Experimental Study Of CTRP9 On Anti-atherosclerosis

1 IntroductionAtherosclerosis is the main cause of cardiovascular disease and stroke. The rupture of vulnerable plaques followed by thrombus formation leads to myocardial infarction, stroke, and sudden death. Therefore, plaque vulnerability has become a hot pot issue of coronary atherosclerotic heart disease at home and abroad.Currently, inflammation is recognized as an important factor involved in the development, progression, and rupture of atherosclerotic plaques, and subsequently, thrombosis. Macrophages are key inflammatory cells that are involved at every phase of atherosclerosis, from its genesis to plaque rupture and coronary thrombosis. During the atherosclerosis pathological process, active macrophages present in plaques of AS, and secret a variety of inflammatory cytokines, such as IL-6, IL-8, MCP-1 and TNF-a, which attract more monocytes to infiltrate into the subendothelial space, propagate inflammatory response and subsequently advance atherosclerotic plaques. Therefore, macrophages contribute to size-independent changes in plaque morphology, notably necrotic core formation and fibrous cap thinning, which characterize the "vulnerable" plaque. Thus, it is essential to investigate the mediators of inflammation in macrophages against atherosclerotic heart disease.Recent reports show that, adipose tissue is not only an energy-stored organ, but also an endocrine organ, which could secreted kinds of cytokines, such as Adiponectin, Resistin, and CTRPs. CTRP9 is a novel adipocyte-derived cytokine, which belongs to the CTRPs family and has the highest homology to adiponectin. Recent reports show that adiponectin has the biological function of anti-inflammation, anti-atherosclerosis, anti-apoptosis and anti-diabetes etc., especially, APN is associated with plaque vulnerability. Similar with adiponectin, recent studies show that CTRP9 is related with cardiovascular disease. Delivery of CTRP9 significantly attenuates vascular smooth muscle cell proliferation and neointimal formation, and induces vascular relaxation. Furthermore, serum CTRP9 concentrations are associated with favorable glucose utilization and arterial stiffness in patients with type 2 diabetes. In our previous research, we find that Serum level of CTRP9 is negative related with acute coronary syndrome. All the information indicates that CTRP9 may be involved in the development of atherosclerotic plaque. However, the biological function of CTRP9 in carotid plaque stability and anti-inflammation remains unclear.These scientific issues contribute to the research objectives and methods.2 Objectives(1) to detect whether CTRP9 can stabilize atherosclerotic plaque in ApoE-/-mice;(2) to elucidate the mechanisms responsible for the anti-atherosclerosis effects of CTRP9.3 Methods3.1 Animal model of atherosclerosis 8-week-old male ApoE-/-mice were obtained from Peking University, and all mice received a high-fat diet (0.25% cholesterol and 15% cocoa butter) until sacrifice. A constrictive collar (0.30 mm inner diameter,0.50 mm outer diameter, and 2 mm long) was placed around right carotid artery to induce plaque formation. Eight weeks after surgery, the LV-CTRP9 or LV-eGFP was injected into the right jugular vein respectively, the titers averaged 2×107 TU/mL, and total volume was 100μL. Equal volume of PBS was used as a vehicle control. Another four weeks of high-fat diet was maintained. Mice were sacrificed for further analysis thirty days after lentivirus injection.3.2 Serum glucose and lipidsMice were fasted overnight and blood samples were collected at the end of the experiment. Serum level of glucose(GLU), total cholesterol(TC), Nonestesterified fatty acid (NEFA), Triglycerides (TG), High-density lipoprotein cholesterol(HDL-C), Low-density lipoprotein cholesterol (LDL-C).3.3 The vulnerable indexThe right carotid arteries were removed and perfusion-fixed with 4% paraformaldehyde overnight and then embedded in optimal cutting temperature compound. The cryosections were cut in 5-μm thick for further histological and morphological staining. Plaque contents, including the lipid core, macrophages, collagen, and