| BackgroundAbout75%of patients with acute coronary syndrome (ACS), and60%of patients with recent symptoms of carotid artery disease are due to the rupture of vulnerable plaque VP) during atherosclerosis (AS). Although the progress from Fibrous plaque without any symptom to VP is related to several facts,more and more evidence that the formation of new blood vessels and Intraplaque hemorrhage is an important factor in plaque instability. Inhibition of angiogenesis has become a new means of treatment As. Studies have shown that simvastatin inhibit angiogenesis by lowering the expression and activity of Cyclooxygenase-2(COX-2) and matrix metalloproteinase protease-9(MMP-9) in arterial atherosclerotic plaques, as well as vascular endothelial growth factor (VEGF) and factor Ⅷ-related antigen (FVⅢRAg). However, because of its serious adverse reactions in liver damage and rhabdomyolysis, the use of simvastatin was limited in clinical application to a certain extent. Prostaglandin E1(PGE1), an efficient, safe and biologically active substances, is widely used due to its effect on expansion of the coronary artery and peripheral vascular, inhibition of platelet aggregation.Recent studies have found that,PGE1also has significant anti-inflammatory, anti-oxidative stress, Increasing the stability of VP. However, it has not been addressed whether the ability PGE1stability VP is related to r the inhibition of angiogenesis within the plaque and reducion of plaque hemorrhage.This study, therefore, was conducted to examine the effects and mechanisms of PGE1on neovascularization and intraplaque haemorrhages of atherosclerotic vulnerable plaque in a rabbit model.Objectives(1) To establish rabbit model of atherosclerotic vulnerable plaques.(2) To test the hypothesis that PGE1inhibit the neovascularization and intraplaque haemorrhages of vulnerable plaque dose-dependently. Simvastatin was chosen as a therapeutic standard to compare with different doses of PGE1.(3) To elucidate the molecular mechanism of PGE1in reduction of neovascularization.Methods1. Establishment of the rabbit model of vulnerability plaque:Fifty male New Zealand White rabbits were fed a1%cholesterol diet2weeks prior to and7weeks after balloon injury of the abdominal aorta. At the end of week9, the rabbits were switched to regular diet, and randomly divided into5groups for treatment for4weeks: control group that received no treatment, low-dose PGE1group that received intravenous injection PGElof0.3μg·kg-1·day-1, moderate-dose PGE1group that received intravenous injection PGE1of0.6μg·kg-1·day-1, high-dose PGE1group that received intravenous injection PGE1of1.2μg·kg-1·day-1, and simvastatin group that received oral simvastatin of5mg·kg-1·day-1, respectively. At the end of week13, all rabbits underwent pharmacological triggering with Chinese Russell’s viper venom and histamine. Then all rabbits were sacrificed for pathological studies. 2. Body weight:At the baseline, week9and week13, body weight of all rabbits was measured, respectively.3. Serum lipid assay:At the baseline, week9and week13, Serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by enzymatic assays.4. Histopathological analysis:The abdominal aorta were processed and examined by HE stain. The distribution of plaque hemorrhage and thrombosis were observed.5. The vulnerability index:The content of lipids and collages in plaques were examined by oil-red O and sinus red staining; the content of macrophages and SMCs were examined by RAM11and a-actin immunohistochemical staining. The vulnerability index was calculated by the following formula:(macrophage%+lipid%)/(smooth muscle cell%+collagen%).6. Immunohistochemical analysis:Immunohistochemical staining was performed and the expressions of CD31, HIF-1α and VEGF were detected.7. RT-PCR assays:The mRNA expressions of HIF-1α and VEGF in the abdominal aorta tissue were analyzed using RT-PCR technique.8. Statistical analysis:Statistic analysis were performed using the with SPSS vl3.0. Quantitative variables are expressed as mean±SD and shown by one-sample Kolmogorov-Smirnov test to be in normal distribution. The data were analyzed by a one-way ANOVA and unpaired Student’s t-test. A P<0.05was considered statistically significant.Results1. General state of the experimental animals:7rabbits did not complete the study. Two rabbits died during anesthesia, three died of high cholesterol induced diarrhea and two died of respiratory infection. Data were available for analysis for9rabbits in the control group,8in low-dose PGE1group,9in moderate-dose PGE1group,9in high-dose PGE1group and8in simvastatin group.2. Body weight:Body weight of rabbits at week9and week13were significantly higher than that at baseline (both p<0.01). There was no significantly difference between all rabbits at week9and week13, and there was no significantly difference in all the treatment gorups by the end of13week.3. Serum lipid profile:Serum lipid profile at week9and week13were significantly higher than that at baseline (all p<0.01). At the end of week13, the serum levels of TC, TG, HDL-C and LDL-C in PGE1groups of low, moderate and high dose did not exhibit significantly difference as compared with the control group.There were no significant differences among the three groups of PGE1. Thus PGE1did not alter the serum lipid levels in atherosclerotic rabbits.4. Histological evaluation:Five groups of rabbit abdominal aorta appearance rugged, and the light yellow plaque-like protrusions were seen in intima, scattered or confluent.Compared with model group, the treatment groups deffer in plaque size, distribution and thickness. The model group shows a ruptured plaque attached to the thrombus.Compared with the model group, the treatment groups in plaque microvessel density decreased (P<0.01), with reduction of intraplaque hemorrhage in term of distribution and range(P<0.01). High dose PGE1group reduce the magnitude stronger than the simvastatin group (P<0.01), and PGE1each treatment group work in a dose-dependentce(P<0.01).5. The vulnerability index:PGE1significantly decreased the plaque contents of lipid and macrophages, and increased the plaque contents of SMCs and collagen. Consequently, the vulnerability index of plaque was significantly reduced in four treatment groups compared with the control group (all p<0.01). The vulnerability index was lower in the high-dose PGE1group than that in the low-dose PGE1group and simvastatin group (both p<0.01). Thus, PGE1dose-dependently reduced the vulnerability index of plaque and high-dose PGE1was more effective in reducing the accumulation of macrophages and increasing the contents of SMCs and collagen in plaques.6. Immunohistochemical analysis:The expression levels of CD31and HIF-1α and VEGF were lower in plaques of all treatment groups than in the control group (all p<0.01). High-dose PGE1was more effective than simvastatin (p<0.01, p<0.05). There were dose-dependent difference in three PGE1groups. 7. RT-PCR:The mRNA expression levels of HIF-1a and VEGF mRNA were lower in plaques of all treatment groups than in the control group (all p<0.01). with the level in moderate-dose PGE1group being similar to that in simvastatin group, but the levels in the high-dose PGE1group were lower than that in simvastatin group (all p<0.01).Conclusions1. The method of a cholesterol-rich diet together with balloon-induced abdominal aortic wall injury and drug triggering is a efficient way to establish rabbit vulnerable plaque model,in which we proved that there were relations between intraplaque haemorrhages,neovascularization and the vulnerability index. which is mimic to human disease, time-saving and useful for interventions.2. PGE1dose-dependently inhibits angiogenesis within the vulnerable plaque, reduces intraplaque hemorrhage,and promotes stability of atherosclerotic vulnerable plaque, and high-dose PGE1was more effective than simvastatin.3. PGE1did not alter serum lipid levels. PGE1effectively inhibit angiogenesis in vulnerable plaque in terms of reducing the synthesis of VEGF following the inhibition HIF-1α expression. |
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