Composition for preventing or treating vascular diseases containing HAPLN1

Abstract

The present invention relates to a composition for preventing or treating vascular regression, repairing damaged blood vessels, and vascular diseases, comprising HAPLN1 as an active ingredient, and more specifically, provides a pharmaceutical composition and a functional health food composition for preventing or treating vascular diseases, comprising HAPLN1 as an active ingredient. HAPLN1 inhibits and improves elastin fibrosis damage and transgelin loss in vascular walls, and effectively inhibits phosphorylation of NF-κB or FAK in vascular smooth muscle cells (VSMCs), and can be used to prevent, improve, or treat vascular damage and various vascular diseases caused by aging and a high-fat diet.

Description

Technical Field

[0001] The present invention relates to a composition for promoting vascular regression, repairing damaged blood vessels, and preventing or treating vascular diseases, which contains HAPLN1 (hyaluronan and proteoglycan link protein 1) as an active ingredient.

Background Art

[0002] Blood vessels are tubes of various sizes present in the body of animals. They play a role in flowing blood carrying oxygen and nutrients to every corner of the body and receiving carbon dioxide and waste excreted by peripheral cells and tissues. Blood vessels are classified into arteries, veins, and capillaries according to their structure and function. Arteries are tubes that send blood containing oxygen and nutrients from the heart to the whole body and gradually become thinner and transition into capillaries. Substance exchange occurs between capillaries and tissues. Capillaries gather again to form veins and return to the heart. The total length of human blood vessels reaches 120,000 km.

[0003] While the aging process is accelerating globally and the interest in healthy longevity is increasing, the demand for the prevention and treatment of aging-related geriatric and chronic diseases is also increasing. Among the top 10 causes of death in the Republic of Korea, 8 are due to chronic diseases such as cancer, cardiovascular and cerebrovascular diseases, diabetes, and chronic respiratory diseases. Among them, vascular diseases are the second leading cause of death in the Republic of Korea and the first leading cause of death worldwide. Such vascular diseases further increase due to population aging, but increase rapidly after the age of 50, peak at the age of 75-79 corresponding to the average lifespan of Koreans, and then remain at a relatively high level until the age of 85. Vascular diseases are closely related to aging to the extent that "humans age with their blood vessels" and "aged blood vessels are fertile soil for cultivating the seeds of cardiovascular and cerebrovascular diseases."

[0004] Vascular diseases are diseases that induce blood supply disorders in tissues while the blood vessels are clogged or ruptured, causing various diseases such as arteriosclerosis, aneurysm, hypertension / bleeding, ischemia, and vascular wall disorders, leading to cerebral infarction, cerebral hemorrhage, ischemic heart disease, myocardial infarction, hypertension, etc. Representative vascular diseases include arteriosclerosis and hypertension. Arteriosclerosis is a disease in which cholesterol accumulates inside the blood vessels and becomes narrow or clogged, just like foreign substances accumulating and clogging an old water pipe. It is a chronic inflammatory cardiovascular disease that can cause angina pectoris or myocardial infarction, and in severe cases, sudden death may occur.

[0005] According to the "Baltimore Longitudinal Study of Aging" (BLSA), established in 1958 by the National Institutes of Health (NIH) in the United States, the main changes in blood vessels that occur with age are called "vascular aging" or "vascular aging", and its main characteristics include thickening of the blood vessel wall, decreased elasticity, and abnormal vascular endothelial cell function. Such structural and functional changes are presented as the ultimate main factors of arteriosclerosis and hypertension. In 2003, Dr. Martin of the University of Washington emphasized the importance of delaying aging (Aging delay) along with the importance of prevention rather than treatment while associating such vascular diseases with aging (aging). Therefore, in order to overcome vascular diseases, research on developing technologies and drugs that can delay or prevent vascular aging is very important. Also, in 2016, Peter et al. emphasized the necessity of prevention against cardiovascular diseases and proposed early vascular aging as its core concept.

[0006] The discovery and development of drugs that can prevent or reverse vascular media thickness and elasticity loss, which are the major changes in vascular aging, are known as the key to overcoming vascular-related diseases. However, at present, no such drugs have emerged. The cells that play a central role in such pathological changes are precisely vascular smooth muscle cells (VSMCs). VSMCs are a type of muscle cells that are most abundant in the normal vascular wall, especially in the media, and play a central role in the contraction and distensibility of blood vessels, and are mainly involved in the generation of vascular lesions.

