| IntroductionCoronary atherosclerotic heart disease is a kind of very import cardiovascular diseases. Studies have confirmed the formation of atherosclerosis plaques can cause coronary heart disease, and the rupture and thrombus formation of plaques can lead to myocardial infarction, stroke, and sudden death. So it has always been a hot topic of coronary atherosclerotic heart disease at home and abroad that researches studied on the atherosclerotic lesion formation and the stability of advanced atherosclerosis.The rein angiotensin system (RAS) plays a crucial role in the pathogenesis of cardiovascular diseases. Type 4 angiotensin receptor (AT4R), a new member of RAS, is a type II integral membrane protein and belongs to the Ml family of zinc-dependent metallopeptidase. It works as a specific receptor of angiotensin IV, and is also known as Insulin-regulated aminopeptidase (IRAP).AT4Rhas been identified in several cardiovascular tissues and cells, including vascular smooth cells, cardiac fibroblasts, coronary and aortic endothelial cells. And it was involved in several physiological and pathological functions in various tissues and cells of cardiovascular system. Precious studies have found that AT4R could induce the activation of eNOS and participated in the regulation of the vasorelaxation of pulmonary arteries. AT4R can also involved in vascular remodeling with the synergy of fibroblast growth factor (bFGF). In addition, research showed that angiotensin IV (Ang IV) via binding to AT4R could counterwork the improvement of cardiac function disorder and damage caused by Ang Ⅱ and ischemia-reperfusion. Recent reports confirmed the cooperation of Ang Ⅳ and AT4R showed a vasoprotective effect evidenced by improving aortic endothelial function through decreasing the superoxide production and increasing eNOS expression in advanced atheroma. All these results suggested that AT4R may provide protection against cardiovascular diseases. However, the role of AT4R in the atherosclerotic lesion formation and the stability of progressive atherosclerosis remains unclear.These scientific issues contribute to the ideas and direction of our research.Objectives1. To explore the effects of AT4Ron atherosclerotic lesion formation.2. To explore the role of AT4R in the stability of advanced atherosclerosis plaques.3. To elaborate the possible mechanism responsible for the anti- atherosclerosis effects of AT4R.Methods1. Animal model for atherosclerosis(1) Part Ⅰ:Male ApoE-/- mice on a C57BL/6J background at 8 weeks old were fed with a high-fat diet (0.25% cholesterol and 15% cocoa butter) during the whole experimental period. At the 4th week, mice were randomly divided into three groups: the control group, the Ad-EGFP and the Ad-AT4R group (n=20). Mice in the control group received infusion of PBS through caudal vein. Mice in the Ad-EGFP and the Ad-AT4R groups received infusion of 2×109 PFU adenovirus vectors of green fluorescence protein (Ad-EGFP) or AT4R through caudal vein respectively. Treatment was repeated two weeks later. Animals were sacrificed at the 8th week.(2) Part Ⅱ:8-week-old male ApoE-/- mice were fed with a high-fat diet (0.25% cholesterol and 15% cocoa butter) for 14 weeks. A constrictive silastic tube (0.30 mm inner diameter,0.50 mm inner and 2mm length) was placed around the right common carotid artery at the 2nd week to induce the formation of carotid plaque. Ultrasound examination was done to view the formation of carotid plaque in mice at the 10th week. Then animals were randomly divided into three groups:the control group, the Ad-EGFP and the Ad-AT4R group (n=20). Mice in the control group received infusion of PBS through caudal vein. Mice in the Ad-EGFP and the Ad-AT4R groups received infusion of 2×109 PFU adenovirus vectors of green fluorescence protein (Ad-EGFP) or AT4R through caudal vein respectively. Treatment was repeated two weeks later. Animals were sacrificed at the 14th week.2. Measurement of body weight and Serum lipids assayAt the end of the experiment, mice were starved overnight and blood samples were collected from left ventricle to measure serum levels of glucose (GLU), triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C).3. Measurement of aortic lesion formationThe aortic root was harvested and placed in 4% formaldehyde for at least 24h. The paraffin sections with thickness of 5μm were cut for hematoxylin and eosin staining. The cryosections with thickness of 5 μm were cut for oil red staining. In addition, the whole aorta was stained by oil red. Image-Pro Plus software 6.0 was used to calculate respectively positive staining area percentage of corresponding active vascular cavity area.4. Measurement of advanced atherosclerosis plaques(1) The vulnerability index:At the end of experiment, the whole aorta including the carotid arteries were harvested and placed in 4% formaldehyde for at least 24h. The cryosections of carotid arteries with thickness of 5 μm were cut for further histopathology and immunohistochemistry experiments, including hematoxylin and eosin staining, oil red staining, Sirius red staining. Immunohistochemistry staining was performed by use of relative primary antibodies of macrophage-specific antigen (MOMA-2) and a smooth muscle cell actin. Histopathological slides were analyzed by use of Image-Pro Plus Software 6.0.The relative content of lipids, VSMCs, collagen, macrophages was quantitated as the ratio of the positive staining area to the total carotid plaque area in 20 high power fields. The vulnerability index was calculated by the formula as follows:(macrophage staining %+lipid staining%)/ (VSMC staining% +collagen staining%).