiment of the present invention, synovial cells obtained from patients with rheumatoid arthritis and osteoarthritis After treatment with inflammatory cytokines TNF-a and IL-1β and NaCl to induce osmotic stress, the expression and migration of NFAT5 in cells were analyzed by Western blot and confocal microscopy. It was found that stress further increased the expression of NFAT5 in angiogenic diseases, and it was found that the NFAT5 protein expressed by this treatment was transferred to the nucleus in the cell (see Example 2).

Furthermore, the present inventors anticipated that the inhibition of NFAT5 expression or activity could prevent or treat angiogenesis-related diseases. In order to elucidate such an expectation, in one embodiment of the present invention, mice lacking the NFAT5 gene were identified. Phosphorus NFAT5 ^+/- and NFAT5 ^{+ / +} mice as controls, intravenous anti-type II collagen antibodies were used to induce arthritis, an angiogenesis-related disease. NFAT5 ^+/- mice showed significantly decreased disease severity compared to NFAT5 ^{+ / +} mice, and the degree of inflammation, synovial proliferation, and joint destruction were significantly reduced compared to controls. See Example 3).

Therefore, the results indicate that only a partial defect of the NFAT5 gene in angiogenesis-related disease cells can reduce the severity of the disease.

In addition, according to another embodiment of the present invention, after transducing NFAT5 siRNA to synovial cells of rheumatoid arthritis and osteoarthritis to inhibit the expression of the NFAT5 gene, cell survival rate and cell proliferation of synovial cells according to NFAT5 siRNA As a result, in the synovial cells into which NFAT5 siRNA was introduced, apoptosis was increased compared to the control group, and cell survival rate was decreased, whereas cell death was increased. Factors affecting cell proliferation, cyclin D and The expression of E was also shown to be reduced (see Example 4). Therefore, inhibiting the expression or activity of NFAT5 inhibited the cell proliferation of pathological arthritis synovial cells, while inducing apoptosis was found to have a therapeutic effect.

Furthermore, the inhibition or expression of NFAT5 according to the present invention is characterized by inhibiting the proliferation and migration of vascular endothelial cells.

On the other hand, in the case of cancer caused by angiogenesis abnormality, the supply of oxygen and nutrients is required in order to grow cancer tissue, and angiogenesis is involved to make such a supply passage. In the angiogenesis process in cancer tissues, cancer cells secrete angiogenesis inducers, and these inducers bind to receptors present in endothelial cells to form new blood vessels. Stimulated endothelial cells begin to grow and secrete proteolytic enzymes, which cause degradation of the base membrane, which causes endothelial cells to move toward the angiogenesis inducer. It is called cell migration. The endothelial cells thus migrated between the blood vessels (cell attachment), then moved to the place to penetrate, then penetrated the basement membrane (cell infiltration) and connected to each other to form a tube, and finally the ends of the hollow sprouts. This combination creates a loop where blood enters. In other words, when any one of these processes is blocked, angiogenesis may be reduced, and thus an effect of treating diseases induced by angiogenesis including cancer may be expected.

The present inventors investigated whether the inhibition or expression of NFAT5 can inhibit the proliferation and migration of endothelial cells. According to one embodiment of the present invention, after introducing NFAT5 siRNA into HUVEC cells, which are vascular endothelial cells, As a result, endothelial cell apoptosis and cell proliferation were confirmed. As a result, in the endothelial cells transduced with NFAT5 siRNA, cell viability and cell proliferation were reduced compared to the control group into which scrambled siRNA was introduced. Cell chemotaxis was shown to decrease with migration and tube formation (see Example 5).

Therefore, suppressing the expression or activity of NFAT5 has the effect of reducing the proliferation, migration and tube formation of endothelial cells has the effect of treating diseases caused by angiogenesis, including cancer.

 Therefore, the present invention can provide a composition for inhibiting angiogenesis, including an inhibitor of NFAT5 expression or activity as an active ingredient.

In the present invention, the expression or activity inhibitor of NFAT5 may be an antisense oligonucleotide, RNAi, siRNA or shRNA for the NFAT5 gene represented by SEQ ID NO: 1, or may be a specific antibody to NFAT5 protein.

In the present invention, the term "antisense oligonucleotide means a DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, and binds to the complementary sequence in the mRNA to translate the mRNA into a protein. The antisense sequence of the present invention refers to a DNA or RNA sequence complementary to the mRNA of the NFAT5 gene and capable of binding to the mRNA, the translation of the mRNA, translocation into the cytoplasm, maturation ( maturation) or any other vital biological function.

In addition, antisense nucleic acids may be modified at the position of one or more bases, sugars or backbones to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol ., 5, 3, 343-55, 1995). The nucleic acid backbone can be modified with phosphorothioate, phosphoroester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic intersaccharide linkages and the like. In addition, antisense nucleic acids may comprise one or more substituted sugar moieties. Antisense nucleic acids can include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-methylpyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acids of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense nucleic acids. Cholesterol moieties, cholesteryl moieties, cholic acid, thioethers, thiocholesterols, fatty chains, phospholipids, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexylamino-carbonyl-oxy Fat-soluble moieties such as a cholesterol ester moiety, and the like. Oligonucleotides comprising fat-soluble moieties and methods of preparation are already well known in the art (US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid can increase stability to nucleases and increase the binding affinity of the antisense nucleic acid with the target mRNA.

Antisense oligonucleotides can be synthesized in vitro in conventional manner to be administered in vivo or to allow antisense oligonucleotides to be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is to use RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin is in the opposite direction of the recognition site (MCS). Such antisense RNA is desirable to ensure that there is a translation stop codon in the sequence so that it is not translated into the peptide sequence.

In the present invention, the "RNAi" refers to the RNA Interference, in English, it means the meaning of RNA interference. RNA interference is a specific gene suppression phenomenon that is well conserved among most organisms. It is thought to be a type of gene monitoring mechanism used by cells to defend against viral infections, to suppress transposons, or to remove abnormal mRNAs. In particular, gene suppression by small RNA is called RNA interference in a broad sense, and RNA interference in a narrow sense means mRNA degradation by siRNA. In addition, RNA interference may refer to a technique for inhibiting genes using siRNA.

In the present invention, the term "siRNA" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (International Patent Nos. 00/44895, 01/36646, 99/32619, 01/29058, 99/07409 and 00/44914) siRNAs are provided as efficient gene knock-down methods or gene therapy methods because they can inhibit the expression of target genes.

The siRNA molecule of the present invention may have a structure in which a sense strand (a sequence corresponding to the mRNA sequence of the NFAT5 gene) and an antisense strand (a sequence complementary to the mRNA sequence) are positioned opposite to each other to form a double strand. The siRNA molecules of the present invention may have a single chain structure with self-complementary sense and antisense strands. Furthermore, siRNAs are not limited to completely paired double-stranded RNA moieties paired with RNA, but can be paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. Parts that do not achieve may be included. In addition, the siRNA terminal structure can be either blunt or cohesive, as long as the expression of the marker gene can be suppressed by the RNAi effect, and the adhesive terminal structure has a 3'-terminal protrusion structure and a 5'- end. Both terminal protruding structures are possible.

In addition, the siRNA molecules of the present invention may have a form in which a short nucleotide sequence is inserted between self-complementary sense and antisense strands, in which case the siRNA molecule formed by expression of the nucleotide sequence is subjected to intramolecular hybridization. As a result, a hairpin structure is formed, and as a whole, a stem-and-loop structure is formed. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

The method for preparing siRNA is to directly synthesize siRNA in vitro and then introduce it into the cell through a transformation process, and to siRNA expression vector or PCR-derived siRNA expression cassette prepared to express siRNA in the cell. There is a method of conversion or infection.