[0007] In normal blood vessels, VSMCs produce elastin fibers, which are polymers of elastin, a major protein required for contraction and dilation. Transgelin (SM22α, TAGLN), a biomarker of healthy normal cells, differentiates into rich cells while simultaneously halting proliferation. Cells in this form are called contractile phenotype of VSMC. However, if chronic abnormalities occur in vascular function due to aging or degeneration, leading to a de-differentiated state and a return to a pre-differentiation stage, the cells are switched to the synthetic or proliferative phenotype of VSMC, restarting proliferation and migration, which becomes the starting point for the development of atherosclerosis. Such pathological changes are well known to be mainly caused by the loss of transgelin and are particularly important key biomarkers. As a result of such biochemical changes, the vascular wall at the lesion site thickens and hardens, and at the same time, various inflammatory cells such as monocytes are mobilized and activated. After going through such a series of processes, it causes various vascular diseases such as atherosclerosis, hypertension, aneurysm, and restenosis.

[0008] Regarding the prior art related to this HAPLN1 protein, by measuring the level of the protein, it has been proposed as a biomarker in terms of its use and role, or reported as a substance that may induce diseases rather than suppressing vascular diseases. Therefore, there is still no report presenting the possibility of this HAPLN1 as a pharmaceutical for treating or preventing related diseases by suppressing vascular diseases such as hypertension and atherosclerosis.

Summary of the Invention

Problems to be Solved by the Invention

[0009] An object of the present invention is to provide a therapeutic composition for vascular diseases using a protein having an effect of suppressing vascular aging and damage.

Means for Solving the Problems

[0010] To achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating vascular diseases containing HAPLN1 as an active ingredient.

[0011] In addition, the present invention provides a health functional food composition for preventing or improving vascular diseases containing HAPLN1 as an active ingredient.

Effects of the Invention

[0012] HAPLN1 according to the present invention suppresses and improves the damage of elastic fibrosis and the loss of transgelin in the vascular wall, and effectively suppresses the phosphorylation of NF-κB or FAK (Focal Adhesion Kinase) in vascular smooth muscle cells (VSMCs). Therefore, it can be used as a pharmaceutical composition or a health functional food composition for preventing or treating vascular damage caused by aging and high-fat diet and various vascular diseases.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0014] Hereinafter, the present invention will be described in detail.

[0015] The present inventor completed the present invention by confirming that the HAPLN1 protein can treat vascular diseases through various mechanisms.

[0016] The present invention provides a pharmaceutical composition for preventing or treating vascular diseases containing HAPLN1 as an active ingredient.

[0017] As used herein, "HAPLN1" is a protein that links hyaluronic acid to proteoglycans to stabilize hyaluronic acid, and is a constituent protein in the extracellular matrix first found in the joints of vertebrates.

[0018] Desirably, the HAPLN1 is a protein fragment consisting of the amino acid sequence represented by SEQ ID NO: 1, and is recombinant human HAPLN1 (rhHAPLN1).

[0019] SEQ ID NO: 1:

[0020] DHLSDNYTLDHDRAIHIQAENGPHLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEAQQACLDQDAVIASFDQLYDAWRGGLDWCNAGWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYN

[0021] In the present invention, the composition can prevent or treat vascular damage or various vascular diseases caused by reasons such as aging or a high-fat diet.

[0022] As used herein, "prevention" means any act of suppressing the occurrence of a vascular disease or at least one or more symptoms of the disease, or delaying the onset thereof, by administration of a pharmaceutical composition or a health functional food composition according to the present invention. Further, it includes treatment of a subject who is improving from the disease in order to prevent or prevent recurrence.

[0023] As used herein, "treatment" means any act that improves or advantageously modifies the symptoms of a vascular disease, such as alleviating, reducing, or eliminating at least one or more symptoms of the disease, by administering the pharmaceutical composition according to the present invention.

[0024] More specifically, the composition can suppress and improve the damage to the elastic fiber layer of the vascular wall, and can suppress and improve the loss of transgelin. Further, the composition can suppress the phosphorylation of NF-κB or FAK in vascular smooth muscle cells (VSMCs).

[0025] According to one embodiment of the present invention, it can be confirmed that the arterial vascular wall of a mouse damaged by a high-fat diet is restored to a shape substantially similar to that of the arterial vascular wall of a normal diet mouse by the administration of HAPLN1, the decreased transgelin area increases, and the phosphorylation of NF-κB and FAK is suppressed in human aortic smooth muscle cells, which are a type of vascular smooth muscle cells.

