Effect of Oxidized High Density Lipoprotein on Endothelial Progenitor Cells Function and Its Mechanism
|School||Second Military Medical University|
|Keywords||Endothelial progenitor cells Oxidation of high- density lipoprotein CD36 Angiogenesis|
Background: high-density lipoprotein cholesterol (high density lipoprotein, HDL) in addition to promote reverse cholesterol transport, protect the vascular endothelium, anti-inflammatory, antioxidant and anti-thrombosis and many other beneficial effects, as well as findings, HDL can improve the number and function of endothelial progenitor cells (endothelial progenitor cells, EPCs), which helps to repair after vascular injury. However, HDL levels in many cases they are not mediated protective effect, on the contrary would lead to cardiovascular events increased, this phenomenon may be due to elevated HDL does not have cardiovascular protective effects high levels of HDL \Currently known under conditions such as diabetes, coronary heart disease, HDL easy oxidative modification generate oxidation of high-density lipoprotein (ox-HDL) occurred reversal become pathogenic factors, ox-HDL on EPCs function and role as well as the mechanism of how I do not know. Objective: This study aimed to in about pre EPCs research on the basis of further observation ox-HDL on EPCs function and to explore the mechanism of which lay the foundation for the HDL function improved treatment and reduce EPCs dysfunction. Methods: First isolated from human peripheral blood by density gradient centrifugation, culture, identification get EPCs. Next, we EPCs function mechanism ox-HDL from three aspects: (1) ox-HDL on EPCs surface receptor gene expression screening: a gene expression microarray ox-HDL intervention EPCs gene change of expression, and further validate the differential gene; (2) ox-HDL EPCs function by realtime PCR and Western Blot method: In the first part of the study results on the basis of the EPCs divided into control group, HDL intervention group (50ug / ml), different concentration of ox-HDL intervention group (10ug/ml, 20ug/ml, 50ug/ml), and antibodies of CD36 and antibody blocking group and CD36 blocking plus OX-HDL intervention group, wherein CD36 and antibody added 2 hours before intervention ahead, and cell proliferation was measured by MTT assay, Annexin V / PI assay the level of apoptosis, ROS Fluorescence Detection of intracellular oxidative stress levels Transwell chamber migration assay EPCs migration, Matrigel detection of angiogenesis in vitro ELISA was used to detect the cell culture supernatant cell factor (VEGF and TSP-1), by ligation from off the right femoral artery to establish nude mice with unilateral lower limb ischemia model by model observation of fluorescence angiography, immunohistochemistry tail vein infusion given after the intervention of EPCs, two weeks after the feeding EPCs in vivo vascular repair ability. (3) the impact of ox-HDL on EPCs function signaling pathway: intervention grouped with the second part, after the end of the intervention, the collection of total cellular protein and nucleoprotein detected by Western Blot MAPK pathway (p38, JNK, ERK ) related to the level of phosphorylation of the protein, the EMSA (Electrophoretic Mobility Shift Assay, EMSA) was used to detect NF-κB activity. Results: (1) ox-HDL screening of the related EPCs surface receptor gene expression: ox-HDL intervention EPCs and cell transport, transcriptional regulation, signal transduction, stress response, metabolism, exercise, differentiation, cell cycle , adhesion, and other related gene expression in changes in HDL or ox-HDL receptor, CD36 expression after ox-HDL intervention significantly increased to 2.3751 times the untreated group, the remaining receptor expression was no significant change in . Subsequent realtime PCR and Western Blot results further validate the ox-HDL can promote increased CD36 expression (P lt; 0.05) in a concentration-dependent manner, the corresponding HDL no role. (2) ox-HDL EPCs function: ox-HDL in a concentration-dependent manner to promote EPCs apoptosis, inhibition of their migration, angiogenesis in vitro as well as in nude mice limb ischemia in vivo angiogenesis ability to promote EPCs ROS level (P lt; 0.05), while the impact of the angiogenesis factor mainly in the promotion of TSP-1 expression and secretion (P lt; 0.05), but had no significant effect on VEGF expression, the role of such damages in advance to give the CD36 antibody treatment has partially mitigated. (3) the impact of ox-HDL on EPCs function signaling pathway: ox-HDL a dose-dependent manner the phosphorylation of p38 MAPK, while the other two aspects of the MAPK pathway ERK and JNK phosphorylation showed no significant impact, EMSA The results suggest that, ox-HDL was concentration-dependent increase of NF-κB activity and pretreated CD36 antibody, ox-HDL role of p38 MAPK and NF-κB significantly reduced. Conclusions: Our results reveal a contrast with HDL, ox-HDL has role of damage EPCs function, the damage effect mediated through the CD36 receptor pathway, which is closely related with the p38 MAPK and NF-κB signaling pathway, which may ischemic One of the important mechanisms of disease impaired angiogenesis.