In addition, the compositions of the present invention comprising an NFAT5 gene specific siRNA may comprise an agent that promotes intracellular influx of siRNA. Agents that promote the influx of siRNA into the cell generally can be used agents that promote the influx of nucleic acids. Examples of these include the use of liposomes or the lipophilic of one of many sterols, including cholesterol, cholate and deoxycholic acid. It can be combined with a carrier. Also, poly-L-lysine, spermine, polysilazane, polyethylenimine, polydihydroimidazolenium, polyallylamine Cationic polymers such as chitosan, succinylated PLL, succinylated PEI, polyglutamic acid, and polyaspartic acid. anionic polymers such as polyaspartic acid, polyacrylic acid, polymethacylic acid, dextran sulfate, heparin, hyaluronic acid, etc. It is available.

Preferably siRNA that can specifically bind to the NFAT5 gene to inhibit the expression of the gene may have a base sequence selected from the group consisting of SEQ ID NO: 2 to 7 and SEQ ID NO: 12 to 17.

In addition, when using an antibody specific for the protein as a substance for reducing the expression and activity of the NFAT5 protein, the antibody is directly coupled to an existing angiogenesis-related disease therapeutic agent or indirectly through a linker or the like (eg, Covalent bonds). Therapeutic agents that can be bound to antibodies include, but are not limited to, radionuclide such as 131I, 90Y, 105Rh, 47Sc, 67Cu, 212Bi, 211At, 67Ga, 125I, 186Re, 188Re, 177Lu, 153Sm, 123I, 111In, etc. ); Biological response variants or drugs such as methotrexate, adriamycin, and lympokine such as interferon; Toxins such as lysine, abrin, diphtheria and the like; Heterofunctional antibodies, ie antibodies that bind to other antibodies and the complex binds to all of the target cells; And natural, ie non-associated or non-complexed antibodies.

Furthermore, the composition for inhibiting angiogenesis comprising the expression or activity inhibitor of NFAT5 according to the present invention as an active ingredient may be used as a pharmaceutical composition for inhibiting angiogenesis or angiogenesis.

The composition for inhibiting angiogenesis according to the present invention is an antisense oligonucleotide, siRNA (small interference RNA) oligonucleotide or NFAT5 complementary to the expression or activity inhibitor of NFAT5, that is, a nucleotide sequence of the NFAT5 gene in a pharmaceutically effective amount It may comprise an antibody specific for a protein alone or include one or more pharmaceutically acceptable carriers, excipients or diluents.

The pharmaceutically effective amount herein refers to an amount sufficient for the physiologically active ingredient to be administered to an animal or human to exhibit the desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed depending on the age, weight, health condition, sex, route of administration and duration of treatment.

In addition, the above pharmaceutically acceptable means physiologically acceptable and when administered to a human, usually does not cause gastrointestinal disorders, allergic reactions such as dizziness or the like. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal, and can be formulated for various oral or parenteral administration. It may be formulated in a form.

 Representative of parenteral formulations are injectable formulations, preferably aqueous isotonic solutions or suspensions. Injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, oral dosage forms include, for example, intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers, and these formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, dextrose). , Sucrose, mannitol, sorbitol, cellulose and / or glycine) and a suspending agent such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. The tablets may comprise binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid or its Disintegrants such as sodium salts, absorbents, colorants, flavors and / or sweeteners may be further included. The formulations may be prepared by conventional mixing, granulating or coating methods.

In addition, the composition of the present invention may further include auxiliaries such as preservatives, hydrating agents, emulsifiers, salts or buffers for controlling osmotic pressure and other therapeutically useful substances, and may be formulated according to conventional methods.

The composition according to the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, the dosage of the active ingredient being determined by several factors such as the route of administration, the age, sex, weight and severity of the patient. It may be appropriately selected depending on. In addition, the composition of the present invention can be administered in parallel with known compounds that can enhance the desired effect.

The route of administration of the pharmaceutical composition according to the present invention can be administered to humans and animals orally or parenterally, such as intravenous, subcutaneous, intranasal or intraperitoneal. Oral administration also includes sublingual application. Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection.

In the composition of the present invention, the total effective amount of the NFAT5 expression or activity inhibitor according to the present invention may be administered to the patient in a single dose, and the divided therapy in which multiple doses are administered for a long time. Administration by a fractionated treatment protocol. The composition of the present invention may vary the content of the active ingredient according to the degree of disease, but can be repeatedly administered several times a day at an effective dose of 100 μg to 3,000 mg once administered based on an adult. However, the concentration of the inhibitor may be determined in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the drug. . Therefore, in view of the above, one of ordinary skill in the art can determine an appropriate effective dosage according to the specific use of the NFAT5 expression or activity inhibitor as an angiogenesis inhibitor or an angiogenesis related disease. The composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is exhibited.

Therefore, the present invention may provide an agent for inhibiting angiogenesis, that is, an angiogenesis-related disease treatment agent for preventing or treating a disease caused by angiogenesis containing the composition for inhibiting angiogenesis of the present invention as an active ingredient. It is possible to provide a method for inhibiting excessive angiogenesis comprising administering the composition according to the present invention to a mammal other than a human whose abnormal angiogenesis continues.

In the present invention, the disease caused by the angiogenesis or abnormal angiogenesis is not limited to this, but rheumatoid arthritis, osteoarthritis, sepsis arthritis, psoriasis, corneal ulcer, macular degeneration, diabetic retinopathy, proliferative Vitreoretinopathy, Immature Retinopathy, Ophthalmic Inflammation, Conical Corneal, Sjogren's Syndrome, Myopia Eye Tumor, Corneal Transplant Rejection, Abnormal Wound Fusion, Bone Disease, Proteinuria, Abdominal Aortic Aneurysm, Degenerative Cartilage Loss Due to Traumatic Joint Injury, Nervous System Myelin deprivation disease, cirrhosis, renal glomerular disease, inflammatory bowel disease, periodontal disease, arteriosclerosis, restenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging and cancer may be selected from the group consisting of.

Furthermore, the present invention can provide a method for screening a substance for preventing or treating angiogenesis-related diseases using NFAT5. Preferably, the candidate substance to be analyzed is contacted with a biological sample containing NFAT5 gene or NFAT5 protein. Making a step; Measuring the expression level of the NFAT5 gene, the amount of NFAT5 protein or the activity of NFAT5 protein; And when the amount of NFAT5 gene expression, NFAT5 protein or NFAT5 protein activity is reduced compared to the control group without contact with the sample, the sample may be used as a substance capable of preventing or treating a disease caused by excessive angiogenesis. And determining.

In the present invention, the expression amount of the NFAT5 gene or the amount of NFAT5 protein may be the mRNA amount of the NFAT5 gene, or may be a polypeptide produced through a translation process from the mRNA.

The method of measuring the expression amount of NFAT5 gene, the amount of NFAT5 protein or the activity of NFAT5 protein is not limited thereto, but reverse transcriptase-polymerase chain reaction and real time polymerase chain reaction. polymerase chain reaction, Western blot, Northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion and immunoprecipitation assay It can be done with

As used herein, the term “biological sample” refers to a sample taken from a patient or patient suspected of developing the disease according to the occurrence or progression of an angiogenesis-related disease. The sample includes, but is not limited to, tissues and cells. , Blood, serum, plasma, saliva, urine, and the like, and may be preferably cells.

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

In cells of angiogenesis-related diseases NFAT5 Expression Analysis of

The present inventors obtained fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis and osteoarthritis, respectively, to investigate the expression of NFAT5 gene in cells of diseases caused by excessive angiogenesis (Yoo, SA et al. , Calcineurin is expressed and plays acritical role in inflammatory arthritis. JImmunol 177,2681-2690, 2006), fetal calf serum (FCS) or insulin-transferrin-selenium (ITSA) added to DMEM medium. In addition, HUVEC cells, vascular endothelial cells, were isolated from normal cord blood veins and cultured in M199 medium containing 20% FCS. Thereafter, the amount of NFAT5 expressed in the cells was confirmed by immunochemical staining, that is, the synovial membrane obtained from the patients with rheumatoid arthritis and osteoarthritis was cut into 5 μm, treated with pepsin for 30 minutes, and at room temperature for 30 minutes. Blocking with bovine serum albumin (BSA). The antibody against NFAT5 (purchased from University of Maryland) was then reacted overnight at 4 ° C., using IgG of nonspecific mice purchased from Sigma as a control. The slides containing the sections were then washed three times with TBS buffer and reacted with biotin-bound anti-mouse IgG in a humid chamber. NFAT5 positive cells were then detected using peroxidase-linked streptavidin with 3'3-diamino-bendidine tetrahydrochloride (DAB), and the slides were stained with hematoxylene.