[0026] The composition having such effects is used for the prevention or treatment of vascular diseases, including vascular damage caused by aging or a high-fat diet, etc. For example, the vascular diseases are one or more selected from the group consisting of arteriosclerosis, hypertension, aneurysm, bleeding, cerebral infarction due to vascular wall disorder, cerebral hemorrhage, ischemic heart disease, myocardial infarction, and peripheral vascular disease, but are not limited thereto.

[0027] The pharmaceutical composition according to the present invention can be manufactured by ordinary methods in the pharmaceutical field. In addition to the active ingredient, the pharmaceutical composition is formulated with a pharmaceutically acceptable appropriate carrier depending on the dosage form, and can be further manufactured including, if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. The appropriate carrier, etc. does not inhibit the activity and characteristics of HAPLN1 according to the present invention and can be selected differently depending on the administration form and dosage form.

[0028] The pharmaceutical composition can be applied in any dosage form, and more specifically, it can be formulated and used in oral dosage forms, external preparations, suppositories, and parenteral dosage forms such as sterile injection solutions by ordinary methods.

[0029] Among the oral dosage forms, the solid dosage forms are in the form of tablets, pills, powders, granules, capsules, etc., and can be prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. In the case of capsule dosage forms, in addition to the substances mentioned above, a liquid carrier such as fatty oil may further be included.

[0030] Among the oral dosage forms, the liquid dosage forms include suspensions, oral solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be included.

[0031] The parenteral dosage forms may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. are used. As the base of suppositories, witepsol, macrogol, Tween 61, cocoa butter, laurin fat, glycerogelatin, etc. are used. Without being limited to this, any suitable preparation known in the technical field can be used.

[0032] In addition, calcium, vitamin D, etc. can be further added to the pharmaceutical composition according to the present invention to enhance the therapeutic efficacy. 3 and the like can be further added.

[0033] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.

[0034] As used herein, the "pharmaceutically effective amount" means an amount sufficient for the treatment of a disease at a reasonable benefit / risk ratio applicable to medical treatment, and that does not cause side effects.

[0035] The effective dosage level of the pharmaceutical composition is determined to vary depending on factors including the purpose of use, the age, sex, weight and health status of the patient, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the administration time, the administration route and excretion ratio, the treatment period, elements including the drugs formulated or used concomitantly, and other elements well known in the medical field. For example, although not constant, it is generally 0.001 to 100 mg / kg, and preferably 0.01 to 10 mg / kg, administered once or several times a day. The above dosage does not limit the scope of the present invention in any way.

[0036] The pharmaceutical composition can be administered to any animal in which a vascular disease occurs, and the animal can include, for example, not only humans and primates, but also livestock such as cows, pigs, horses, dogs, etc.

[0037] The pharmaceutical composition is administered by an appropriate administration route according to the dosage form, and is administered through various oral or parenteral routes as long as it can reach the target tissue. The administration method is not particularly limited and can be administered, for example, by ordinary methods such as oral, rectal or intravenous, intramuscular, topical skin application, subcutaneous, intratracheal inhalation, endometrial or intracerebroventricular injection.

[0038] The pharmaceutical composition is used alone or in combination with surgery or other drug treatments for the prevention or treatment of vascular diseases.

[0039] In addition, the present invention provides a health functional food composition for preventing or improving vascular diseases containing HAPLN1 as an active ingredient.

[0040] Preferably, the HAPLN1 is a protein fragment consisting of the amino acid sequence represented by SEQ ID NO: 1, and is recombinant human HAPLN1 (rhHAPLN1).

[0041] The composition can suppress and improve the damage to the elastic fiber layer of the blood vessel wall, and can suppress and improve the loss of transgelin. In addition, the composition can suppress the phosphorylation of NF-κB or FAK in vascular smooth muscle cells (VSMCs), and can prevent or improve vascular damage or various vascular diseases caused by factors such as aging or a high-fat diet.

[0042] As used herein, "improvement" means any act of improving or favorably changing the symptoms, such as alleviating, reducing, or eliminating vascular diseases or at least one or more symptoms of the diseases, by ingesting the health functional food composition according to the present invention.

[0043] The vascular diseases are one or more selected from the group consisting of arteriosclerosis, hypertension, aneurysm, hemorrhage, cerebral infarction due to vascular wall disorder, cerebral hemorrhage, ischemic heart disease, myocardial infarction, and peripheral vascular diseases, but are not limited thereto.

