The Effects And Mechanisms Of Tongxinluo On Atherosclerotic Intraplaque Angiogenesis In Rabbits

BackgroundAtherosclerotic cardiovascular disease(ASCVD)is one of the leading causes of mortality throughout the world.In case of plaque rupture and subsequent intraluminal thrombosis,these common change of cardiovascular disease manifest as acute coronary syndrome,such as myocardial infarction and unstable angina pectoris.The pathologic processes of atherosclerosis(AS)including many different factors and pathogenesis are very complex.Currently,the efficacious intervention on vulnerable plaque is still lacking.Therefore,the effective treatment of drugs targeting different pathogenesis is crucial to stabilization of vulnerable plaques and prevention of plaques rupture.Recent studies show that intraplaque angiogenesis have correlated to atherosclerotic lesion progression and vulnerability.Human vulnerable plaques are characterized by extensive proliferation of vasa vasorum(VV)and subsequent intraplaque neovascularization.In atherosclerotic plaques,the neovessels consist of merely one single layer of endothelial cells lacking of basement membrane and connective tissue.The cellular connections between the endothelial cells are loose and the secretory function of the endothelial cells is abnormal Therefore,neovessles are characterized by high fragility and permeability,resulting in the spillage of erythrocyte and the permeability of inflammatory cells which contributing to the rapid increase of the lipid core of plaque,then promoting the plaque destabilization.The greater the number of neovessels in the plaque,the worse the plaque's stabilityAngiogenesis is induced by various growth factors,such as vascular endothelial growth factor(VEGF)family,fibroblast growth factor(FGF)family and platelet-derived growth factor(PDGF)family.Among them,VEGF-A,FGF-2 and PDGF-BB and their relevant receptors(VEGFR-2,FGFR1 and PDGFR-P)play prominent roles.VEGFs and their tyrosine kinase receptors are the key regulators of angiogenesis.VEGF-A/VEGFR-2 is the main pathway that stimulates angiogenesis by increasing the proliferation,migration and permeation of endothelial cells.FGFs are known to include at least 20 factors including FGF-2,which is the first to confirm the ability to regulate angiogenesis.FGFR-1 is the receptor of high affinity binding with FGF-2.FGF-2/FGFR-1 signaling pathways play an important role in injury repair,angiogenesis and tissue regeneration.PDGF family consists of five kinds of dimers and two corresponding receptors.Previous studies have demonstrated that binding of PDGF-BB and PDGFR-? is crucial for the proliferation and migration of pericytes and the development of functional vessels.Currently,statins play crucial roles in reducing blood lipids,anti-inflammatory,regulating angiogenesis,promoting endothelial cell function and plaque stability.Clinical trials indicated that intensive statin therapy could benefit patients with coronary heart disease.Tongxinluo(TXL),a traditional Chinese compound prescription,has been used for the treatment of coronary artery disease and ischemic stroke treatment.Similar to simvastatin,tongxinluo has multiple pharmacological effects,such as the effect of anti-inflammatory,regulating serum lipid levels,antioxidant and stabilization of vulnerable plaque.However,the studies of TXL on intraplaque angiogenesis,especially on multiple angiogenic factors,are still not been reported.Therefore,compared with atorvastatin,by observing of the influence of tongxinluo on three angiogenesis factors and their corresponding receptors VEGF-A/VEGFR-2,FGF-2/FGFR-1,and PDGF-BB/PDGFR-?,we design this experiment to investigate the possible effects and molecular mechanisms of TXL on atherosclerotic intraplaque angiogenesis and plaque vulnerability in rabbits.Objective1.To observe the effects of Tongxinluo and atorvastatin on angiogenesis and plaque vulnerability in a rabbit model of atherosclerosis.2.To elucidate the possible mechanisms of Tongxinluo and atorvastatin on plaque stabilization.3.To investigate the synergistic effects of Tongxinluo and atorvastatin on stabilizing the plaque.Methods1.Experimental