Studies Of Pathogenesis Of Atherosclerosis By Hypercholesterolemia

IntroductionHypercholesterolemia, a major risk factor for atherosclerosis, cause local inflammation, oxidative stress injury, damage of the metabolism and function of endothelial cells that, in turn, lead to atherosclerosis ulimately. But we do not know yet the decisive mechanisms by which hypercholesterolemia lead to atherosclerosis.VEGF is a glucoprotein secreted by endothelial or smooth muscle cells, and is the major cytokine responsible for physiological or pathological angiogenesis and endothelial permeability. Tt has been reported that hypercholesterolemia may significantly upregulate VEGF expression[1-3]. Wheras other studies have not shown the same effect[4, 5]. Although VEGF upregulation could constitute a vascular homeostatic mechanism for compensating endothelial dysfunction,[6]. there are more evidences to show that sustained vascular VEGF overexpression could contribute to the atherosclerotic process by favoring lipoprotein infiltrationthrough increased cascular permeability, chemotaxis of monocytes/macrophages, which is crucial in early atherosclerosis and inducing angiogenesis in advanced lesion[7-10]..It has been proposed that VEGF-mediated angiogenesis is not only necessary but sufficient for atherosclerotic plaque growth[11]. Up to now, it is still unclear whether hypercholesterolemia could influence the expression and secretion of VEGF inendothelial cells.In the presence of hypercholesterolemia, the density and binding affinity of AT1 receptors in endothelial and smooth muscle cells are increased [12]. The antiatherogenic actions of ARB, independent of antihypertensive or antilipidemic effects, have been reported[13]. IStrawn et al observed a marked reduction in atherogenesis in hypercholesterolemic monkeys treated with losartan and the fatty streak composition was likely to be composed of macrophage-derived foam cells [14]. However, the mechanisms of ARB of anti-atherosclerotic action is unknown. and indecisive.Tight junction is the connection between endothelial cells. It is arranged along the borderline of cells and formed a net-like structure, which constitute the major structure to control the permeability of macromolecule. The deposition of lipoprotein into intima is the fundamental process in the initiation of atherosclerosis and the mechanisms of which is still unknown. The previous studies connectioning the risk factors of atherosclerosis with deposition of lipoprotein in the arterial endothelium focused on the impact of hypercholesterolemia on vessel permeability, not on the mechanisms by which the LDL transported across vascular endotheliai barrier.. And the molecular biological studies about the relationship between risk factors of atherosclerosis and endothelial tight junction is very scarce.Based upon above research background, the aim of our study were1. To observe the effect of hypercholesterolemic serum on the expression and secretion of VEGF in endothelial cells, and explore the mechanism of ARB antiatherosclerotic action.2. To observe the impact of hypercholesterolemic serum on tight junction in endothelial cells, and explore the mechanism of lipoprotein to penetrate through endothelium in the initiation of atherosclerosis.Methods1. Establish immortalized endothelial cell linePrimary human umbilical vascular endothelial cells (HUVECs) were purchasedfrom Digestech Co. and were cultured in matrigel. Recombinant retrovirus containing SV40LT or hTERT fragment respectively were used to transfect primary HUVECs. G418 and puromycin were used for selection. Drug resistant cells were selected and expanded for identification and further study. The expression of SV40LT and hTERT were detected with western blot. The morphology of the cells was observed with microscope and scanning electron microscope. Immunohistochemistry, ELISA and RT-PCR were used for identification of the tranfected cell line.2. Observe the change of VEGF expression in endothelial cellsRabbits were fed with a diet containing 1% cholesterol for 4 weeks. Hypercholesterolemic serum was isolated from the rabbits. Cultured HUVECs were treated with hypercholesterolemic serum of various cholesterol concentrations or combination with telmisartan and/or PI3K specific inhibitor wortmannin. VEGF expression were determined by ELISA and western blot respectively. PI3K activity was detected[15]. Angiogenesis in vitro was performed to confirm the change of