[0044] In the health functional food composition according to the present invention, the health functional food is for the purpose of preventing or improving vascular diseases, and can be manufactured in the form of powder, granule, tablet, capsule, syrup, or beverage, etc. There is no limitation on the form that the food can take, and it can include any food in the ordinary sense. For example, beverages and various drinks, fruits and their processed foods (canned fruits, jams, etc.), fish, meats and their processed foods (hams, bacons, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.), etc. are possible, and it can include any functional food in the ordinary sense. Also, foods used as feed for animals can be included.

[0045] The health functional food composition can be further manufactured by including food additives (food additives) and appropriate other auxiliary components that are commonly used by those skilled in the art and are acceptable in food science.

[0046] The suitability as the food additive can be determined by the standards and criteria regarding the item according to the general rules and general test methods of the Food Additive Codex approved by the Ministry of Health, Labour and Welfare, unless there are other regulations. Examples of the items listed in the "Food Additive Codex" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, cinnamic acid; natural additives such as indigo carmine, licorice extract, crystalline cellulose, safflower pigment, guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, tar pigment preparations, and the like.

[0047] The other auxiliary components can further contain, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents, and the like. In particular, as the natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol can be used, and as the sweeteners, natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used.

[0048] The effective volume of HAPLN1 contained in the health functional food according to the present invention is appropriately adjusted according to its intended use, such as the prevention or improvement of vascular diseases. The composition has the advantage of having no side effects such as those occurring during long-term administration of general drugs when using food as a raw material, is excellent in portability, and is ingested as an adjuvant for the prevention or improvement of vascular diseases.

[0049] Hereinafter, in order to assist the understanding of the present invention, examples will be given for detailed description. However, the following examples illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more fully explain the present invention to those skilled in the art.

[0050] <Example 1> Production of Recombinant Human HAPLN1 Protein (rhHAPLN1)

[0051] 1-1. Amino acid sequence of recombinant human HAPLN1 protein

[0052] The amino acid sequence constituting the recombinant HAPLN1 protein is as follows.

[0053] DHLSDNYTLDHDRAIHIQAENGPHLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEAQQACLDQDAVIASFDQLYDAWRGGLDWCNAGWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYN (SEQ ID NO: 1)

[0054] 1-2. Expression, purification and storage of recombinant human HAPLN1 protein (rhHAPLN1)

[0055] For the production of recombinant human HAPLN1 (rhHAPLN1; gene number 2678736), the DNA vector encoding the recombinant human HAPLN1 amino acid sequence was used as the host cell Expi TM293 cells (ThermoFisher Scientific Co., Waltham, MA, USA) were used. For purification, it was expressed with a secretion signal peptide and 10 histidines (H, His, histidine) at the amino terminus, and a TEV protease-recognition site inserted. Three days after vector infection, the culture medium was collected and purified through a HisTrap column (GE Healthcare, IL, USA). Then, the TEV protease-recognition sequence was cleaved with TEV protease, and the molecules containing histidine were removed using DynaBeads (Thermo Fisher Scientific). The solution thus obtained was dialyzed against 40 mM Tris-HCl, 1 M NaCl, pH 8.0 for 16 hours. The protein concentration of the final purified product was 0.11 mg / ml, and the solvent was 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0, 50% glycerol. After aliquoting as a single dose, it was stored in a -20°C refrigerator for use.

[0056] <Example 2> Confirmation of the effect of repeated intraperitoneal administration of recombinant human HAPLN1 protein (rhHAPLN1) on improving the arterial vascular structure of high-fat diet mice

[0057] 2-1. Preparation and breeding of experimental animals

[0058] Experimental animals were set up with 9-week-old female C57BL / 6J (Korea Basic Science Institute (KBSI), Daejeon, Republic of Korea) mice as the control group (PBS) and the administration group (rhHAPLN1). Water was changed every two days to allow free access to food. The mice were placed in cages of four each, and 22 g of high-fat diet was given daily. The temperature in the breeding room was maintained at 21 - 24 °C, the humidity at 40% - 60%, and the day and night cycles were each set to 12 hours. The control group and the administration group were each assigned eight mice. The administration group (rhHAPLN1) was intraperitoneally injected (IP injection) with recombinant rhHAPLN1 diluted in phosphate buffered saline (PBS) at a dose of 0.1 mg / kg in a volume of 100 μl once every three days for a total of 10 times over 30 days. The remaining two control groups were administered the same amount of PBS in the same manner.