protocolSixty adult male New Zealand White Rabbits underwent balloon-induced endothelial injury in the abdominal aorta and then were fed a high-cholesterol diet(1%cholesterol and 99%standard rabbit diet)for 12 weeks to estabilish an animal model of athersclerosis in rabbits.2.Experimental Group and Drug InterventionSubsequently,the high-cholesterol diet was replaced by a general diet for another 12 weeks,and rabbits were randomly divided into 6 groups:control group(without treatment,n=10),group TXL-L(low-dose Tongxinluo,0.15 g/kg/d,n=10),group TXL-M(moderate-dose Tongxinluo,0.3 g/kg/d,n=10),group TXL-H(high-dose Tongxinluo,0.6 g/kg/d,n=10),group A(atorvastatin,5 mg/kg/d,n=10)and group AT(combination of high-dose Tongxinluo and atorvastatin,TXL of 0.6 g/kg/d and atorvastatin of 5 mg/kg/d,n=10).3.Serum lipid measurementBlood was drawn from the auricular vein of rabbits after fasting overnight.The serum levels of TC,TG,LDL-C and HDL-C were measured by enzymatic assays.4.Histopathological analysisParaffin sections underwent general histological staining with H&E to measure IMT,and sirius red staining to determine the collegan content.Frozen sections were stained with Oil Red O to determine the lipid deposition.5.Immunohistochemical analysisParaffin sections were stained with immunihistochemical staining.The expression of RAM-11,a-SMC actin,VEGF-A,VEGFR-2,FGF-2,FGFR-1,PDGF-BB and PDGFR-? were determined.Histology images were analyzed by Image-Pro Plus 6.0.6.Statistical analysisAll data are presented as mean ± SEM.Comparison of multiple groups was performed by analysis of variance with one-way ANOVA followed by the Dunnett's T3 test(equal variances not assumed)or least-squares difference test(with equal variance assumed).Spearman's rank correlation coefficient was used for correlation analysis.All data analyses was carried out by the SPSS software 22.0.Two-tailed P<0.05 was considered statistically significant.Results1.Serum lipid analysisCompared with control group,the serum levels of TC,TG and LDL-C were significantly lower,and HDL-C levels were significantly higher in all treatment groups.The level of TC in group AT was lower than that in group TXL-H or A;and the level of HDL-C in group AT was higher than that in group TXL-H or A.2.The maximum IMT measurementCompared with the control group,the maximum IMT of plaque were significantly reduced in group TXL-M,TXL-H,A and AT.3.Vasa vasorum density measurementCompared with the control group,the vasa vasorum density of plaque were significantly reduced in group TXL-M,TXL-H,A and AT.4.Histopathological examinationThe macrophages and lipid content in group TXL-H,A and AT were lower than that in the control group.The SMCs content in all treatment groups were higher than that in the control group.Compared with the control group,the collagen content was increased only in group AT.The vulnerability of the plaques was significantly reduced in group TXL-M,TXL-H,A and AT,and the reduction of the combined drug group was significantly lower.5.Immunohistochemical examinationThe expression of VEGF-A,VEGFR-2,FGF-2,FGFR-1,PDGF-BB and PDGFR-? were reduced in all treatment groups than that in the control group.The expression of VEGF-A and PDGFR-? in group AT were lower than that in group TXL-H or A.6.Correlation analysisThe correlation analysis results showed that all correlations between the VVD in plaque and VEGF-A,VEGFR-2,FGF-2,FGFR-1,PDGF-BB and PDGFR-? were positive.The expression of VEGF-A,VEGFR-2,FGF-2,FGFR-1,PDGF-BB and PDGFR-P was positively correlated with the vulnerability index of plaque.The correlation between VVD and ? was also positive.Conclusions1.This study found that both the Tongxinluo and atorvastatin could decrease vasa vasorum density and vulnerability index of plaque in the abdominal aorta of rabbits;2.This study confirmed that Tongxinluo and atorvastatin might inhibit intraplaque angiogenesis and promote the stability of plaque via inhibiting the expression of VEGF-A/VEGFR-2,FGF-2/FGFR-1 and PDGF-BB/PDGFR-p.3.Tongxinluo and atorvastatin might have the synergistic effects on inhibiting the expression of VEGF-A,FGF-2 and PDGFR-? and lowering the vulnerability index.