In addition, the present inventors reported that NFAT5 is regulated by surrounding osmoticity. After treating NaCl with FLS cells, the present inventors analyzed the expression of NFAT5 through Western blot, wherein the Western blot was After dissolving FLS cells treated with NaCl at a concentration of 100 mM using a conventional lysis buffer known in the art, insoluble materials were removed by centrifugation at 12000 g for 20 minutes at 4 ° C. The concentration of the final protein obtained was then quantified by Bradford analysis, followed by electrophoresis on SDS-PAGE and electrophoresis onto nitrocellulose membranes, followed by reaction with an antibody to NFAT5 (purchased from University of Maryland) and ECL solution. Was visualized on the film.

As a result, as shown in Fig. 1, NFAT5 was found to be expressed in the synovial tissue of patients with rheumatoid arthritis and osteoarthritis very much, and especially in the synovial tissue of rheumatoid arthritis, more NFAT5 is expressed than osteoarthritis. (See FIGS. 1A-1H).

In addition, as a result of measuring the expression level of NFAT5 after treating NaCl at each concentration of the cells, the expression of NFAT5 was increased in NaCl concentration-dependently in both synovial cells of patients with rheumatoid arthritis and osteoarthritis (see FIG. 1I).

Therefore, the present inventors found that the NFAT5 gene was overexpressed in the angiogenic disease, and furthermore, when the surrounding osmotic environment was established, the expression amount was increased.

< Example  2>

Caused by inflammatory cytokines NFAT5 Expression Analysis of

To investigate whether the expression patterns of NFAT5 are altered by inflammatory cytokines, the amount of pro-inflammatory cytokines TNF-a and IL-1β, respectively, was 10ng / ml in synovial cells obtained from patients with rheumatoid arthritis and osteoarthritis. And then incubated for 12 hours, Western blot was performed by the same method as described in Example 1 using an antibody against NFAT5. In addition, the present inventors treated TNF-a and IL-1β at 10 ng / ml, respectively, in order to observe intracellular migration of NFAT5 protein following treatment with TNF-a, IL-1β and NaCl, at a concentration of 100 mM. Cells treated with NaCl were each dispensed into glass chamber slides, fixed with cold methanol, treated with PBS solution containing 0.25% Triton X-100, and then reacted at 4 ° C. with an antibody against NFAT5. The Alexa 488-bound anti-rabbit IgG was then reacted for 30 minutes and the location of NFAT5 protein in the cells was confirmed by confocal microscopy, where the nucleus contained 4,6-diamino-2-phenylridol (DAPI). And stained.

As a result, as shown in Figure 1j, when treated with inflammatory cytokines TNF-a and IL-1β, the expression of NFAT5 was significantly increased compared to the untreated control group, the degree of expression is increased osteoarthritis Rheumatoid arthritis was found to be significantly increased. In addition, when the position of the NFAT5 protein was confirmed by confocal microscopy, it was confirmed that the NFAT5 protein was moved to the nucleus when treated with TNF-a, IL-1β and NaCl (see FIG. 1K).

Therefore, the above results showed that the present inventors found that inflammatory cytokines and osmotic environments increase the expression of NFAT5 in angiogenic diseases, and that the expressed NFAT5 has been transferred to the nucleus within the cell. Could.

< Example  3>

NFAT5  Severity alleviation of angiogenic diseases due to gene deficiency

The above examples confirmed that NFAT5 is overexpressed in the case of angiogenic diseases, and thus, whether the NFAT5 gene is deficient and the NFAT5 protein is not expressed, the severity of the angiogenic disease may be reduced. To this end, 8-week-old heterozygous mice NFAT5 ^+/- deficient in the NFAT5 gene obtained from the University of Maryland University School of Medicine and anti-type II collagen antibodies (5 mg) inducing arthritis in NFAT5 ^{+ / +} mice as controls. ) 3 days after intravenous injection, 50 ug of LPS was administered intraperitoneally. Afterwards, the mice were observed daily, and the degree of pain was observed by the limbs of the mice to be given 0 to 4 points according to the degree of pain, and the maximum score for each mouse was 16 points. In addition, the degree of pain was analyzed by measuring the thickness of the foot of the mouse, that is, the forefoot and hind feet were measured using a microcaliper daily, and at a specific time the diameter of the ankle divided by the thickness measurement on day 0 after the antibody injection The value was used as the thickness index and expressed in%. Severity scores were measured from 0 to 3, with 0 being no pain, 1 being mild, 2 being moderate, and 3 being severe.

In addition, the present inventors observed the degree of inflammation, proliferation of the synovial membrane and the degree of joint destruction of the tissues of the joints in the NFAT5 ^+/- and NFAT5 ^{+ / +} mice using hematoxylene and eosin staining methods known in the art.

As a result, as shown in Figure 2, the symptoms of arthritis induced by collagen in the case of NFAT5 ^+/- NFAT5 compared to ^{+ / +} mice that normally NFAT5 gene expression, was found to be a moderate decrease (Fig. 2a), measure the thickness of the mouse to analysis of the degree of abnormality of the joints, the thickness of the ankle joint also compared to NFAT5 ^{+ / +} mice showed that the NFAT5 ^+/- remarkably thin (see Fig. 2b), via immunological staining NFAT5 ^In the case of ^+/- , the degree of inflammation, synovial proliferation, and joint destruction were significantly reduced compared to NFAT5 ^{+ / +} mice.

Therefore, the present inventors have found that only a partial defect of the NFAT5 gene can significantly reduce the symptoms of angiogenic diseases including arthritis.

< Example  4>

NFAT5 of Synovial cells  Role analysis

The results confirm that the defect of the NFAT5 gene can reduce the symptoms of angiogenic diseases, the present inventors investigated how the NFAT5 gene actually plays a role in the survival and proliferation of cells in synovial cells. To this end, NFAT5 siRNA having a nucleotide sequence shown in Table 1 as a siRNA capable of inhibiting the expression of NFAT5 and a control NFAT5 siRNA for use as a control were prepared, respectively. Subsequently, NFAT5 siRNA (SEQ ID NOs: 2-7 and SEQ ID NOs: 12-17) were injected into synovial cells of rheumatoid arthritis or osteoarthritis, and then cultured for 72 hours, and then the survival rate and morphology of the cells were observed. In this case, the control siRNA (SEQ ID NOS: 8-11) described in Table 1 was used as a control group, respectively. The control siRNAs for the NFAT5 siRNAs of SEQ ID NOS: 2-5 and SEQ ID NOs: 12-17 were SEQ ID NO: And siRNAs of 9 were used, and siRNAs of SEQ ID NOs: 10 and 11 were used as controls for the NFAT5 siRNAs of SEQ ID NOs: 6 and 7. Cell morphology was observed under a microscope, cell viability was performed using MTT assay methods or cell counting kit-8 (cck-8) known in the art, and apoptosis was performed via TUNEL analysis. TUNEL analysis is performed using Apotag peroxidase, in which synovial cells of arthritis are cultured in four chamber slides, NFAT5 siRNA is transduced into the cells and washed with PBS 24 hours later, followed by 4% PFA. Cells were fixed for 10 minutes. The immobilized cells were then incubated with digoxigenin-bound dUTP at 37 ° C. for 1 hour to react with TdT catalase. The reaction stop solution was then treated at room temperature, reacted with an anti-digoxigenin antibody bound to peroxidase for 30 minutes, and then DNA fragments cleaved by apoptosis were measured by staining with DAB, a substrate of peroxidase. .

