WO2014084433A1 - Composition comprising peroxisome proliferator-activated receptor delta agonist, as active ingredient, for promoting treatment of myocardial injury after myocardial infraction - Google Patents

Abstract

The present invention provides a composition comprising (PPAR) peroxisome proliferator-activated receptor (PPAR) delta agonist, as an active ingredient, for promoting a treatment of a myocardial injury after a myocardial infraction so as to prevent and treat cardiovascular diseases.

Description

Composition for promoting myocardial wound healing after myocardial infarction containing peroxysome growth factor activating receptor delta agonist as an active ingredient

The present invention relates to a myocardial injuries treatment composition after myocardial infarction, and more particularly, to a myocardial injuries treatment composition comprising a PPAR-delta agonist as an active ingredient.

Although mortality from coronary heart disease has decreased due to the development of reperfusion therapy, mortality and re-hospitalization rate due to heart failure in survivors have increased relatively. The myocardium constantly beats, causing pressure on the necrotic heart wall while the wound heals. If the necrotic tissue is not replaced quickly with the appropriate connective tissue, the heart wall may rupture prematurely and progress to heart failure by ventricular remodeling at the end. In addition, delayed healing of myocardial infarction can also lead to ventricular remodeling by deforming the wall through pressure on the heart wall in other healthy myocardium. Therefore, it is most important to heal quickly into connective tissue that can withstand the appropriate wall pressure in a short time.

Treatment strategies to prevent progression to heart failure after myocardial infarction include acute treatment strategies to prevent early reperfusion and reperfusion injury. Recently, stem cell therapy is the main method, but there are few treatments and drugs that promote myocardial healing after reperfusion. . There is a lot of research around the world, but the development of therapeutics has not been done yet. This is because most treatments and studies for acute myocardial infarction focus on the treatment of early inflammation. Too much inflammation, or too much inflammation, has led to a lack of proper myocardial healing, and new procedures, therapeutics, and basic research are reaching their limits.

Conventional drugs used after myocardial infarction have been suggested to reduce collagen production in acute myocardial healing, and there is a problem that can not be used in hypotension, bradycardia or contraindications.

The present invention is to solve a number of problems, including the above problems, in order to properly control the initial inflammatory stage, to provide a therapeutic agent that acts on the proliferation and regeneration of fibroblasts, activated by myocardial infarction It is an object of the present invention to provide a therapeutic composition that targets PPAR-δ (delta), which promotes the healing of damaged myocardium after myocardial infarction and prevents complications caused by delay in healing. However, these problems are exemplary, and the scope of the present invention is not limited thereby.

According to an aspect of the invention, there is provided a composition for promoting myocardial wound healing after myocardial infarction, which contains a peroxisome proliferator-activated receptor (PPAR) -delta agonist as an active ingredient.

In the composition, the PPAR-delta agonist is GW610742 (GlaxoSmithKline), GSK-0660 (CAS 1014691-61-2), GSK 3787 (CAS 188591-46-0), and GW 501516 (CAS 317318-70-0 It may be one or two or more selected from the group consisting of). In particular, the composition can be used to promote healing of damaged myocardium after acute myocardial infarction.

The composition can be administered orally or parenterally during clinical administration, and is expected to be possible intracoronary administration after the first reperfusion procedure of acute myocardial infarction.

Each oral composition according to one embodiment of the present invention further comprises an inert ingredient, including a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier refers to a composition, specifically an ingredient except for the active substance of the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include binders, disintegrants, diluents, fillers, glidants, solubilizers or emulsifiers and salts.

In addition, the pharmaceutical composition according to the embodiment of the present invention uses a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention belongs. Can be prepared in unit dose form or incorporated into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

Suitable dosages of the compositions vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity of the patient, food, time of administration, route of administration, rate of excretion and response sensitivity, and are usually The physician can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, a suitable daily dose may be between 100 μg / kg and 1 mg / kg body weight, which dose can be adjusted by further study results.

According to another aspect of the present invention, there is provided a method for treating myocardial wound after myocardial infarction, comprising administering the above-described composition to an individual suffering from cardiovascular disease.

