Dissertation > Excellent graduate degree dissertation topics show

HDL Induces COX-2 Expression and PGI₂ Production Through SR-B1 Mediated PI3K-Akt-eNOS Pathway in Endothelial Cells

Author: ZhangQingHai
Tutor: YiGuangHui
School: Nanhua University
Course: Pathology and Pathophysiology
Keywords: Atherosclerosis Thrombosis ECV304 endothelial cells High-density lipoprotein Scavenger receptor class B type I Phosphatidylinositol 3 - kinase Endothelial nitric oxide synthase Cyclooxygenase-2 Prostacyclin
CLC: R363
Type: Master's thesis
Year: 2010
Downloads: 116
Quote: 0
Read: Download Dissertation

Abstract

Background: Atherosclerosis thrombosis (atherothrombosis) has a myocardial infarction, thrombotic stroke, and peripheral vascular disease, a variety of clinical diseases common basic pathological processes. Atherosclerosis as a chronic pathological process in the early formation of atherosclerosis can be no clinical symptoms, and other factors in chronic inflammatory stimulation, especially the vulnerable plaque atheroma plaque erosion or rupture can occur and further hair platelet adhesion, activation, accumulation, acute thrombus formation. The presence of natural anti-vivo platelet activation, accumulation inhibitor, these inhibitors in preventing atherosclerosis and thrombosis plays an important role. Prostacyclin (prostacyclin, PGI2) is arachidonic acid by cyclooxygenase (cyclooxygenase, COX) pathway catalyzed cascade generated by a lipid media. Study found that PGI2 has a powerful vasodilator and anti-platelet aggregation effect. PGI2 and TXA2 is by far the regulation of platelet function known to have the strongest one endogenous substance, PGI2 and TXA2 is to maintain a balance between the environment is relatively stable within the normal blood necessary condition. Currently elevated blood levels of PGI2 has become clinically effective antithrombotic therapy target. Cyclooxygenase is the arachidonic acid cascade catalytic reaction process is an important rate-limiting enzyme, there are two subtypes: structure type (COX-1) and a fast-growing type (COX-2). Study found that COX-2 and atherosclerosis are closely related, in atherosclerotic plaques of COX-2 expression was significantly increased, and plaques COX-2/PGES overexpression of atherosclerotic plaque is not stability related, but there are more and more evidence that COX-2 is not entirely harmful to the body, it can promote inflammation and also has anti-inflammatory, anti-fibrotic and anti-thrombosis effect. HDL (high density lipoprotein, HDL) mainly by the composition of lipids and apolipoproteins. At present, more and more studies show HDL has anti-atherosclerosis and vascular protective effects of plasma high density lipoprotein cholesterol (HDL-C) levels and the risk of clinical cardiovascular events significantly negative correlation exists between and research also found that the plasma levels of HDL-C and plasma PGI2 stable product 6-keto-PGF1 levels were positively correlated. In vitro experiments also confirmed HDL2 and HDL3 could be dose-dependent induction of endothelial cell PGI2 release after inhibition of COX-2 activity decreased production of PGI2 and phospholipid composition of HDL, one - phosphoric acid - sphingosine (Sphingosine-1-phosphate , S1P), through the cell membrane p38MAPK/CREB S1P receptor-mediated smooth muscle cells upregulate the expression of COX-2 and PGI2 release. Scavenger receptor class B type 1 (Scavenger receptor class B type I, SR-B1) as relevant in vivo physiological HDL receptor, which can be carried out with the specific recognition of HDL apoA1, combined, and thus HDL-mediated anti-inflammatory, anti-thrombotic, anti-oxidation, anti-endothelial injury and so on. HDL and SR-B1 receptor binding via PI3K-Akt/MAPK pathway activation of endothelial nitric oxide synthase (endothelial nitric oxide synthase, eNOS), increased NO production and activation of Rac, culminated in the formation of endothelial cell pseudopods migration, thereby repairing endothelial injury. The first part of SR-B1 in HDL-induced endothelial cell PGI2 release Role purpose: SR-B1 as a physiological relevant HDL receptor, it also generates HDL-induced endothelial cell PGI2 play a role. Therefore, the purpose of this study is the observation of SR-B1 receptor in endothelial cells induced PGI2 release HDL role in the process. Method: 1. Different concentrations (30μg/ml, 60μg/ml, 120μg/ml) apoA1 treated ECV304 cells 24 hours; 30μg/ml apoA1 handle different time (3,6,12,24 hours); 30μg/ml apoA1 and 1μmol / L S1P treatment of endothelial cells for 24 hours. 