The Role And Mechanism Of EndMT In Atherosclerotic Endothelial Injury Induced By Hyperlipidemia

Background and Objective:Atherosclerosis is the leading cause of morbidity and mortality in the world,and endothelial cell dysfunction plays an important role in its pathogenesis.Hyperlipidemia is one of the recognized risk factors for atherosclerosis and has been shown to have a damaging effect on endothelial cells,but the underlying mechanism is still unclear.End MT is a special state in which endothelial cells are transformed into mesenchymal cells under hyperlipidemia and other stimulating factors,and physical barrier function and secretion function of endothelial cells are impaired.In recent years,study after study shows that End MT can promote the development of atherosclerosis.However,most of these studies focus on the state of endothelial cells in the interior or adjacent endothelial layers after the formation of atherosclerotic plaques,and less to study the extent of End MT in vascular endothelial cells before plaque formation.The purpose of this study is to examine whether hyperlipidemia causes endothelial cells to develop End MT before atherosclerotic plaque formation,and,if so,to further explore the mechanism of this process.Methods:Part 1 EndMT was induced by hyperlipidemia in endothelial1.Clinical vascular specimens were collected and classified into non-hyperlipidemia group(N group)and hyperlipidemia group(HLP group)according to the condition of blood lipids.Immunofluorescence experiments were performed on frozen sections of collected blood vessels using CD31 as endothelial cell marker and ?SMA as mesenchymal cell marker to detect the degree of End MT.Leica SP8 laser confocal microscopy was used to photograph and analysis of images was made by its analysis module;2.The aorta was isolated of the apoE-/-transgenic mice at different yearlings and oil red staining was used to select the mice with early atherosclerosis.For the selected mice,eyeball blood samples were taken to measure blood lipids and plasma ox-LDL,and the aorta was isolated and immunofluorescence enface was performed after segmentation,using ?-SMA as the target protein for detecting the occurrence of End MT.After several consecutive photographs were taken with a Leica SP8 laser confocal microscope,the images were 3D reconstructed using the confocal software and then analyzed using the 3D analysis module.3.HAECs were intervened with LDL and ox-LDL,and the expression of End MT-related proteins such as VE-cadherin,CD31,fibronectin,and ?SMA were detected using WB to determine what kind of hyperlipidemia factors could be used for follow-up cell intervention experiments,then the concentration gradient experiments were performed to determine the appropriate intervention concentration.Part 2 The mechanism of End MT induced by ox-LDL in endothelial cells1.The expression of TGF?,smad2/3 and p-smad2/3 proteins in the TGF? pathway were detected by WB after ox-LDL intervention in HAECs.2.The expression of e NOS and p-e NOS were detected in HAECs treated with ox-LDL.The myograph system was used to detect the vasodilation ability of apo E-/-mice and the wild control mice to evaluate the endothelial e NOS function.Then the expression of End MT related protein was detected in HAECs after enhancing or inhibiting e NOS function.Results:Part 1 EndMT was induced by hyperlipidemia in endothelialThe EndMT levels in human mesenteric artery endothelial cells and apoE-/-mice aortic endothelial cells were significantly increased under hyperlipidemia conditions,and the End MT level of ox-LDL-treated HAECs was significantly increased in a concentration-dependent manner.Part 2 The mechanism of End MT induced by ox-LDL in endothelial cellsThe increase of TGF?,smad2/3 and p-smad2/3 expression after Ox-LDL intervened with HAECs proved that TGF? pathway was involved in the regulation of this process.This study also found that the expression and function of e NOS in HAECs treated with ox-LDL,and the vasodilation function test also confirmed that hyperlipidemia can inhibit endothelial e NOS function.In addition,the extent of End MT was found to be inversely related to the function of e NOS through interfering HAECs with enhancing or inhibiting the release of NO from e NOS,demonstrating that e NOS can inhibit End MT by releasing NO in endothelial cells.Conclusion:Prior to atherosclerotic plaque formation,hyperlipidemia caused damage to endothelial cells by inducing End MT,which may be caused by increased ox-LDL in hyperlipidemia condition.TGF?pathway and e NOS/NO pathway were mainly invoved in promoting End MT in endothelial cells,which may provide new therapeutic ideas for reversing the early endothelial injury in atherosclerosis.