According to one embodiment of the present invention made as described above, it can be used as a composition for promoting myocardial wound healing after myocardial infarction by a composition containing a PPAR-delta agonist (agonist) as an active ingredient.

1 is a photo of confirmed through tissue staining after the administration of the PPAR-delta agonist GW610742 according to an embodiment of the present invention to myocardial infarction, tissue fibrosis and collagen deposition (Fig. 1a), collagen protein Western blot confirming the expression (Fig. 1b), angiogenesis (CD31) and endothelial cell proliferation (Ki-67) is an illustration showing schematically the picture confirmed through tissue staining (Fig. 1c).

FIG. 2 shows the results of performing echocardiography (FIG. 2A) and the extent of recovery of the left ventricle after administration of a PPAR-delta agonist GW610742 according to an embodiment of the present invention to a myocardial infarction model. Fig. 2b shows a graph (Fig. 2b) confirmed through LVIDS, LVEDV, LVESV, FS and EF-AL values.

FIG. 3 is a Western blot photograph confirming fibrosis and neovascularization of the left ventricle through expression of TGF-beta and MMP-9 after administration of GW610742, a PPAR-delta agonist, according to an embodiment of the present invention, to a myocardial infarction model. (FIG. 3A), fluorescence micrograph (FIG. 3B) confirmed through double staining of alpha-SMA and TGF-beta, and photographs confirming that expression of TGF-beta and MMP-9 increased around the cardiovascular vessel (FIG. 3C). It is a figure which shows.

4 is a Western blot photograph (FIG. 4A) confirming the differentiation into myofibroblasts using alpha-SMA after administration of the PPAR-delta agonist GW610742 according to an embodiment of the present invention to myocardial infarction model, alpha It is a figure which shows the histochemical staining photograph (FIG. 4B) using SMA, and the Western blot photograph (FIG. 4C) which confirmed the expression of TIMP.

Figure 5 is a result of confirming the myocardial infarction treatment effect of the PPAR-delta agonist GW610742 according to an embodiment of the present invention using a cell, cell number calculation method that increases the proliferation of fibroblasts and endothelial cells (HUVEC) Graph confirmed through (Fig. 5a), Western blot image (figure 5b) confirmed by using alpha-SMA fibroblasts differentiation into myofibroblasts, endothelium conditioned medium (conditioned medium) and TIMP- by GW610742 Western blot photograph (Fig. 5c) confirms that the expression of 3 is increased, and optical micrograph (Fig. 5d) confirms that the fibroblasts are differentiated into myofibroblasts and the mobility is increased.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the present invention is not limited to the examples and experimental examples disclosed below, but may be embodied in various different forms. The following examples and experimental examples are provided to make the disclosure of the present invention complete and the general knowledge. It is provided to fully inform those who have the scope of the invention.

Example 1 Breeding of Mice

Male SD (Sprague Dawley) rats (230-250 gm; Koatech Animals Inc., Peongtaek, Gyeonggi-Do, Korea) were used, and all mice were controlled in temperature, light / dark cycle, and free diet. And water were supplied. All animal experiments were performed in accordance with the guidelines of the Gyeongsang National University Animal Care and Use Committee (GLA-110324-R0022).

Example 2: Preparation of a Myocardial Infarction Model

The left anterior coronary artery (LAC) of anesthetized SD rats was induced by ligation using nonabsorbable 4-0 silk ligation. After performing the ligation as described above, vascular occlusion was confirmed through a left ventricular musculature blanching test.

The PPAR-δ agonist GW610742 (GlaxoSmithKline, Stevenage, UK) was directly administered through the peritoneum to the myocardial infarction model prepared as described above. GW610742 (1 mg / kg) was prepared by diluting in dimethyl sulfoxide (DMSO) and then administered every 3 days. The myocardial infarction model was randomly divided into 5 groups: (1) control group without left anterior coronary artery ligation (sham control group: 1 week control, n = 2 and 2 weeks control, n = 2); After coronary artery ligation, 10% DMSO twice a week (1 week MI group, n = 10); (3) After left anterior coronary artery ligation, 10% DMSO four times for 2 weeks ( 2 weeks MI group, n = 10); And (4) GW610742 administered twice a week after left anterior coronary artery ligation (1 week MI + GW group, n = 10). (5) GW610742 administered four times for 2 weeks after left anterior coronary artery ligation. Group (1 week MI + GW group, n = 10). The control group was sacrificed at the same time as the experimental group.