NFAT5 siRNA sequence NFAT5 siRNA Types Sequence SEQ ID NO: 297R NFAT5 siRNA (S) 5'-augcccucggacuucaucucauu-3 ' 2 297R NFAT5 siRNA (AS) 5'-ugagaugaaguccgagggcauuu-3 ' 3 569R NFAT5 siRNA (S) 5'-augggcggugcuugcagcuccuu-3 ' 4 569R NFAT5 siRNA (AS) 5'-ggagcugcaagcaccgcccauuu-3 ' 5 Control inv569R (S) 5'-ccucgacguucguggcggguauu-3 ' 8 Control inv569R (AS) 5'-uacccgccacgaacgucgagguu-3 ' 9 NFAT5 siRNA (S) 5′-cccucucagcaaggguuau (dtdt) -3 ′ 6 NFAT5 siRNA (AS) 5′-auaacccuugcugagaggg (dtdt) -3 ′ 7 Control siRNA (S) 5′-uucuccgaacgugucacgu (tt) -3 ′ 10 Control siRNA (AS) 5′-acgugacacguucggagaa (tt) -3 ′ 11 A_NFAT5 siRNA (S) 5′-GCAAAGAAGUGGACAUUGA (tt) -3 ′ 12 A_NFAT5 siRNA (AS) 5'-UCAAUGUCCACUUCUUUGC (tt) -3 ' 13 B_NFAT5 siRNA (S) 5′-GCAUAGCUGUUCAGUGAAA (tt) -3 ′ 14 B_NFAT5 siRN (AS) 5'-UUUCACUGAACAGCUAUGC (tt) -3 ' 15 C_NFAT5 siRNA (S) 5′-GCAAGUAACUCUCUUCUUA (tt) -3 ′ 16 C_NFAT5 siRNA (AS) 5′-UAAGAAGAGAGUUACUUGC (tt) -3 ′ 17

In addition, the degree of cell proliferation was transduced with NFAT5 siRNA in the cells, and then treated with 10 ng / ml of TNF-a or TNF-β, respectively, followed by [ ³ H] thymidine incorporation. Measured using, the extent of proliferation was expressed in cpm, as a control was used for cells treated nothing, the assay was calculated by the average value obtained by performing the same method three times. In addition, the expression level of cyclin D, E, which is a factor involved in cell proliferation, was also confirmed by Western blot, and Western blot was used in the same manner as described in the above example except that antibodies of cyclin D and E were used. .

As a result, when the NFAT5 siRNAs of SEQ ID NO: 2-7 and SEQ ID NO: 12-17 were transduced into the synovial cells of arthritis, the cell viability of the synovial cells was reduced compared to the control group (see FIG. 3A). , TUNEL analysis showed that the synovial cells treated with NFAT5 siRNA significantly increased apoptosis compared to the cells treated with nothing and the group treated with scrapable siRNA (FIG. 3B). Therefore, the results showed that the present inventors found that NFAT5 plays a role in regulating apoptosis of synovial cells. In particular, when the expression of NFAT5 gene is inhibited, apoptosis is induced in synovial cells of arthritis disease. there was.

Furthermore, synovial cells treated with NFAT5 siRNA were shown to inhibit cell proliferation of synovial cells despite treatment with inflammatory cytokines (see FIGS. 3C and 3D), and expression of cyclins D and E, factors affecting cell proliferation. It was also shown to be reduced in synovial cells treated with NFAT5 siRNA (see FIG. 3E).

< Example  5>

NFAT5 Of the effects on endothelial cell proliferation and migration

In order to investigate whether NFAT5 affects the proliferation and migration of endothelial cells that play an important role in angiogenesis in addition to synovial cells, the present inventors transduced the NFAT5 siRNA used in Example 4 on HUVEC cells, which are vascular endothelial cells. The degree of apoptosis and cell proliferation was examined at each time, and cell death and cell proliferation were performed in the same manner as described in Example 4 above. In addition, after introducing NFAT5 siRNA into HUVEC cells, the migration and tube formation of HUVEC cells were examined. First, cell migration measurements were performed on HUVEC cells in which NFAT5 siRNA was introduced into a 60 mm cell culture dish and cells not treated with siRNA. After dispensing and incubating the dish to full, the dish was scraped using pipette tips, and then treated with VEGF ₁₆₂ (20ng / ml) in M199 medium containing 1 mM thymidine and 1% FBS and incubated for 12 hours. Then, the number of cells was counted to measure the degree of movement of the cells. In addition, tube formation was analyzed by dispensing HUVEC cells into polymerized Matrigel (BD Bioscience, CA) added with VEGF ₁₆₂ (20 ng / ml), incubating for 12 hours, and then measuring the length of the formed tubes. Observed using -Pro Plus v4.5.

In addition, the present inventors investigated cell chemotaxis by NFAT5 in HUVEC cells, which were analyzed using a transwell chamber equipped with a 6.5 mm diameter polycarbonate filter (8 μm pore size). That is, VEGF (50 ng / ml) prepared in DMEM medium containing 1% FBS was added to the lower wells, and 5 x 10 ⁴ cells / ml of cells suspended in DMEM containing 1% FBS were added to the upper wells. After that, the cells were incubated at 37 ° C. for 12 hours. Cells that did not migrate on the filter top surface were removed using a cotton swab, and the migrated cells were fixed and stained using the Diff-Quik kit. Chemotaxis assays were performed by counting the number of cells migrated at x 200 magnification using a microscope.

As a result, as shown in Figure 4, when the NFAT5 siRNA of SEQ ID NO: 2 to 7 and SEQ ID NO: 12 to 17 transduced into HUVEC cells, vascular endothelial cells, cell survival rate and the degree of cell proliferation was reduced compared to the control group Cell proliferation analysis showed that cell proliferation was reduced by treatment with NFAT5 siRNA, regardless of the presence of growth factor VEGF (see FIGS. 4A and 4B). In addition, the tube formation of vascular endothelial cells also showed a decrease in the degree of tube formation in the group treated with the NFAT5 siRNA of SEQ ID NOS: 2 to 7 and SEQ ID NOs: 12 to 17 (see FIG. 4C). Analysis of cell migration also showed that when NFAT5 siRNA according to the present invention was treated, cell migration by VEGF was inhibited (see FIG. 4D). As a result of cell chemotaxis assay, when the NFAT5 siRNA was transfected into HUVEC cells, chemotaxis of vascular endothelial cells by VEGF ₁₆₂ was also significantly reduced (see FIG. 4E).

Therefore, the present inventors showed that when NFAT5 siRNA, an inhibitor of NFAT5, according to the present invention was treated to vascular endothelial cells, cell proliferation, tube formation, cell migration and cell chemotaxis of vascular endothelial cells were inhibited. , Cell death was shown to increase.

Therefore, the inventors have found that the inhibitor of NFAT5, in particular NFAT5 siRNA, according to the present invention can inhibit the proliferation of vascular endothelial cells and can prevent or treat diseases that may be caused by neovascularization or angiogenesis. .

< Example  6>

NFAT5 end Macrophage  Analysis of effects on migration and cytokine production

In addition, the present inventors injected 3% of thioglycolate into NFAT5 ^+/- and NFAT5 ^{+ / +} mice intraperitoneally to confirm that NFAT5 also affects macrophage migration and cytokine production. I was separated from the peritoneal macrophages (Kreckler, LM, Wan, TC , Ge, ZD & Auchampach, JA Adenosine inhibits tumor necrosis factor-alpha release from mouse peritoneal macrophages via A2A and A2B but not the A3 adenosine receptor. JPharmacolExpTher 317, 172 -18, LPS (1ug / ml) or IL-1β (10ng / ml) to the macrophages for 12 hours, and counted the number of cells to determine the degree of macrophage migration Investigate.