2 transfected pcDNA3.1 (-)-hSR-B1 expression plasmid for 48 hours, 30μg/ml HDL incubated for 24 hours. 3 Transfection SR-B1 siRNA 48 hours later, 30μg/ml HDL incubated for 24 hours. Let blank as control group, treated with 30μg/ml HDL as a positive control, identified under a fluorescence microscope transfection efficiency; RT-PCR and / (or) Western blot detection of SR-B1 mRNA and protein expression; ELISA assay medium of 6-keto-PGF1 formation. Results: 30μg/ml apoA1 for 12 hours can make increased production of PGI2 and 24 hours and reached the peak; while the concentration of the experiment can be found at 30μg/ml concentration PGI2 see increased production, but then did not generate PGI2 apoA1 concentration increased with the increase; 30μg/ml HDL, 1μM S1P could significantly increased endothelial release of PGI2 and apoA1 could increase the release of PGI2 endothelial cells, but its higher amplitude than HDL, S1P must be low. Transfected with pcDNA3.1 (-)-hSR-B1 recombinant plasmids overexpressing SR-B1 receptor, Western blot detection of SR-B1 protein successfully regulated, but not over-expression of SR-B1 can significantly increase HDL-induced endothelial cell PGI2 formation. RT-PCR, Western blot detection of SR-B1 three pairs of siRNA sequences could decrease SR-B1 mRNA and protein expression, but to SR-B1 siRNA-S2 most obviously transfected SR-B1 siRNA-S2 48 hours after and then incubated with HDL for 24 hours, can significantly reduce the HDL induced endothelial PGI2 production. The second part of the SR-B1 HDL-mediated induction of endothelial cell PGI2 release mechanism Purpose: preliminary experimental results show that SR-B1 in HDL-mediated induction of endothelial cell PGI2 generation, so the present study focuses on SR-B1 affect HDL-induced endothelial cell PGI2 release possible mechanisms. Method: 1. Were transfected with pcDNA3.1 (-)-hSR-B1 expression plasmid or SR-B1 siRNA 48 hours later, 30μg/ml HDL 24 hours of incubation; 2. 30μg/ml apoA1 ECV304 endothelial cells were incubated with 24 hours; 3 respectively 25μmol / L PI3K inhibitor LY294002 or 50μmol / L eNOS inhibitor L-NAME pretreated for 3 hours, 30μg/ml HDL incubated for 24 hours. Blank group as a negative control, 30μg/ml HDL treated as a positive control, RT-PCR and / (or) Western blot detection of PGIS, COX-2, CREB and phospho-CREB expression; immunocytochemistry assay of COX endothelial cells -2 expression; ELISA assay medium 6-keto-PGF1 formation. Results: either mRNA levels or protein levels in the overexpression of SR-B1 after, HDL were not significantly increased COX-2 expression, however, interestingly siRNA silencing SR-B1, the visible HDL-induced endothelial cells of COX-2 expression was significantly reduced, and immunocytochemistry to get similar results. 30μg/ml apoA1 endothelial cells were incubated for 24 hours, COX-2 expression also appears to increase. However, regardless of the separate treatment or over-expression of HDL or silenced SR-B1, PGIS expression have not changed significantly. Inhibitor treated cells alone did not affect the formation of PGI2, but after inhibition of PI3K or eNOS activity can significantly reduce HDL-induced PGI2 production, after which the activity to inhibit PI3K, PGI2 production decreased more significant; Similarly, LY294002 or L-NAME on HDL-induced endothelial cell COX-2, phosphorylated CREB expression and influence the generation of PGI2 showed the same effect, specific inhibition of PI3K and specific inhibition of eNOS activity can lower HDL-induced COX-2 expression and CREB phosphorylation, and equally to inhibit PI3K have dropped more obvious after. Conclusions: 1. HDL-induced endothelial cell COX-2 expression and PGI2 release partially dependent HDL receptor SR-B1. 2. Inhibit endothelial cell PI3K or eNOS activity can be lowered HDL-induced CREB phosphorylation in endothelial cells, COX-2 expression and PGI2 formation, and to inhibit PI3K activity after a downward effect is more significant. 3. HDL-induced endothelial cell PGI2 release its dependence SR-B1-mediated intracellular PI3K-Akt-eNOS signaling pathway activation.

HDL Induces COX-2 Expression and PGI2 Production Through SR-B1 Mediated PI3K-Akt-eNOS Pathway in Endothelial Cells

Abstract

Related Dissertations

HDL Induces COX-2 Expression and PGI₂ Production Through SR-B1 Mediated PI3K-Akt-eNOS Pathway in Endothelial Cells