Example 3: Cell Culture

3-1: Fibroblast Culture

Fibroblasts were donated by Professor Sang-Soo Kang of Gyeongsang National University, and were planted in a 6 cm petri dish at a ratio of 4 x 10 ⁵ and in DMEM medium (Lonza, Swiss) containing 10% fetal bovine serum (FBS). The cells were incubated for 3 days in a cell incubator, and maintained at 37 ° C., 20% oxygen, 5% carbon dioxide, and 20% bovine.

3-2: Endothelial Cell Culture

Human endothelial cells (ATCC CRL-1730) were planted at a ratio of 3 x 10 ⁵ in a 6 cm petri dish and 3 in EGM-2 medium (Clonetics, Swiss) added with 10% FBS, rhEGF, FGF and VEGF. The cells were incubated in a cell incubator for one day, and the incubator was maintained at 37 ° C., 20% oxygen, 5% carbon dioxide, and 20% bovine.

Experimental Example 1: Measurement of changes in fibrosis and angiogenesis levels by PPAR-delta agonists in myocardial infarction model

In order to confirm the treatment effect of myocardial infarction of PPAR-delta according to an embodiment of the present invention, after administering the PPAR-delta agonist GW610742 to the myocardial infarction model of Example 2, the most observed in the remodeling process of the ventricles Fibrosis level, an important phenomenon, was observed.

1-1: Confirmation of increased fibrosis by PPAR-delta agonist using tissue staining

In order to analyze the effect on fibrosis, Masson's trichrome tissue staining method and tissue staining for collagen I were performed. Masson's trichome tissue staining method is a useful method for detecting collagen that is differentiated. After staining by the method, the collagen appears blue. Specifically, after 1 week or 2 weeks of the PPAR-delta agonist 1 mg / kg GW610742 in the myocardial infarction model of Example 2, perfusion with 1xPBS solution, heart tissue is separated by 4 ℃, 4% PFA The tissue was fixed in (phosphate-buffered paraformaldehyde) and then embedded in paraffin. Thereafter, tissue sections were made into 5 μm thick coronal tissue (Coronal) using a tissue sectioner. After removing paraffin by using xylene, the tissue sections were rehydrated and washed, and ten infarct tissues were selected at random, followed by Masson's trichrome tissue staining. Each tissue was analyzed under 400 magnification, and trichome positive signal was analyzed using Image-Pro PLUS (Meyer Instruments, Houston, TX).

As a result, as shown in the upper panel of Figure 1a, the degree of fibrosis was significantly increased in the MI + GW group compared to the MI group was confirmed through tissue staining pictures and graphs. The graph quantifies the result of analyzing the trichome positive signal.

Next, the present inventors performed tissue staining using collagen I. The tissue prepared as described above was blocked using 1% normal goat serum and reacted with the collagen I antibody for 16 to 24 hours under humid conditions at 4 ° C. Thereafter, the tissues were washed with PBS and reacted with the secondary antibody at room temperature for 90 minutes. The secondary antibody was used by diluting at a ratio of 1: 200 with anti-rabbit IgG to which biotin was bound. The tissue was then reacted with avidin-bitinylated-HRP complex for 60 minutes at room temperature, washed with PBS and washed with 0.003% hydrogen peroxidase and 0.027% DAB (3,3′-diaminobenzidine tetrahydrochloride, Sigma -Aldrich, USA) and staining was performed using hematoxylin for visualization.

As a result, as shown in FIG. 1B, the collagen I signal was strongly observed in the MI + GW group treated with GW610742 as compared to the MI group.