In addition, the production of cytokines was treated with LPS (1ug / ml) or IL-1β (10ng / ml) for 12 hours after intraperitoneal macrophages (3X10 ⁴ cells / ml) isolated by the above method, and then the supernatant of the medium. After collection, the production of LPS or IL-12 was measured using ELISA. In addition, the cultured cells were reacted with FITC-labeled anti-TNF-a antibody for 30 minutes, diluted with PBS buffer containing 0.1% FBS and 0.01% NaN ₃ , and then FACS caliber (Becton dickinson, mountain view, The amount of TNF-a produced was measured using a CA) instrument.

As a result, as shown in Figure 6, in the case of mice lacking the NFAT5 gene in the macrophage migration involved in angiogenesis and vascular inflammation, the NFAT5 gene is normally expressed in the macrophage migration by treatment with inflammatory cytokines. It was shown to be reduced compared to the mouse group (see FIG. 5A).

In addition, as a result of comparative analysis of the amount of cytokines produced, the production of IL-12 and TNF-a was found to be significantly reduced in mice lacking the NFAT5 gene compared to mice in which the NFAT5 gene is present (FIG. 5B). To FIG. 5D).

Therefore, the above results suggest that the present inventors inhibited the production of cytokines that stimulate angiogenesis and inhibited the migration of macrophages involved in angiogenesis when cells, especially cells with excessive blood vessel formation, inhibited the expression of the NFAT5 gene. Inhibition revealed that angiogenesis could be suppressed, and ultimately, inhibitors of NFAT5 could be used as therapeutic agents for preventing or treating angiogenic diseases.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

In addition, the research on the present invention was carried out with the support of the Ministry of Health, Welfare and Family Health Technology Promotion Project (Task No .: A84948).