(2)Immunohistochemistry analysis:Immunohistochemistry analysis was used to measure the expression level of matrix metalloproteinases (MMP-2, MMP-9) and pro-inflammatory cytokines such as TNF-a, IL-6. Histopathological slides were analyzed by use of Image-Pro Plus Software 6.0.The expression level of inflammatory cytokines and MMPs were quantitated as the ratio of the positive staining area to the total carotid plaque area in 20 high power fields.(3)Western Blot analysis:Total proteins were extracted from aorta. And the protein expression of matrix metalloproteinases (MMP-2, MMP-9) and pro-inflammatory cytokines (TNF-a, IL-6) were analyzed by western blot for detection. Relative protein expression levels were quantified by using Image J Software. B-actin immunoblot analysis was conducted to ensure equal protein loading.(4) Gelatin zymography assay:Activities of matrix metalloproteinases in aortic plaque were assayed by gelatin zymography. Protein concentrations were determined by use of a BCA protein assay kit. Gelatin zymography assay was done using a MMP Gelatin Zymography Kit according to the manufacturer’s instruction. Data were assayed by using Image J Software.5. Statistical analysis:SPSS 20.0 was applied for statistical analysis. Data were presented as mean ± SEM by use of Graph Pad Prism version 5.0. The comparisons among groups were done by one-way ANOVA analysis with LSD post-hoc analysis. P＜0.05 was considered statistically significant.RESULTS1. Parti1.1Body weight and serum lipid measurementThere was no significant difference in body weight and serum lipid levels among the three groups (P>0.05), suggesting that overexpression of AT4R had no effect on these hematologic characteristics.1.2 Measurement of the adenovirus transfection efficiencyFluorescence of adenovirus and expression of AT4R were used to measure the adenovirus transfection efficiency. The protein expression of AT4R analyzed by Western blot was significantly increased in Ad-AT4R group compared to the control group or the Ad-EGFP group (P<0.05), suggesting that the model of overexpression of AT4R was successfully established.1.3 The effect of AT4R on atherosclerotic lesion formationCompared to the control group, the Ad-EGFP group showed no difference in the area percentage of plaque (P>0.05), whereas, AT4R overexpression markedly reduced the area percentage of atherosclerotic lesion (P＜0.05).2. Part Ⅱ2.1 Body weight and serum lipid measurementThere was no significant difference in body weight and serum lipid levels among the three groups (P>0.05), suggesting that overexpression of AT4R had no effect on these hematologic characteristics.2.2 Measurement of the adenovirus transfection efficiencyFluorescence of adenovirus in the carotid plaque was observed obviously. The protein expression of AT4R analyzed by Western blot was significantly increased in Ad-AT4R group compared to the control group or the Ad-EGFP group(P<0.05), suggesting that the model of overexpression of AT4R was successfully established.2.3 The effects of AT4R on the stability of carotid plaqueCompared to the control group, the Ad-EGFP group showed no difference in the components of carotid plaque, whereas, AT4R overexpression markedly reduced the lipids and macrophages in carotid plaque (P<0.05), with obvious increase of SMCs and collagen content (P<0.05).We further calculated the vulnerability index to assess the stability of carotid plaque. There was no significant difference in vulnerability index between the control and Ad-EGFP groups, while in the Ad-AT4R group, vulnerability index was markedly lower than in the other two groups(P<0.05).2.4 The effects of AT4R on expression of pro-inflammatory cytokinesWestern blot and immunohistochemistry assays were used to measure the protein expression of IL-6 and TNF-a. In the Ad-AT4R group, the expression of IL-6 and TNF-a was significantly decreased compared to the control or the Ad-EGFP group (P<0.05).2.5 The effects of AT4R on expression and activity of MMPsThe protein expression levels of MMP-2 and MMP-9 in the carotid plaque assayed by immunohistochemistry and western blot were markedly reduced in the Ad-AT4R group compared to the control group or the Ad-EGFP group (P＜0.05). In addition, the activities of MMP-2 and MMP-9 detected by gelatin zymography were obviously lower in the Ad-AT4R group than in the control group or the Ad-EGFP group (P<0.05).2.6 The effects of AT4R on expression of AT1R, AT2R, MasR, ACE and ACE2To estimate the influence AT4R overexpression on the other members of RAS system, we measured the expression of AT1R, AT2R, MasR, ACE and ACE2 by western blot. Their expressions were no remarkably different among the three groups (P>0.05).CONCLUSION1. The overexpression of AT4R inhibits the atherosclerotic lesion formation by reducing the level of lipids.2. The overexpression of AT4R significantly decreased the plaque vulnerability index and stabilized the vulnerable plaques by reducing the content of lipids and macrophages and increasing of SMCs and collagen content.3. AT4R acts as an anti-inflammation role by reduction of IL-6 and TNF-a. Thus, AT4R stables the vulnerable plaques.4. The overexpression of AT4R could suppress the expression and activity of MMPs, Thereby suppressing the degradation of ECM and increasing the stability of plaques.5. The overexpression of AT4R probably had no effect on the other components of RAS system (AT1R, AT2R, MasR, ACE and ACE2). |
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