[0059] 2-2. Confirmation of the improvement effect of recombinant human HAPLN1 on vascular structure by H&E staining of the arterial wall of high-fat diet-fed mice

[0060] The high-fat diet mice were fed an atherogenic Paigen diet (PD; 15.8% fat: about half cocoa butter, 1.25% cholesterol and 0.5% cholate) diet for 16 weeks (product number TD88051). After intraperitoneally injecting PBS or rhHAPLN1 into the high-fat diet mice, the mice in each group were anesthetized, and then the abdomen was incised to collect the mouse thoracic artery blood vessels to observe the arterial wall.

[0061] As a result, as shown in Figure 1, it can be confirmed that typical atherosclerotic lesions are clearly shown in the thoracic arterial wall of the high-fat diet PBS group in the middle drawing compared to the thoracic arterial wall of normal diet mice and the thoracic arterial wall of high-fat diet administration group mice (rhHAPLN1). In particular, it can be confirmed that the elastic fiber layer between the media and the intima is decomposed or destroyed. On the other hand, it was confirmed that the thoracic arterial wall at the same site in the group administered with human recombinant HAPLN1 protein was significantly restored to a shape almost similar to that of the arterial wall of normal diet mice.

[0062] 2-3. Confirmation of the improvement effect of rhHAPLN1 on elastic fibers in the arterial wall of high-fat diet-fed mice

[0063] When there is aging and disease of the arterial wall, elastic fibers are gradually degraded and broken down (Cardiovascular Research, Volume 110, Issue 3, 1 June 2016, Pages 298 - 308).

[0064] Thus, changes in the elastic fiber layer were confirmed in the thoracic aorta samples of high - fat diet mice produced as described in 2 - 2 above using the Verhoeff’s Van Gieson / EVG (ab150667, USA) staining method. After the aortic samples slides underwent deparaffinization and re - hydration processes, elastic fiber staining reagents and differentiation reagents were used. After staining with sodium thiosulfate solution and Van Gieson solution, they were finally dehydrated in alcohol. Finally, they were fixed using Canada balsam (C1795, Sigma).

[0065] To maintain the accuracy of the measurement, the same region was selected and measured in the arterial staining part. Considering that the elastic lamina can vary depending on the cross - section of the aortic wall, the same cross - section at about 40% from the top to the bottom was selected, and a magnification of 400 times was used to evaluate the degradation and recovery of the elastic fiber layer. Mice fed a normal diet were used as the negative control group, and mice fed a high - fat diet were used as the positive control group for the elastic fiber layer.

[0066] In addition, the evaluation criteria for the elastin degradation stage were implemented in four stages as follows:

[0067] Stage 1: There is no elastin degradation, and the elastin layer is completely present in the tissue.

[0068] Stage 2: There is slight degradation or rupture of the lamina elastin.

[0069] Stage 3: Moderate degradation or some rupture phenomena of thin-film elastin occur.

[0070] Stage 4: Elastin is severely degraded and lost, or the aortic wall ruptures.

[0071] The black area (elastin+Area) of the elastin fibers observed from FIG. 3 was used to judge the recovery effect of the elastin fibers using Image J software. The data were presented as mean ± SD ( ** P<0.01).

[0072] As a result, as shown in FIGS. 2 to 4, it was confirmed that the rhHAPLN1 treatment group statistically significantly suppressed the elastin fiber layer degraded by the high-fat diet compared with the control group.

[0073] 2-4. Transgelin (SM22α) staining and quantification of the mouse arterial wall

[0074] Quantitative changes in transgelin (SM22α), an important biomarker of atherosclerotic arterial vessel walls, were observed. To observe the changes in transgelin levels in another rhHAPLN1 intraperitoneal administration group of mice (n = 8) fed a high-fat diet for 4 months, staining was performed using a red transgelin antibody labeled with a fluorescent substance (anti-transgelin antibody labeled with Alexa Fluor). In addition, the red area (Transgelin+Area) of the red transgelin region was measured using Image J software.

[0075] As a result, as shown in FIGS. 5 and 6, it was confirmed that the area of transgelin increased in the rhHAPLN1 treatment group.

[0076] <Example 3> Confirmation of the effect of changes in the signal transduction pathway by administration of recombinant human HAPLN1 protein (rhHAPLN1) to vascular smooth muscle cells (VSMCs)

[0077] 3-1. Cell culture of human aortic smooth muscle cells (HASMC; a type of VSMC)

[0078] Human aortic smooth muscle cells (304 - 05a, Cell Applications, USA) were cultured in HASMC growth medium (#311 - 500) using a T75 flask. The cells were cultured in a humidified incubator at 37°C with 5% CO 2 and incubated. When the HASMC cells grew to about 80 - 90%, they were subcultured. Cells with passage numbers 4 - 8 were used in the experiment.