smooth muscle cells were identified respectively by Oil Red O staining, MOMA-2(diluted 1:200), Sirius red and a-actin(diluted 1:200). The vulnerable index was calculated by the formula as follows:the relative positive staining areas of (macrophages%+lipid %)/the relative positive staining areas of (a-SMCs%+ collagen%).3.4 Immunohistochemical AnalysesImmunohistochemical analyses involved the use of primary antibodies for TNF-a (diluted 1:200)and MCP-1 (diluted 1:100). Followed with secondary antibody. To detect cell nuclei, sections were rinsed in water and counter-stained with Hematoxylin. Data were analyzed by Image Pro-Plus software.3.5 Cell culture and lentiviral transfectionRAW264.7 cells were cultured in DMEM medium supplemented with 10% fetal calf serum at 37℃ in 5% CO2. For overexpression of CTRP9, recombinant LV-CTRP9 or recombinant LV-eGFP was used to transfect RAW264.7 cells, the transfection efficiency was detected by fluorescence microscope, and then cells were treated with oxLDL for another 24 hours for further experiments.3.6 Western blotsTotal proteins were extracted from RAW264.7 Cells and culture medium. And the protein expression of CTRP9 from cell lysates and culture medium were detected by western blot. Also the expression of TNF-a (diluted 1:1000) and MCP-1 (diluted 1:1000) from cell lysates were analyzed by western blots for detection. Relative protein levels were quantified by using the Image J.3.7 StatisticsSPSS 13.0 statistical software package was used for data analysis. Data are presented as means±SEM. Group differences were analyzed by Student’s unpaired t test or one-way analysis of variance.p value<0.05 was considered statistically significant.4 RESULTS4.1 In vivo study4.1.1 Serum level of CTRP9 is significantly elevated in LV-CTRP9 group Mice infected with LV-CTRP9 showed a 3.9 or 3.5-fold increase in serum level of CTRP9 (p<0.05 vs. LV-CTRP9 group), but there was no significant difference between the LV-eGFP group and the vehicle group4.1.2 CTRP9 decreases serum glucose level The LV-CTRP9 group displayed markedly reduced glucose levels compared with the LV-eGFP group or the vehicle group (p<0.05). However, no significant differences were found in serum lipid levels among the three groups.4.1.3 CTRP9 enhances the carotid plaque stability The relative contents of lipids and macrophages were lower in the LV-CTRP9 group than in the LV-eGFP group or the vehicle group (p<0.05). In contrast, the relative contents of collagen and smooth muscle cells were higher in the LV-CTRP9 group than the other two groups (p<0.05). However, there was no difference of the relative contents between the LV-eGFP group and the vehicle group. The vulnerability index of the LV-CTRP9 group was lower than the LV-eGFP group or vehicle group (p<0.05). Again the vulnerability index between the LV-eGFP group and vehicle group has no significant difference.4.1.4 CTRP9 reduces pro-inflammatory cytokines secretion in the carotid plaque the expression levels of TNF-a and MCP-1 in the LV-CTRP9 group were apparently lower than those in other two groups (p<0.05). However, there was no significantly difference between the LV-eGFP group and the vehicle group.4.2.1 CTRP9 is detected in both the cell lysates and the culture medium of RAW264.7 macrophages after LV-CTRP9 transfectionThe transfection efficiency was measured by fluorescence with infusion protein GFP (green light). We found that when MOI was at 60, almost 90% cells were transfected successfully. In LV-CTRP9 group, CTRP9 exists both in the cell lysates and the culture medium, but there is no CTRP9 expression in the LV-eGFP group.4.2.2 Pro-inflammatory cytokines are decreased in RAW264.7 macrophages after LV-CTRP9 transfection the expression of MCP-1 and TNF-a was significantly decreased in the LV-CTRP9 group compared with the LV-eGFP group(p<0.05).5 CONCLUSION(1) CTRP9 could stabilized the plaque by alters the components of carotid plaque and reduces the index of vulnerable plaque.(2) CTRP9 enhance carotid plaque stability and act as an anti-inflammation role by reducing TNF-a and MCP-1 in vivo.(3) CTRP9 enhance carotid plaque stability by reducing TNF-α and MCP-1 in vitro.