1-2: Increased fibrosis by PPAR-delta agonist using Western blot

PPAR-delta according to an embodiment of the present invention through the expression level of collagen protein in the heart tissue of the myocardial infarction model of Example 2 administered with PPAR-delta for 1 week under the same conditions as in Experimental Example 1-1 By quantitatively confirm the regeneration of myocardial infarction heart.

The PPAR-delta agonist was administered to the myocardial infarction model of Example 2 for one week, the animal was sacrificed, the right ventricular portion was surgically removed, the left ventricle was separated into the infarct area and the non-infarct area, and frozen. For protein isolation, frozen heart tissues at the infarct or non-infarct sites were placed in liquid nitrogen, respectively, and protein lysis buffer containing 10 μM leupeptin and 200 μM phenylmethylsulfonyl fluoride (1% Triton X-100). and 1 mM ethylene diamine tetra acetic acid in 1 × phosphate buffered saline (PBS; pH 7.4)]. The tissue sample was homogenized and the lysate was centrifuged at 13,000 rpm for 20 minutes. Then, the supernatant was obtained, and the protein concentration of each lysate was measured using a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL), and bovine serum albumin was used as a control.

Then, 40 μg protein sample was mixed with the same amount of sample buffer [2% Sodium Dodecyl Sulfate, 100 mM Tris, 0.2% bromophenol blue, 20% glycerol, and 200 mM dithiothreitol], then boiled for 10 minutes and boiled for 10-12 Proteins were isolated by loading onto% polyacrylamide gel. The protein separated from the gel was transferred to a nitrocellulose membrane and reacted with anti-collagen I (Calbiochem, Germany), and the membrane was subjected to a stripping reaction and a-tubulin as a control. Reacted.

As a result, as shown in FIG. 1B, the MI + GW group administered with GW610742 increased compared to the MI group, which is consistent with the result of FIG. 1A (see FIG. 1B). These results indicate that the PPAR-delta agonist according to an embodiment of the present invention affects the regeneration or remodeling process of infarcted heart tissue. An increase in fibrosis levels in infarcted heart tissue was first demonstrated by the inventors.

1-3: Increase of neovascularization by PPAR-delta agonist using tissue immunostaining

After induction of myocardial infarction, neovascular changes are one of the pathophysiology of heart failure. Angiogenesis, along with collagen deposition and myofiber accumulation, is known to promote heart recovery following myocardial infarction. Thus, in order to confirm whether the PPAR-delta agonist according to an embodiment of the present invention is involved in neovascularization, tissue staining for CD31 and Ki-67 was performed. One week after administration of GW610742 to the myocardial infarction model of Example 2, the obtained heart tissue was used, and Masson's trichrome tissue staining was performed to check the fibrosis level of the tissue.

First, to confirm whether the PPAR-delta agonist is involved in neovascularization, the cardiac tissue of the myocardial infarction model was stained using CD31, a vascular endothelial cell marker. As a control group, a sham control group that did not cause infarction was used. After one week after GW610742 was administered to the myocardial infarction model of Example 2, the obtained heart tissue was stained using CD31, a vascular endothelial cell marker.

As a result, as shown in Figure 1c, CD31 immunoreactivity was observed in the revascularized endothelium and mature blood vessels, more observed in the MI + GW group than the MI group (see Figure 1c). Importantly, the regenerating small blood vessels in which the CD31 signal is observed are more present in the infarcted heart tissue administered GW610742.

Next, the present inventors stained the heart tissue of the myocardial infarction model using Ki-67, which is known as a marker of proliferating cells, to confirm whether the endothelial cell proliferation is accompanied by PPAR-delta administration. The control group was a sham control group that did not cause infarction.

As a result, as shown in Figure 1c, Ki-67 immune response was not observed at all in the Siamese control cardiac tissue. Most Ki-67 immunoreactivity was observed in the MI group which induced myocardial infarction and the MI + GW group administered with GW610742, and the Ki-67 positive signal was markedly strong in the MI + GW group (see FIG. 1C).