<110> Industry Academic Cooperation Foundation of Catholic University <120> Compositions comprising NFAT5 inhibitor as an active ingredient          for preventing or treating of angiogenesis-related diseases <160> 17 <170> KopatentIn 1.71 <210> 1 <211> 14174 <212> DNA <213> Artificial Sequence <220> NFAT5 gene sequence (transcript varient 6) <400> 1 tgcaacggaa acttttggct ccacgaacag aaaagcaaga gcaaaaactc agttaaatgc 60 gttcttcctc catctttgcc tagtccaaaa ggaaaaaaaa agaaacagaa aaaagaaaaa 120 ctgtttggaa gagtagcgtt gaggtttgct ttttacctgt ttttcctcta gaacagccag 180 gtggattcac atgatccaat tttttaattg tcttctatgt cccactccga ataacccgga 240 tgccccagca caatcagaga gatcgttttt tttaaaaaaa atttcagcct ccaaaggtag 300 gaggggagtg ttaggggaga aagtacttta attaaaaatc aataactcga ggtttttggg 360 atacggtttg ccatttccta attaagaaat gggtttgaca gtccctttgt acacactgct 420 atgcaaaccc caaagggttg gcggctgtcc gggcgatgac actccggtcc cctgcgagac 480 cccgggccag ccaggcccgt ccgccgccgg cctctggggt ccgtccccgg ctcgcgcaga 540 cctctcgctt ctctcggctc tgtctcctgc gctcagctct gctcggggcc ggccgcctca 600 ggctcgccgc caccaggtcg ttgcaaatac cttttccctc cccggggccc cagcgcgcgg 660 ccacctccca gcctcccccc ctcccaccct ggcagcgggg ccctttcccg gctcaggaac 720 agcagcagcc cgggccgcgc cggcaggaag cgaggcccat gttgctgctg ttccctggcg 780 cgcctccccg ccctccgggg gccgccacgg ctcttccgcg ctcccgggca cccccctccg 840 cgcctgcgct gtgccccacg ggggcggggc tcagattcct gtcagcggcg gcggcggtgg 900 cggcgaccgt cagttttcgc tgaggagaaa cacgaaacgg accctttggc tctccccctt 960 ccccttcccc gtcctgaacc cctctcctgg tcaccgagaa tcagtccccg tggagttccc 1020 cctccacctc gccatcgttt cctcggtcct cggcccagtg gaagtcacta ccctcgagga 1080 ggaggcagcg gcagccgccc tcgcgtcgcc gcccccggtt cggtgcccgc ggtcccggag 1140 aggaggtgcc gccgccaccg ccgctccccc cctcccgctg ccctcgggcc gggctgggtc 1200 gagctgcgat gccctcggac ttcatctcat tgctcagcgc ggacctagac ctggaatcgc 1260 ccaagtccct ctactcgcga gattctctga agttacaccc atcacagaat tttcatagag 1320 ctggactatt ggaagaatct gtctatgatc ttctcccaaa ggagttacag ttacctccat 1380 ctagagaaac atctgtagca tcaatgagtc agacaagcgg tggtgaggca ggctcgcctc 1440 ctccagctgt tgttgctgct gatgcttctt cagctccctc ctcttcctcc atgggcggtg 1500 cttgcagctc ctttaccacc tcttccagcc ctaccattta ttctacctca gtcaccgaca 1560 gcaaggctat gcaagtggag agctgctcct cagccgtggg ggtaagtaac agaggggtaa 1620 gtgaaaagca gttaaccagt aacacagttc agcagcatcc atcaacaccg aagaggcaca 1680 cagtcttgta catctcacca ccacctgagg acttgctgga taacagtcgg atgtcctgcc 1740 aggatgaggg gtgtggattg gaatctgagc agagctgcag tatgtggatg gaggattccc 1800 cctccaactt cagtaacatg agcaccagtt cctacaatga taacactgag gtacctcgta 1860 aatcacgaaa acgaaatcca aagcagaggc cgggggtcaa acgacgagat tgtgaagaat 1920 ctaatatgga tatatttgat gccgacagtg ccaaagcacc tcactatgtg ctttctcagc 1980 ttaccacgga caacaaaggc aactcaaaag cgggaaatgg aacattggaa aaccaaaaag 2040 gaactggagt aaagaagagc cctatgttgt gtggacaata tcctgttaaa agtgagggaa 2100 aggagctgaa gatagttgta caacctgaga cacagcaccg agctcggtac ctgactgagg 2160 gcagccgtgg ctcagtgaaa gatagaacac agcaaggctt tcctacagta aagctggaag 2220 gccataatga acctgtagtg ttgcaagtgt ttgtgggcaa cgactctgga cgagtgaaac 2280 cacatggatt ttatcaggcc tgcagagtaa ctggacgaaa tacaactcct tgcaaagaag 2340 tggacattga aggcactact gttatagaag tcggccttga tcctagcaac aacatgacac 2400 tggcggtgga ctgcgtaggg atattgaaat tgaggaatgc tgatgtcgaa gccagaatag 2460 gaattgctgg ttccaagaag aaaagcactc gtgccagatt ggtttttcga gttaatatca 2520 tgaggaaaga tggctccact ttgacactgc aaacaccctc ttctccaatt ttgtgtactc 2580 agccagcagg agtgccagaa atcttaaaga aaagcttgca tagctgttca gtgaaaggag 2640 aagaagaagt gtttttaatc ggcaagaact ttctgaaagg aactaaagtt attttccaag 2700 aaaatgtttc tgatgaaaac tcttggaagt cagaagctga aattgatatg gaactatttc 2760 atcagaatca tcttattgtg aaggttcctc cctatcatga ccaacatata actttgcctg 2820 tgtcagtggg aatatatgta gtgacaaatg ctggaagatc tcatgatgtt caaccattca 2880 cttacactcc agacccagca gctggtgctt tgaatgtaaa tgtgaagaag gaaatatcta 2940 gtccagcaag accttgctct tttgaagagg ccatgaaagc aatgaaaact actggatgta 3000 atttagataa ggtaaatatt atccctaatg ccctgatgac tccactcata ccaagcagta 3060 tgattaagag tgaagatgtt actccaatgg aagtaacagc agaaaaaaga tcttccacta 3120 tttttaagac tacaaagtct gttggatcaa ctcagcaaac attagaaaac atctcaaaca 3180 tagcaggaaa tggctctttt tcatcaccat catcttccca cctaccttct gaaaatgaaa 3240 aacagcagca gattcagccc aaggcataca acccagagac cctgacaact attcaaaccc 3300 aggacatctc acagcctggt acttttccag cagtttctgc ttctagtcag ctgcccaaca 3360 gcgatgcact attgcagcag gctacacagt ttcagacaag agaaactcag tctagagaga 3420 tattacagtc agatggtaca gtggttaatt tgtcacaact gactgaggca tcacaacaac 3480 agcagcagtc accactacaa gaacaagcac agactttaca gcagcagatt tcatcaaata 3540 tttttccatc accaaatagt gtgagtcagc ttcagaatac tattcagcag ctgcaagcag 3600 ggagtttcac aggcagtact gctagtggca gcagtggaag tgttgacttg gtccaacaag 3660 ttttagaggc acagcagcag ttatcttcag ttttattttc tgctccagat ggtaatgaga 3720 atgttcaaga gcagcttagt gcagatattt ttcaacaagt cagtcaaatt cagagtggtg 3780 taagccctgg aatgttttcc tcaacagagc caacagtcca taccagacca gataatttat 3840 tacctggaag agctgaaagt gttcatccac agtctgaaaa cacgttatct aatcaacagc 3900 agcagcagca gcagcaacag caagtgatgg aatcttcagc cgcaatggtg atggagatgc 3960 aacagagtat ctgccaggca gctgcccaga ttcagtcaga gttattccct tcaactgctt 4020 cagcaaatgg aaaccttcag caatcgccag tttaccagca gacttctcac atgatgagtg 4080 cattgtctac caatgaggat atgcaaatgc agtgtgaatt gttttcttct cctcctgcag 4140 tttctggaaa tgaaacttct acaactacca cacagcaggt tgcaacccct ggcactacca 4200 tgtttcagac atcaagttca ggagatggag aagaaactgg aacacaagca aaacagattc 4260 agaacagtgt ctttcagacc atggtccaaa tgcaacatag tggggacaat caacctcaag 4320 ttaacctttt ttcatccaca aaaagtatga tgagtgttca gaatagtggt acccaacaac 4380 aaggtaatgg tttattccag caagggaatg agatgatgtc acttcaatct ggaaattttt 4440 tgcagcagtc ttctcattca caggcccaac tttttcatcc tcaaaatcct attgccgatg 4500 ctcagaacct ttcccaggaa actcaaggtt ctctctttca tagtccaaat cctattgtcc 4560 acagtcagac ttctacaacc tcctctgaac aaatgcagcc tccaatgttt cactctcaaa 4620 gtaccattgc tgtgttacag ggctcttcag ttcctcaaga ccagcagtca accaacatat 4680 ttctttccca gagtcccatg aataatcttc agactaacac agtagcccaa gaagcatttt 4740 ttgcagcacc gaactcaatt tctccacttc agtcaacatc aaacagtgaa caacaagctg 4800 ctttccaaca gcaagctcca atatcacaca tccagactcc tatgctttcc caagaacagg 4860 cacaaccccc gcagcagggt ttatttcagc ctcaggtggc cctgggctcc cttccaccta 4920 atccaatgcc tcaaagccaa caaggaacca tgttccagtc acagcactca atagttgcca 4980 tgcagagtaa ctctccatcc caggaacagc agcagcagca gcaacagcag cagcaacagc 5040 agcagcaaca acaacagagc attttattca gtaatcagaa taccatggct acaatggcgt 5100 ctccaaagca accaccacca aacatgatat tcaacccaaa tcaaaatcca atggctaatc 5160 aggagcaaca gaaccagtca atttttcacc aacaaagtaa catggcccca atgaatcaag 5220 agcaacagcc catgcaattt cagagtcagt ccacagtttc ctcacttcag aacccaggtc 5280 ctacccagtc ggaatcatca cagaccccct tgttccatag ctctcctcag attcagttgg 5340 tacaagggtc acctagttct caagagcagc aagtaactct cttcttatct ccagcatcca 5400 tgtctgcctt gcagaccagt ataaatcaac aagatatgca acagtctcct ctttattccc 5460 ctcagaacaa catgcctgga attcaaggag ccacatcttc gcctcaacca caggctactt 5520 tatttcacaa cacagcagga ggcacaatga accaactgca gaattctcct ggctcatctc 5580 agcagacatc aggaatgttc ttatttggca ttcaaaataa ctgtagtcag cttttaacct 5640 ctggaccagc tacattgcct gatcagttga tggccataag tcagccaggc caaccacaaa 5700 acgagggcca gccacctgtg acaacacttc tttctcagca aatgccagag aattctccac 5760 tggcatcctc tataaacacc aaccagaaca tcgaaaagat tgatttgctt gtttcattgc 5820 aaaaccaagg gaacaacttg actggctcct tttaactgga tataaattcc acgaagaaaa 5880 tcctgattcc aagatgtcct gagatcttgt ggttccatga gaattattac tttaaaaaca 5940 aaacaaaata taaaaaactg tgtttgagta aactgataga ttttactctg actgcaaaag 6000 agcacaccta tgctgcttgt tgcagtaact aaccaccaat gttaacatct tcatatttta 6060 tattcctaat aacagtgatg actgagaatc tatttgagtt tccagctggc agaattaatt 6120 gttattattt tcctaggcgc aatttcctta aacgtacagt ttaaattcaa ggctggacca 6180 ctcagttatt attgctatta gaaaataata tatcatgttt acttttgttc ttcattattt 6240 tctttcctgc attgttttag tcaagtaatg gcttttgaaa aagtaaagtt caataataac 6300 taaggctgtg atttttttca atataaaagg cacagctgtt ggccaaagtg aaggaatctt 6360 ttttcagttt tattggagaa actgaagggt aacattctaa caagtaaact gtatgtgcag 6420 ataaaagtac tcttgattta acacaaaggc agatgataca cttataaaac tgggaacagc 6480 tggaatgctt cttgatttta ttttttcaga gagttgttag ttctctgggt ttctactaag 6540 gggtttagcc ataactgtgc atagaaaaat aattatctgt aaaaaatgaa ggggataata 6600 tatgataaat tatgttctga tatcctccta cagtagttta aattgacaga aaaatttgaa 6660 tgttttcttc ttaacccagt cttaggctgg tattcccttt ttatatatat ctatattact 6720 tttcacctct ttttcacttt actttagaga actattaata tactactggc ttcatgaccc 6780 tgtagcatct ttggccactt taatctaggg tgacctagca atcctgcagc acagggcaga 6840 gagtactgtc ttaggaatta ttaggagttg attcctgaga aacaacacat ttttccccat 6900 gaacggtgct gttctgaagt cttcaaattt ttccctctaa taggaaacag tataaatttt 6960 aattaaaaaa aaaaggcaaa ctaaaatttc ttgaaatatc acttctccct gatctgcagt 7020 gagtataaat tcacttgtca cctcagtgct ttacagtttg aagtggtcac ttacctgatg 7080 gttcccacaa gccttaggct ttacagggtt gtatcattga cttaaaatga agaattaact 7140 tgtgttacat ctataaagag caaaataaca cactccagaa cttggcagtt gtagcattag 7200 ttatacagtt ttgggtgttc ttgccacccg tgggatgcct gcttctcact accacctgtg 7260 tctggacaca tgcttatgtc tcattttcct tttggcatgt ggaaagctgt caatgcagtg 7320 taaggccaac gtgtgtgtgg cttctatgtg ttgagataat gttttggtat ccttgtccgt 7380 ttcatttatt ttttaagtgt acaaaaaata acctgttaat tgttgaaggc tacttttctg 7440 ttcttttttt tttttttttt ctatcctgta catttagttg aactgtgcgg aattgtggtg 7500 ttggttttgt ttacacagcc agatttttcc ttctttttgt tttgtgatga tcttcctttg 7560 ttctttgaat gtgctctttt gtctttttct cttttttctc atgttttctt ccctccacct 7620 ccaccccttt ctttctttct ctctctgatt gagaggcatt gaattacgtt ttcagtagta 7680 caggcttctt gccgatatga agggaacttt tcagaaagag acctactctg ggtcatttaa 