The High Shear Strees And OxLDL By VEGF-VEGFR2 Siginal Pathway Induced Angiogenesis Involved In Rupture-prone Plaque Formation

According to the current studies in the clinical, the death rate of ischemic heart disease and cerebrovascular disease were the highest, which accounted for 21.9% of deaths. Along with the improvement of people living standard, cardiovascular and cerebrovascular disease have become the first killer in China. Coronary atherosclerosis was due to the rupture of unstable plaque of coronary artery and the formation of therombosis. So the early idendification of unstable plaque is particularly important for preventing the acute cardiovascular events. The pathological study showed that the unstable plaque has the characteristics of the following organizations: a large lipid core, a thin fibrous cap with inflammatory cell infiltraction; nevasculation more. Pathological neovacularization occurred in atherosclerotic plaque, which may induce intraplaque hemorrthage and plaque rupture to promote atherosclerotic lesion development.The study reported that the plaque rupture occurred mainly in the proximal part of the plaque( high stess area), but its mechanism had not been able to clarify. Therefore, the angiogensis in the plaque neovacularization, especially immature( unformation of stable integrity membrane and outer membrane) may be the cause of intraplaque hemorrhage leading to vulnerable plaque. Shear stress and oxidized low density lipoprotein is not only the important to stimulate the formation of AS plaque vulnerability, but also the factors to angiogenesis. The view was expressed that the trophoblast vascular proliferation in plaque was the key point. The nourishing blood vessels, which had not complete function and structure, especially the unstable and leaking outer membrane, induced plaque hemorrhage and damage. So it could clarify that stenosis proximal high shear stress stimulated angiogenesis of endothelial cells to cause the formation of vulnerable plaque.VEGF was the potent and specific angiogenic factor involved in a variety of pathological angiogenesis in vivo,which played a key role in the regulation of protein in tumor angiogenesis and plaque. In the study of tumor angiogenesis, it had systematically explored the mechanism of VEGF protein and downstream pathways in tumor lesions. However, in the study of AS vulnerable plaque, VEGF was involved in the process of plaque lesions and angiogenesis, but the mechanism of VEGF and downstream regulatory protein in plaque angiogenesis(iduced by high shear stress and the high-fat), still wasnot accurate concluded. Based on the classical pathway of angiogenesis VEGF-VEGFR2-FAK / paxillin and research in ox LDL and high shear stress-mediated angiogenesis, the mechanism of VEGF and protein pathways downstream to vulnerable plaque formation would be the new AS clinical gene target.In the research, we made a narrow model through the peripheral ring of mouse left common carotid artery.On the basis of the high fat-diet fed to mice(1% cholesterol and 5% lard), it researched the composition and angiogenesis of plaque. It studies the plaque formation and angiogenesis of treatment group through injection of angiogenesis inhibitor(TNP-470). Thus, the animal model proved that angiogenesis stimulated the formation of atherosclerotic vulnerable plaque. Immune stain from angiogenesis related genesVEGF, CD31,we could get that VEGF and CD31 expression are concentrated in the shoulder area and plaque fibrous cap. And by synthesizing VEGFsiRNA, VEGFR1 siRNA, VEGFR2 si RNA, VEGF recombinant protein to stimulate endothelial cells by high fat and shear stress, we observed that downstream pathways protein FAK / paxillin activation(phosphorylation), could regulate the endothelial cell migration and tube formation,which may affect the angiogenesis in plaque mediated by high-fat and high shear stress.The main research contents and conclusions were follows:① Constraction of mice left carotid artery stenosis model, the right carotid artery was sham operation to control. In the ordinary dietary conditions, the constriction ring model dosenot caused the formation of vulnerable plaque; high stress and low shear stress regions had the consistent intimal hyperplasia. But under high fat diet, the left carotid proximal stenosis artery proned to vulnerable plaque; the distal caused intimal hyperplasia. However the right control vessel exposed to high fat condition, had very slight intimal hyperplasia. By HE staining, it showed that the neovascularization of vulnerable plaque in the high stress areas had incomplete outer membrane, apoptosis trend endothelial cell, leakage microvascular with a large number of lymphocytes infiltrating. However, in the TNP-470 treatment group, vascular intimal hyperplasia was weakened, and angiogenesis in the vulnerable plaque was also weakened.② Through the mouse carotid artery angiogenesis-related proteins VEGF, CD31 immunostaining, we found the blood vessels without the expression of CD31 and VEGF in the normal group. In the model set of high shear stress group, VEGF, CD31 are present positive expression in the shoulder and fibrous cap. Studies have been shown that VEGF was involved in the pathogenesis of plaque angiogenesis, and for a targeted gene on angiogenesis studies.③ We constructed ox LDL and sheer stress to stimulate HUVECs, for observing the role of ox LDL and shear stress in the migration and tube formation of HUVECs. The results showed that there were nomigration and tube formation of HUVECs in normal group; lots of migration and tube formation of HUVECs in oxLDL group(20μg/m L); lots of migration and tube formation of HUVECs in shear stress group(25 dyn/cm2). Therefore, ox LDL and shear stress could stimulate the migration and tube formation of HUVECs to angiogenesis.④ We synthesized VEGFsiRNA,VEGF to stimulate HUVECs by oxLDL and shear stress regulated, for observing the role of VEGF in angiogenesis mediated by shear stress and ox LDL. The results showed that overexpression of VEGF could promote the migration and tube formation of HUVECs; oxLDL and shear stress could promote VEGFR1,VEGFR2 overexpression; HUVECs with VEGFsiRNA, stimulated by ox LDL and shear stress, could reduce the migration and tube formation of HUVECs, compared with ox LDL and shear stress group. So, VEGF was the key point of the regulation on the angiogenesis mediated by shear stress and ox LDL.⑤ We synthesized VEGFsiRNA,VEGF,VEFR1 siRNA,VEGFR2siRNA to stimulate HUVECs by oxLDL and shear stress regulated,for observing the role of VEGF- VEGFR2- FAK/ paxillin in angiogenesis mediated by shear stress and oxLDL. The results showed that VEGF could promote phosphorylation of FAK/ paxillin; ox LDL and shear stress could regulate the expression of VEGF- VEGFR2-FAK/paxillin signaling pathway; VEGFR2 could promote phosphorylation of FAK/paxillin; VEGFR2/VEGFR1 was the key point of regulation on the angiogenesis mediated by shear stress and ox LDL. Therefore, VEGF-VEGFR2-FAK/paxillin could promote the angiogenesis mediated by shear stress and oxLDL.In summary, the research elucidated the role of angiogenesis in AS vulnerable plaque, and provided a theoretical and scientific basis for the VEGF- VEGFR2- FAK/ paxillin signal pathway influence to vulnerable plaque formation, that provided new targets for inhibiting AS pathological angiogenesis.