[0079] 3-2. Confirmation of the inhibitory effect on NF-kB phosphorylation in HASMC

[0080] Inflammation and hyperproliferation of HASMC play a central role in various vascular diseases including restenosis, atherosclerosis, and hypertension. In cultured HASMC, the inflammatory and cell - proliferative cytokine IL - 1β suppresses the expression of VSMC anti - atherosclerotic marker genes and induces the expression of inflammatory genes. When NF - κB is activated, it can promote the proliferation of HASMC, induce the migration of HASMC, and up - regulate other matrix metalloproteinases (MMPs) that cause extracellular matrix (ECM) damage and plaque rupture. Therefore, suppressing the expression of NF - κB has important implications for the regulation of atherosclerosis.

[0081] As a result of treating cultured HASMC cells with interleukin - 1β (IL - 1β), which is known as a vascular aging inducer, to confirm the changes in the activation and phosphorylation of NF - κB, as shown in Figure 7, it was confirmed that phosphorylated NF - κB (p - NF - κB) increased by IL - 1β was suppressed by recombinant human HAPLN1 protein (rhHAPLN1). This means that NF - κB is not only related to inflammatory factors but can also affect the proliferation of VSMC cells.

[0082] 3-3. Confirmation of the inhibitory effect on FAK phosphorylation in human aortic smooth muscle cells (HASMC)

[0083] Excessive proliferation and migration of HASMC contribute to occlusive cardiovascular diseases such as arteriosclerosis. HASMC in mature blood vessels have suppressed proliferation and migration activities. However, after vascular injury, inflammatory stimuli increase the secretion of growth factors and matrix synthesis, promoting the proliferation of HASMC and their migration into the vascular intima, thus inducing arteriosclerosis such as arterial stenosis.

[0084] FAK (focal adhesion kinase) regulates the proliferation of vascular smooth muscle cells (VSMC) and the growth of the new intima through GATA4-mediated Cyclin D1 transcription. FAK is a type of protein tyrosine kinase that mediates integrin and growth factor signaling pathways related to cell proliferation and migration, and is involved in the migration, proliferation, and inflammatory response of HASMC. Therefore, suppressing the activation of FAK through phosphorylation means that the onset of arteriosclerosis can be suppressed.

[0085] As a result of treating cultured HASMC cells with interleukin-1β (IL-1β), which is known as an inducer of vascular aging, to confirm changes in FAK activation and phosphorylation, as shown in Figure 8, it was confirmed that phosphorylated FAK (p-FAK) increased by IL-1β was suppressed by recombinant human HAPLN1 protein (rhHAPLN1).

[0086] As described in detail the specific part of the content of the present invention above, it is clear to those skilled in the art that such a specific description is merely a desirable embodiment, and thus does not limit the scope of the present invention. That is, the substantial scope of the present invention is defined by the claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating vascular diseases, containing HAPLN1 as an active ingredient, wherein the vascular diseases are one or more selected from the group consisting of arteriosclerosis, hypertension, aneurysm, hemorrhage, cerebral infarction due to vascular wall disorder, cerebral hemorrhage, ischemic heart disease, myocardial infarction (excluding inflammatory myocardial infarction), and peripheral vascular diseases.

2. The HAPLN1 is a protein fragment, and the pharmaceutical composition according to Claim 1 is characterized by consisting of the amino acid sequence represented by SEQ ID NO:

1.

3. The composition is characterized by suppressing and improving the damage of the elastic fiber layer of the vascular wall, and is the pharmaceutical composition according to Claim 1.

4. The composition is characterized by improving the loss of transgelin, and is the pharmaceutical composition according to Claim 1.

5. The composition is characterized by suppressing the phosphorylation of NF-κB or FAK in vascular smooth muscle cells (VSMCs), and is the pharmaceutical composition according to Claim 1.

6. A health functional food composition for preventing or improving vascular diseases, containing HAPLN1 as an active ingredient, wherein the vascular diseases are one or more selected from the group consisting of arteriosclerosis, hypertension, aneurysm, hemorrhage, cerebral infarction due to vascular wall disorder, cerebral hemorrhage, ischemic heart disease, myocardial infarction (excluding inflammatory myocardial infarction), and peripheral vascular diseases.

7. The HAPLN1 is a protein fragment, and the health functional food composition according to Claim 6 is characterized by consisting of the amino acid sequence represented by SEQ ID NO: 1.

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