These results indicate that the PPAR-delta according to one embodiment of the present invention acts on angiogenesis that promotes left ventricular remodeling after myocardial infarction.

Experimental Example 2: Measurement of left ventricular structure and function change by PPAR-delta agonist in myocardial infarction model

In order to confirm the left ventricle (LV) regeneration effect of the myocardial infarction animal model induced infarction by ligation of the left ventricle in Example 2 by GW610742, a PPAR-delta agonist according to an embodiment of the present invention, the myocardium After administration of GW610742 to the infarct model, the structural changes of the heart tissues were confirmed by echocardiography (Transthoracic echocardiography, TTE). The test was performed after sacrificing the myocardial infarction model for a certain period of time after the myocardial infarction surgery. The animals were lightly anesthetized using ketamine and xylazine together. Two-dimensional and M-mode echocardiograms were measured using a 12-MHz linear transducer (GE Healthcare, Wauwatosa, WI) equipped with a VIVID Q system. The M-mode image was taken at the papillary muscle level of LV. Fractional shortening (FS), ejection fraction (EF), LV internal dimension at diastole (LVIDD), diastolic interventricular septal thickness (IVS), LV diastolic posterior wall thickness (LVPW) and LV volume were measured using M-mode images. . In addition, LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) were calculated through two-dimensional images using a one-sided “area-length” (AL) method. Stroke volume (SV), cardiac output (CO), and EF values (EF-AL) using the AL method were calculated by LVEDV and LVESV measurements.

As a result, as shown in Figure 2, LVEDV, LVESV, LVIDD, and LVIDS values showed a tendency to increase in the MI + GW group compared to the MI group. At baseline, the EF-AL level was higher in the MI group than in the MI + GW group. On the other hand, after one week, no difference was observed between FS and EF-AL levels between the MI and MI + GW groups. In addition, changes in LVEDV and LVESV between 0 and 1 weeks were much higher in the MI + GW group than in the MI group. However, no significant change was observed at 2 weeks (see Table 1). These results indicate that the expansion of LV occurs first, and according to this expansion, functional recovery of LV is accelerated more in MI + GW group than MI group.

Experimental Example 3: Confirmation of left ventricular regeneration effect by PPAR-delta agonist in myocardial infarction model

In order to confirm the LV regeneration effect by GW610742, a PPAR-delta agonist according to an embodiment of the present invention, after administration of GW610742 to the myocardial infarction model of Example 2, changes in fibrosis and angiogenesis of the heart tissue The expression levels of TGF-β and MMP-9 were measured.

Specifically, after sacrificing an animal administered with GW610742 for one week in the myocardial infarction model of Example 2, cardiac tissues were isolated and the expression levels of the TGF-β and MMP-9 proteins were analyzed using Western blot and immunostaining method. It was confirmed through.

As a result, as shown in Figure 3a, it was confirmed that the expression of TGF-β and MMP-9 significantly increased in the MI + GW group administered with GW610742 compared to the myocardial infarction MI group.

These results could also be observed in the immunostaining results of FIG. 3b. In particular, MMP-9-positive cells were observed around the blood vessels at the fibrosis site of myocardial infarction heart tissue, and these signals were more strongly distributed in the MI + GW group than in the MI group. In particular, positive signals of alpha-SMA and TGF-beta2, alpha-SMA and MMP-9 are mainly located in the small blood vessels that are regenerated in myocardial infarction heart tissue of the MI + GW group. Compared with the MI + GW group it was confirmed that there is more strong and a lot present (see Figure 3c).

Experimental Example 4: Confirmation of the effect of promoting the differentiation of fibroblasts into myofibroblasts of PPAR-delta agonists

The most important process in cardiac remodeling is the extensive fibrosis process. Fibrosis is initiated by the proliferation and differentiation of fibroblasts. The fibroblasts are called myofibroblasts when they have muscle-like properties, such as contractile stress fibers containing alpha-SMA. Thus, in order to determine whether the PPAR-delta agonist according to an embodiment of the present invention affects the morphological differentiation of fibroblasts into myofibroblasts, alpha-SMA was analyzed by Western blot and immunostaining using myocardial infarction heart tissue. Was performed. In this case, the myocardial infarction heart tissue refers to a tissue obtained by sacrificing the group administered with the PPAR-delta agonist for a week to the myocardial infarction model of Example 2.