7740 ttttgaatac agttttcaat cgttcaagtt ttggatggtt tatatctaat gtgtgtttca 7800 tttttttgga aagctatatt ttgtatttag gaaatggtat actattttgc tatttgtact 7860 gagtgagtac attggcataa atatagaaat ttatatatat acatatatat aaactattct 7920 tttttgccac acatttttgt ggtaaatttg tgagtttgtc tgatgttcta ccacaacgtg 7980 gcgtctgata acagtgaggg ggggtggggt ttgttatgtc tttattgagt atttaagtat 8040 cttttgaaac aaatgacctg ttcatctgtg gccattccat caggcagtta gttccttgat 8100 gtcagtagtg ggctaaaggc agcttactgt gtgtttgctg gagctttcac tcagccaagt 8160 gttagagtca ggaaacccat tgaggcaatg gcgtcaaatg gtgtttcaca agaatgagcc 8220 attcagtctt tgctcactat atatttaata ttttattatt gttgttattg ttattattaa 8280 ttggctttct gtattctatg ccttttattt ataaagacac taagaaaacc catgtttgta 8340 attttaataa catttttccc atcttgtaat atccagagct actttataaa ttctctgaac 8400 caaaagtatt ttcctcagtg tatctcttct cccccagccc ctattgggaa aaattaccca 8460 gtatagttca ggttatgagg aggatcagcc acacaatcca gtgcttcagt ttgaaaatgt 8520 aaaattctaa ccctaaagta gggttggttg aaatttcaga caaagcaaac ccagcaggta 8580 taaaaagtag tataaataca aatctgtaag ttatttttga attttctgaa cttttttcta 8640 agagattaca taggagacta aagaaatcta tctgttcaag ttctaattag gatgattgtt 8700 aatactgcac tgtggatgaa gtggcgactg gcttgtgtgc tgacttctgt ggtttagcaa 8760 gaggtttatt gttatcaaat gctaattggc aatgccaagt cactgggacc aattttctgt 8820 tttataatat ctaagtttag aacagaatat atacctgaac tgtagtggtt tgatcggatg 8880 gagacagaaa acccgatttt tattctcata aattttgtgg ttatttatac aagggctgtg 8940 ctatgctacc atattcttgt tcaataataa taggtttgtt gtttttttta cattgttaaa 9000 tgttccttac ccctaaaggt caatgttaag tacaacattc tgaaaataca atttggctac 9060 gaagagtatt catcttcttt gaagctcagt ggttgatatt tgtgctaata atgcaatttc 9120 ctgattactg ttacaagtta tagctacata tgggagagac tcagtgagcc agcaaaggcc 9180 atagaaacaa caatttatta aatgtattta tggcagaagg acctaaataa actgtgagcc 9240 accttttctt ctttatattg ttacatttaa gtgttcttgc tttcagcaac tcacattaat 9300 gcttggagct tatctctttc tctctctctc tctctctctc tgtgtgtgtg tgtgtatgtg 9360 tgtgtgtgtg tgtgtgtgtt tccttattgt cattccatta tatatccaca ccaacatggg 9420 tgacgataat tcaaagtcat attttgcctc taagcttgat catgttacct ttatgattaa 9480 agtatcatgt tatttagcca atgcaaatct gttttaaaac aaatagttta aaaaaagaac 9540 aagtttttaa gggctttatt atagaagaag tattaatgaa ggactttcct tcctccctcc 9600 ctttcctccc ctccctgcct cccttcttcc cttccatctc cccctcctcc ctgccttctt 9660 tgtttctcct tcccttattc ctccctccct cctttctccc ttccttcctt tcttccattc 9720 atccttcctt gccttttatt tttatttttt gtaatatcac atgtgctgta gtttggaatt 9780 ttattctagt gcatttcttg ctcatcagaa cctcagctaa tctacctagg aaaaatagta 9840 tcaaaggaaa tgagaaagtt gtatctgagt ccctccagaa ctaagataat tctttttgac 9900 catttaagcc tttataaatg cgttttgacc atttaagcct ttataaatgc ttgttttagg 9960 aaagtgaatc tgttagatgc atcaacaaat aatgaccagg acaaaacgat ttaataatta 10020 aagtctcaaa tcaccatggt tatacatttt caccagaaat agtaatctta caatttttca 10080 tttttctgat gaagatttct gttccaatat ctgtttccta atagattttt taaattaatt 10140 agctttcctc tgctttatga ccacaggttt tatccctaac cgagacagct gtcttatatc 10200 tgcatgcctt agactgtgtg gagggactcc atgaagaaag accataggtt agaaaaataa 10260 ctcatagtat ataccctagt aagtgggtta gtagaatctc ataacatgta ttaaaaagag 10320 gttttcttct ctgcttgttt gtgtcactag agcaaaattg tagagataat gctcataatg 10380 cagtaaatat cagaataata tctacaatat catttgtgga tggtcccagg tcccagtgct 10440 ctagttactt tacttctttt tttttttttg agatggagtc ttgctctgtc tctcaggcta 10500 gagcagtgtg cgatctcagc tcactgcagc ctccacctcc caggttcaag cgattctcct 10560 gcctcagcct cccaagtagc caggattaca ggcaccctcc actaggcccg gctaattttt 10620 tttgtatttt tttagtagag atggggtttt gccatgttgg ccaggctggt ttcgaactcc 10680 taacctccag tgatccacct gcctcggcgt cccaaagtgc taggattaca ggcatgagcc 10740 accacatccg gcctaattac ttctttaatc cccatttatt tttatgccat tctagcctca 10800 tttattaata aaattatgtt tttactttct ctttcaggaa attttttaaa ttaatatttt 10860 atatctagat ctaatgctat ggaaaagtgc ctttttatca tttataattt catttttcac 10920 tatttccaaa aacacataaa caaatagttt cagtaggtcc cagcttttac tttttccatt 10980 taaaccttct tttctccatt tcttcccttt ggcttaagaa taaaagaaaa ggtacattgc 11040 tagaattgtt tctttgggag agggtaaaag attacagaat tagactgttc agcctttata 11100 taaactaaat ttgtcttcat ctcaaccagc taatggtagg tcttatctga atactcatga 11160 gaattttagc atctgtgaaa ctccatgcac cagatgtgtg taaatttcag gaagaaagtg 11220 ttgaaagcat tttctctgat gttaattaga tggaaataaa tcactaaaac atagtttagg 11280 taaagcctga ttatgccact tttttttaac tagacagggc aaagttgttt atgttagtgt 11340 acttcttgtc tatcctcagt taatttacct agacaaaaag tgtcaaagga aatgagaaaa 11400 aggttatatc tgactccctc cagacctaag ataattcctt ttgatcagat acagtcagat 11460 ggagtgcctt ggtttttgtt aattttgcct ctattccagc tccttaccac agcggtggtg 11520 cttaaagaaa ggatcatcag caacaggtca ggatagttct acctttggga tagggctgct 11580 ttccccgtgc tagtatttct gtgactgtta gtggcactga ggactgcaaa cttttatgca 11640 atattcttaa taccctattg atattatgca ctttaatcat tccaaagaag ccaagaatgc 11700 tgtatagtga tgattccttc ctaatgaatt catcttaact atttagaatg ttatgtccct 11760 tttcttttgg atagccaact tggtataaat gttatatgga tttttctaaa atgactatat 11820 aggacttaag actttgaaat gtaatttact tataagggga aataattatg ctttagcaca 11880 tcattttaga aacgtcacat tttagaaaca ttcagcttgc taacctacat gtttgggaat 11940 tcattaaaac cagttgtcta tatattttgt gccatgtata taagaacatt acaatatatc 12000 tttttctaca tatgtagtat gtgcaaccag tggttctcag agtatggttc tcagcccacc 12060 agctagtatc agtatcacct gggaactagt tagaaatgta aattctttgg ccccatccca 12120 gacatactga gtcagaaact ctggaatagg gcccccgcaa tctgttttca caagccctcc 12180 aggtgattct gatgcacact ttaaagttta ggaaccactg ggctaagact ctgttgagat 12240 atagagtttt tcttccactc agactgatat agttatacat tgttcttcat gtaaattcag 12300 cttaacctgg ttatctataa tcttttattg gcaaaagtta attctcagta ctgcctatag 12360 agatacagtg tattttatgt acatacacaa ttagtctaat tcttgataat tcagttaatt 12420 tagtttggca ttttcctacc acttactaaa aggtttacat taaatgactg atttaaatat 12480 ataggtgcaa tgttctatgt ttattttaat tgttatgaca tttaagtagc taatataatt 12540 gaccggtgct aaagtctcct gtttatccat aaaatgggta cattatgggc agtgtaatac 12600 aagctttctt ttcattgcct agtactttac cagcagacca cagttttgcc ctggctagac 12660 caaccctcag aacaaaatca tcattccttg tatttatatt tgtatctgag atagtaaaca 12720 agatggctgg ccaggtcaac atggcacctt aacttatttt tttaataggt aaaacttctt 12780 caaaagtagc ttgctttgta taagaactaa gctatcagta tagatatagc tatccttgga 12840 gcttatgttt cagacaagaa ttatttacta aaataaataa taaacaagat aatgcattat 12900 acaatttggg catttctcgt ttctcaagtg tatgcatcat ggtaaatata aactaaccac 12960 aagataggta gattgattca tttcatttta atctccttgt gtaattcagt acctccataa 13020 ttgttctaat cttcttccca ctgtttacaa attaccagtt aattaactcg tgaaagaaaa 13080 attcacatat cagaataaaa ataaatgtat actcacttta taaaaatcac cactgctgtc 13140 tttccttaat actagcagtg gaaatgtaag tggcttactc tacaaatttt ggtgctggca 13200 aatacatagg caaactgttg ggagctgctc tagttacatt cctcccttct tattcccttt 13260 ttctcttcct cactttattg cataacatat tcctgtaccc aaagcattct accacagttc 13320 tatttgactc ccacttgtaa taactccttt aaaaaattcc atgtttaacc atatgaccct 13380 gcttgcttac tcatattctc cctccctctc cccttccttt ctctctcttc cagaagtcat 13440 ttgcctggtt tgaaatattt tgtagggatt gcttattata ttattttagc tgatgaacct 13500 caggacaacg tctacacaca cacacataca tacacgcaca caaaatctca gctgttgaag 13560 agtgggcttg gaatcagact tctgtgtcca gtaaaaaact cctgcactga agtcattgtg 13620 acttgagtag ttacagactg attccagtga acttgatcta atttcttttg atctaatgaa 13680 tgtgtctgct taccttgttt ccttttaatt gataagctcc aagtagttgc taattttttg 13740 acaactttaa atgagtttca ttcacttctt ttacttaatg ttttaagtat agtaccaata 13800 atttcattaa cctgttctca agtggtttag ctaccattct gccattttta atttttattt 13860 aattttattt gcttgagcac actgatcaac cactgaactg ccttcttcca ttgtcctgca 13920 atgatataag ggttacattt ttgtgtatat ggctttcata gttgggattt cagagcactg 13980 ataccagata ttttcagttt gttctctggg ggaatttcat ttgcatctat gtttttagct 14040 atctgtgata acttgttaaa tattaaaaag atattttgct tctattggaa catttgtata 14100 ctcgcaacta tatttctgta aacagctgca gtcaaaaata aaacactgaa agttaaaaaa 14160 aaaaaaaaaa aaaa 14174 <210> 2 <211> 23 <212> RNA <213> Artificial Sequence <220> 297R NFAT5 siRNA (S) <400> 2 augcccucgg acuucaucuc auu 23 <210> 3 <211> 23 <212> RNA <213> Artificial Sequence <220> 297R NFAT5 siRNA (AS) <400> 3 ugagaugaag uccgagggca uuu 23 <210> 4 <211> 23 <212> RNA <213> Artificial Sequence <220> 569R NFAT5 siRNA (S) <400> 4 augggcggug cuugcagcuc cuu 23 <210> 5 <211> 23 <212> RNA <213> Artificial Sequence <220> 569R NFAT5 siRNA (AS) <400> 5 ggagcugcaa gcaccgccca uuu 23 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> NFAT5 siRNA (S) <400> 6 cccucucagc aaggguuau 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> NFAT5 siRNA (AS) <400> 7 auaacccuug cugagaggg 19 <210> 8 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> inv569R (S) <400> 8 ccucgacguu cguggcgggu auu 23 <210> 9 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> inv569R (AS) <400> 9 uacccgccac gaacgucgag guu 23 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <220> Control siRNA (S) <400> 10 uucuccgaac gugucacgu 19 <210> 11 <211> 19 <212> RNA <213> Artificial Sequence <220> Control siRNA (AS) <400> 11 acgugacacg uucggagaa 19 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> A_NFAT5 siRNA (S) <400> 12 gcaaagaagu ggacauuga 19 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> A_NFAT5 siRNA (AS) <400> 13 ucaaugucca cuucuuugc 19 <210> 14 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> B_NFAT5 siRNA (S) <400> 14 gcauagcugu ucagugaaa 19 <210> 15 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> B_NFAT5 siRN (AS) <400> 15 uuucacugaa cagcuaugc 19 <210> 16 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> C_NFAT5 siRNA (S) <400> 16 gcaaguaacu cucuucuua 19 <210> 17 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> C_NFAT5 siRNA (AS) <400> 17 uaagaagaga guuacuugc 19