VEGF expression.3. Observe the change of ZO-1 expression and distribution in endothelial cellsCultured HUVECs were grown on the surface of plastic slide, fixed with 100% methanol. Immunofluorescence was used to observe the distribution of ZO-1. Western blot and RT-PCR were used to detected the expression of ZO-1. HUVECs permeability was determined with vessel permeability assay. PI3K activity detection and PI3K specific inhibitor wortmannin were used too in the study.Results1. Foundation of immortalized endothelial cell lineEndothelial cells digested by collagenase IV were polygonal, abundant with kytoplasm. The nuclei were round or elliptic. Cell clones were appeared in 8-12 weeks after tranfection of recombinant retrovinis containing SV40LT and hTERT. Identification of cell characteristic: Under scanning electron microscope, the cells were polygonal, round or fusiform, with many apophysis at cell surface whichconnected with each other. Ⅷ factor was detected with immunohistochemistry. Endothelial specific E-selectin was detected with ELISA. And endothelial lipase mRNA was detected with RT-PCR. The expression of SV40LT and hTERT were detected by Western blot. In the test of PI3K activity , it is found that the activity of telomerase was 0.36 at 12 population doublings and 0.38 at 50 population doublings in transfected cells; compared with 1.12 at first population doubling, 0.06 at third population doublings in primary endothelial cells. Till now, it had been cultured more than 60 population doublings, and no malignant appearence could be found.2. The change of VEGF expression in endothelial cellsVEGF expression in endothelial cells treated with hypercholesterolemic serum(cholesterol concentration ≥0.08mMol/L) was significantly enhanced and moreover,increased VEGF protein expression in endothelial cells was in a concentration-dependent manner. Treated with PI3K specific inhibitor wortmannin, VEGF expression could be significantly inhibited. And PI3K activity was positively correlated with VEGF expression. Telmisartan alone seemed to increase VEGF expression and PI3K activity, but there is no statistically significant different compared with control. When telmisartan cooperated with hypercholesterolemic serum, VEGF expression and PI3K activity were significantly enhanced compared with that of hypercholesterolemic serum alone. PI3K activity was positively correlated with VEGF expression too. PI3K activity and VEGF expression could also be attenuated by wortmannin. When placed on matrigel matrix in the presence of hypercholesterolemic serum, angiogenesis in vitro by endothelial cells was enhanced. In the presence of hypercholesterolemic serum plus telmisartan, the appearence of angiogenesis was evident compared to hypercholesterolemic serum alone. Telmisartan alone had no obvious effect on angiogenesis. Wortmannin could significantly suppress angiogenesis induced by hypercholesterolemic serum or combined with telmisartan, and no significant difference could be seen compared with control.3. The change of ZO-1 expression and distributionThe expression of ZO-1 in endothelial cells treated with hypercholesterolemicserum was not changed significantly compared with control. In control, ZO-1 was arranged along with borderline of the cells and formed a net-like structure under microscope. Hypercholesterolemic serum could disturb the distribution of ZO-1 in endothelial cells, and the net-like structure was interrupted. The ZO-1 redistribution induced by hypercholesterolemic serum could be inhibited by wortmannin. Vessel permeability assay showed that hypercholesterolemic serum could significantly increase the permeability of endothelial cells to albumin, which could be attenuated by wortmannin.Conclusion1. Ectopic co-expression of SV40LT and hTERT within cells could extend the life span of endothelial cells markedly.2. Hypercholesterolemic serum could upregulate the expression of VEGF in endothelial cells through PI3K signal pathway.3. There is no effect of telmisartan alone on VEGF expression in endothelial cells.4. Telmisartan can accelerate overexpression of VEGF by hypercholesterolemic serum in endothelial cells.5. Hypercholesterolemic serum has no effort on expression of ZO-1 in endothelial cells, and it can alter the distribution of ZO-1 within cells, leading to increase in permeability of endothelium. The effect of hypercholesterolemic serum mentioned above was at least partly due to the overexpression of VEGF.