Western blot analysis of protein using cardiac tissue revealed alpha-SMA in the MI + GW group administered with PPAR-delta agonists compared to the control (C) and myocardial infarction model (MI), as shown in FIG. 4A. The protein expression of was increased, the alpha-SMA protein level was significantly higher in the MI + GW group than the MI group (see Figure 4a).

Subsequently, the present inventors performed immunostaining for alpha-SMA in cardiac tissue. As a result, no alpha-SMA immunogenicity was observed in the Sham control heart tissue except blood vessels. In the MI group, alpha-SMA positive signals were observed in the fibrotic part and in the blood vessels. Moreover, the signal increased significantly in the MI + GW group (see FIG. 4B).

Next, the inventors confirmed the expression of TIMP, which is known to be an important factor for differentiating fibroblasts into myofibroblasts. Cardiac tissue obtained from the same myocardial infarction model was used as a sample to confirm the expression of TIMP-1, TIMP-2 and TIMP-3, and alpha-tubulin was used as a control for this. .

As a result, as shown in Figure 4c, TIMP-3 expression was reduced in the MI group causing myocardial infarction, but TIMP-2 and TIMP- by treatment with GW610742, a PPAR-delta agonist according to an embodiment of the present invention. An increase in expression of 3 could be observed (see FIG. 4C). These results demonstrate that, after myocardial infarction, PPAR-δ agonists promote differentiation of myofibroblasts during LV remodeling.

Experimental Example 5: Myofibroblast differentiation and migration promoting effect of PPAR-delta agonist

5-1: PPAR-delta Agonist Promotes Differentiation of Myofibroblasts and Endothelial Cells

Through the above experimental results, it was demonstrated that the PPAR-delta agonist according to one embodiment of the present invention promotes angiogenesis and differentiation into myofibroblasts during LV remodeling after myocardial infarction. Subsequently, the present inventors attempted to study the relationship between angiogenic effects of PPAR-delta agonists and differentiation and function into myofibroblasts. We used an in vitro cell culture method using fibroblasts and endothelial cells (HUVEC). PW-delta agonist GW610742 was treated with 0, 0.1, 1, and 5 μM for each of the cells for 72 hours, and then, the cells were obtained, and then the cells were obtained using a MTT assay. The degree of differentiation of was confirmed.

As a result, as shown in Figure 5a, GW610742 increased the proliferation of fibroblasts, but was not statistically significant. In endothelial cells (HUVECs), proliferation was dependent on throughput. In particular, an increase in the number of endothelial cells was observed in the group treated with 0.1 μM in the group treated with 0.1 μM compared with the group treated with GW610742. (See FIG. 5A).

5-2: Confirmation of the promotion of differentiation of myofibroblasts by PPAR-delta agonists using Western blot

Next, the inventors observed the expression of alpha-SMA protein, which is known to express in differentiating muscle cells, in order to confirm the effect of GW610742 on differentiation into myofibroblasts. Myofibroblasts were treated with PPAR-delta agonist GW610742 for 0, 0.1, 1, and 5 μM, respectively, for 72 hours, after which the cells were obtained and subjected to western blot. In this case, anti-alpha-SMA (Sigma-Aldrich, Saint Louis, MO) as a primary antibody and alpha-tubulin (alpha-tubulin) was used as a control thereto.

As a result, as shown in FIG. 5B, as the treatment concentration surpassed GW610742, it was confirmed that the expression of alpha-SMA was increased in myofibroblasts. These results demonstrate that, unlike its effect on the proliferation of fibroblasts, GW610742 itself activates metastasis to fibroblasts-myofibroblasts (see FIG. 5B).

