Basic Study On Intraplaque Angiogenesis

BackgroundAtherosclerotic plaque rupture with subsequent intraluminal thrombosis is the major cause of acute coronary syndrome (ACS). As atherosclerosis (AS) progresses, the reduced oxygen diffusion and perfusion of inflammatory factors within the plaque trigger a continual release of angiogenic growth factors, such as VEGF, FGF, PDGF and Ang-TIE. Angiogenesis, the formation of new capillary vessels, is known to occur in response to a hypoxic and inflammative environment. Neovessels within the plaque are characterized by fragility and high perfusion, resulting in increasing inflammatory cells permeability and intraplaque hemorrhage (IPH), all of these leading to the plaque destabilization.We investigated the expression of RAM-11ICAM-1, VCAM-1, TNF-a, CRP, FGF-2, VEGF-A, PDGF-BB, CD31and HIF-1a in rabbit’s abdominal aorta plaque from week4till week24after balloon injury. We also investigated the correlation between the expression of adhesion molecules, RAM-11, CRP, TNF-a, VEGF-A, FGF-2,, HIF-1a, CD31and PDGF-BB.Objective:1. To make elucidate the correlation between hypoxia and intraplaque angiogenesis o2. To elucidate the correlations between inflammation and intraplaque angiogenesis. Methods:1. Animal modelNew Zealand white male rabbits underwent balloon-induced endothelial injury in the abdominal aorta and were then fed a high-cholesterol diet (1%cholesterol) for12weeks. Sbusequently, the atherogenic diet was replaced by a regular diet for another12weeks.Every2weeks from the week4after the balloon injure,5rabbits were euthanized..2. Biochemical studiesBlood was drawn from rabbits fasting overnight to measure lipid profile at the beginning of the study and every two weeks from the4week after balloon injury to the24th week. 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.3. Histopathological and immunohistochemical stainingSerial cross-sections underwent general histological staining with hematoxylin&eosin (H&E) and specific immunohistochemical staining Slides were stained with antibodies against RAM11and CD31to identify macrophages and neovassels. The primary antibodies were also included ICAM-1, VCAM-1, CRP, TNF-α,VEGF-A, FGF-2, PDGF-BB and HIF-1a. Nucleus counterstaining involved within hematoxylin, which can be dyed blue.Slides were scanned by optical microscopy, a morphometric analysis system IPP6.0was used to analysed histopathological parameters of these slides. The area of positive staining of ICAM-1, VCAM-1, RAM-11, CRP, TNF-α,VEGF-A, FGF-2, PDGF-BB, HIF-1a and CD31were expressed as a percentage of stained area divided by plaque area in at least5high-power fields (×400). Neovascularization density on digitized images was quantified by counting the total number of CD31-positive microvessels per200×magnification microscopic field.4. Statistical analysisAll numeric data were expressed as mean±SD. Differences in continuous variables between two groups were assessed by unpaired t-test, and comparison among multiple groups was performed by analysis of variance with ANOVA. Linear regression analysis was performed to evaluate the relationship. A p value<0.05was considered significantResults:All animal underwent initial balloon injury without complications and showed full recovery.1. Biochemical studiesThe mean plasma level of TC and LDL-C were significantly increased after12weeks of a high-cholesterol diet but returned to the baseline level after10weeks of normal diet. The baseline level of TC and LDL-C in5rabbits at week4was23.1980±3.19094mmol/L and6.0300±1.38948mmol/L. After a high-cholesterol diet for12weeks at week12, the level of TC and LDL-C increased to40.7180±3.39382mmol/L,7.7480±0.68525mmol/L respectively. Withdraw of a high-cholesterol diet result in a significant reduction of TC and LDL-C levels to25.8020±3.49010mmol/L and4.1240±0.52539mmol/L at week24, still higher than those at week4. On the other hand, there is no significant change of TG and HDL-C were found in all rabbits at week4, week14, and week24.2. Immunohistochemical StainingFrom week4to week12, the expression of ICAM-1, VCAM-1, RAM-11, CRP, TNF-α, HIF-1α, VEGF-A, FGF-2, PDGF-BB and CD31were all significant increased. But what the different is the expression of CRP and VCAM-1were still in a high level from week14to week24, other factors gradually decreased. And the expression level of PDGF-BB was significant increased from week4to week24.Conclusion:Our research indicated that there is a positive correlation between HIF-1a and CD31from week4to week12, and the correlation between HIF-1a and VEGF-A is also positive, VEGF-A has been regard as the most important angiogenesis growth factor, so We suggested that hypoxia may perform the role of promote plaque angiogenesis through the effect of HIF-1α-VEGF-A. But there is no correlation between HIF-1a and CD31from week14to week24, we hypothesis that with the growth of intraplaque neovasseles, the anoxia condition within the plaque has been ameliorate, so hypoxia may not been an inducer of intraplaque angiogenesis any more. So we indicated that hypoxia may be a novel promoter for early AS plaque neovascularization.Our research indicated that inflammatory molecules TNF-α、 CRP、ICMA-1and VCAM-1could promote plaque angiogenesis through various factors such as VEGF-A, FGF-2and PDGF-BB. Among these inflammatory molecules ICAM-1and VCAM-1have been recognized as the most potent maker to predict vulnerable plaque in the early stages of AS. ICAM-1and VCAM-1both contribute to the form of plaque neovessles in the early stages of AS from week4till week12, there was a positive correlationship between CAMs and CD31(r=0.550,P<0.05; r=0.762,P<0.05), but have no effect in the late stage of AS.