Claims (8)

Angiogenesis comprising, as an active ingredient, an NFAT5 expression or activity inhibitor selected from the group consisting of antisense oligonucleotides, siRNA (small interference RNA) oligonucleotides and NFAT5 protein-specific antibodies that complementarily bind to the nucleotide sequence of the NFAT5 gene. Inhibiting composition. The method of claim 1,
The NFAT5 gene is an angiogenesis inhibiting composition, characterized in that it has a nucleotide sequence represented by SEQ ID NO: 1. The method of claim 1,
The siRNA specific to the NFAT5 gene has an nucleotide sequence selected from the group consisting of SEQ ID NO: 2 to 7 and SEQ ID NO: 12 to 17. A therapeutic agent for angiogenesis-related diseases comprising the composition of claim 1. The method of claim 4, wherein
The disease includes rheumatoid arthritis, osteoarthritis, sepsis arthritis, psoriasis, corneal ulcer, macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ocular inflammation, cone cornea, Sjogren's syndrome, myopia ophthalmic tumor, cornea Transplant rejection, abnormal wound union, bone disease, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, demyelination of the nervous system, cirrhosis, renal glomerular disease, inflammatory bowel disease, periodontal membrane disease, arteriosclerosis, restenosis , Inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging and cancer, characterized in that selected from the group consisting of cancer therapeutic agents. Contacting a candidate to be analyzed with a biological sample comprising the NFAT5 gene or NFAT5 protein;
Measuring the expression level of the NFAT5 gene, the amount of NFAT5 protein or the activity of NFAT5 protein; And
When the amount of expression of NFAT5 gene, the amount of NFAT5 protein or the activity of NFAT5 protein is reduced in comparison with the control group which did not contact the candidates, the sample is a substance capable of preventing or treating a disease caused by excessive angiogenesis. A method for screening a substance for preventing or treating angiogenesis-related diseases, comprising the step of determining. The method of claim 6,
The measurement is reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA). : Method for screening a substance for preventing or treating angiogenesis-related diseases, characterized in that selected from the group consisting of radioimmunoassay, radioimmunodiffusion, and immunoprecipitation assay. A method of inhibiting excessive angiogenesis comprising administering the composition of any one of claims 1 to 3 to a mammal other than a human whose abnormal angiogenesis persists.

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