5-3: Confirmation of the association between the neovascularization effect of PPAR-delta agonist and the promotion of differentiation into myofibroblast using Western blot

Subsequently, the present inventors used a conditioned medium used for culturing endothelial cells in fibroblast culture, and treated GW610742, a PPAR-delta agonist, to differentiate angiogenesis into myofibroblasts. It was confirmed whether or not to be related to. To this end, we measured the expression of tissue inhibitor of metalloprotease (TIMP) and matrix metalloproteinases (MMP), which are important factors for the differentiation of fibroblasts into myofibroblasts.

Specifically, the inventors treated fibroblasts and endothelial cells with PPAR-delta agonist GW610742 for 0, 0.1, 1, 5 μM, respectively, for 72 hours, and then obtained cells and performed Western blot analysis. In addition, the fibroblasts were cultured using a conditioned medium used for culturing the endothelial cells, and after treatment with GW610742 for 0, 0.1, 1, 5 μM for 72 hours, cells were obtained and Western blot analysis was performed. (Fibroblast + HUVEC_CM). In this case, anti-MMP-9 and TIMP-3 (Millipore, Temecula, CA) were used as primary antibodies, and alpha-tubulin was used as a control for this.

As a result, as shown in Figure 5c, the expression of MMP-9 in fibroblasts slightly increased when treated with 0.1 μM GW610742, TIMP-3 expression was little changed. In addition, the expression of MMP-9 in endothelial cells was increased when 1 μM GW610742 was treated, and there was little change in the expression of TIMP-3. In addition, the protein expression of TIMP-3 and alpha-SMA increased in a dose-dependent manner when GW610742 was treated by culturing fibroblasts using a conditioned medium used for endothelial cell culture. Not observed (see FIG. 5C). These results indicate that the PPAR-delta agonist according to one embodiment of the present invention increases the expression of MMP-9 in the blood vessel side, and this increased expression of MMP-9 is related to the expression of TIMP-3 related to the differentiation of fibroblasts. As a result, it is believed that PPAR-delta agonists promote differentiation of fibroblasts. However, further studies on the expression of MMP-9 on the vascular side and the expression of TIMP-3, which increases expression in differentiation of fibroblasts, should be further studied. That is, the possibility of having other mechanisms than MMP-9 and TIMP-3 cannot be excluded. It can be interpreted to promote differentiation into myofibroblasts through other isolated mechanisms.

5-4: Confirmation of migration of myofibroblasts by PPAR-delta agonists

Next, the inventors confirmed the mobility of myofibril cells. The moving properties of myofibroblasts are known as important mediators of fibrosis in cardiac remodeling (Shi Q et al. , 2011, PLos One).

A cell culture vessel capable of observing the movement of fibroblasts using a conditioned medium treated with PPAR-delta agonist GW610742 used at the endothelial cell culture at a concentration of 0, 1, 5 μM for 72 hours ( After 24 hours of incubation in a transwell migration assay, the mobility of fibroblasts was observed. To observe the mobility, the transferred cells were stained with hematoxylin and the cells were observed using an optical microscope.

As a result, as shown in FIG. 5D, it was observed that the migration of fibroblasts was increased depending on GW610742 treatment concentration (see FIG. 5D). These results indicate that GW610742 affects the function of fibroblasts and that angiogenic conditions promote the differentiation and migration of fibroblasts.

Although the present invention has been described with reference to the embodiments shown in the drawings, this is merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

PPAR-delta agonist according to an embodiment of the present invention, in the myocardial infarction animal model, has the effect of promoting fibrosis, the growth and differentiation of myofibroblasts, promote the remodeling process of the ventricles, myocardial infarction It can be usefully used for improvement and treatment.

Claims (2)

1. A composition for promoting myocardial wound healing after myocardial infarction, comprising a peroxisome proliferator-activated receptor (PPAR) -delta agonist as an active ingredient.
2. The method of claim 1,

   The PPAR-delta agonist is from the group consisting of GW610742 (GSK), GSK-0660 (CAS 1014691-61-2), GSK 3787 (CAS 188591-46-0) and GW 501516 (CAS 317318-70-0). At least one selected is a composition for promoting myocardial wound healing after myocardial infarction.

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