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

BACKGROUND: Atherothrombosis has become a common pathological process in many diseases such as myocardial infarction, thrombotic stroke, and peripheral vascular disease. Because of atherosclerosis as a chronic pathological process, the individuals might have no clinical symptom in the early stage of atherosclerosis. With the development of atherosclerosis, some stimuluses such as chronic inflammatory factors might erode or rupture plaque lesions to promote platelet adhesion and aggregation, and then contribute to thrombosis. Nevertheless, there also exist natural anti-platelet molecules which are significant in the prevention of atherosclerosis and thrombosis in vivo.Prostacyclin (PGI2) is a lipid mediator derived from arachidonic acid after a cascade reaction catalyzed by the cyclooxygenase (COX). It has been well known so far that PGI2 is a powerful vasodilator and an anti-platelet aggregation molecule. PGI2 and TXA2 as a pair of endogenous substances which exert the strongest regulation on platelet function, the balance between them is necessary to maintain normal blood homeostasis in vivo. At present increasing plasma levels of PGI2 has become a therapeutical target on against thrombosis.Cyclooxygenase is the rate-limiting enzyme in arachidonic acid cascade reaction, and there are two main subtypes: COX-1 and COX-2. Numerous studies found that COX-2 expression significantly increased in atherosclerotic plaques and COX-2/PGES overexpression within atherosclerosis plaques might affect plaque stability. Besides, there have been many increasing evidences that COX-2 was not entirely harmful in vivo, it could play an anti-inflammatory, anti-fibrosis and anti-thrombosis role.Recent findings have indicated that HDL might exert a powerful cardioprotective effect, and the plasma levels of high-density lipoprotein cholesterol (HDL-C) were negatively correlated with the risk of clinical cardiovascular events. Studies also found there was a positive correlation between the plasma levels of HDL-C and 6-keto-PGF1αwhich was a stable metabolite of PGI2. Experiments in vitro also confirmed that HDL2 and HDL3 could induce PGI2 release in endothelial cells in a dose-dependent manner, and inhibited COX-2 activity significantly decreased PGI2 production. Apart from these, the phospholipid composition of HDL, sphingosine-1-phosphate (S1P), binding to S1P receptors could raise COX-2 expression and PGI2 release through p38MAPK/CREB pathway in vascular smooth muscle cells.Scavenger receptor class B type 1 (SR-B1) as a HDL physiological receptor, it might specifically bind with apoA1 composition of HDL and then mediate multiple roles of HDL like anti-inflammatory, anti-thrombotic, anti-oxidant and repairing endothelial injury. Findings showed that HDL associated with SR-B1 might activate eNOS to increase NO production, and activate Rac to form pseudopodia, migrate and repair endothelial injury through the PI3K-Akt/MAPK pathway.Part I The role of SR-B1 in HDL induced PGI2 release in endothelial cellsObjective: SR-B1 as a physiological HDL receptor, whether it also play a role in the process of PGI2 induction by HDL in endothelial cells is still unclear. Thus, in this part we are to observe the role of SR-B1 in PGI2 induction by HDL in endothelial cells.Methods: 1. Different concentrations of apoA1 (30μg/ml, 60μg/ml, 120μg/ml) incubated with ECV304 cells for 24 hours. 30μg/ml apoA1 treated for different time (3, 6, 12, 24 hours). 30μg/ml apoA1 and 1μmol/L S1P treated endothelial cells for 24 hours. 2. Transfected with recombined expression plasmid, pcDNA3.1(-)-hSR-B1, for 48 hours, then incubated with 30μg/ml HDL for 24 hours. 3. Transfected with SR-B1 siRNA 48 hours later, and 30μg/ml HDL treated for 24 hours. 30μg/ml HDL-alone-treated group as a positive control. Subsequently, fluorescent microscope identification of transfection efficiency, RT-PCR and (or) Western blot detection of SR-B1 mRNA and protein expression and ELISA assay of 6-keto-PGF1αin medium were performed.Results: With 30μg/ml apoA1 treated for 12 hours, the PGI2 production increased significantly, and peaked after 24 hours. In concentration experiments, we found that treated with apoA1 30μg/ml could induce PGI2 production obviously, but PGI2 release did not rise significantly later with the increasing in apoA1 concentration. Both 30μg/ml HDL and 1μM S1P treatment could notablely increase the release of PGI2 in endothelial cell, and apoA1 could also slightly increase the release of PGI2, but the increase was smaller than HDL and S1P treated group. After transfected with pcDNA3.1 (-)-hSR-B1 for 48 hours, Western blot detection turned out SR-B1 protein were successfully raised, but the over-expression of SR-B1 did not affect PGI2 generation induced by HDL. Transfected SR-B1 siRNA sequence for 48 hours, then RT-PCR and Western blot detection of SR-B1 mRNA and protein expression showed three pairs of siRNA sequence all could down-regulate SR-B1 expression, but SR-B1 siRNA-S2 group turned out the most obvious down-regulation. Interestingly, transfected SR-B1 siRNA-S2 for 48 hours later then incubated with 30μg/ml HDL for 24 hours, the PGI2 levels of the medium was significantly reduced.Part II The possible mechanism of SR-B1 mediated PGI2 release induced by HDL in endothelial cellsObjective: Our previous findings showed that SR-B1 receptor might participate in HDL induced PGI2 release in endothelial cells. Thus, in this part we are to investigate the possible mechanism of SR-B1 mediated PGI2 release induced by HDL in endothelial cells.Methods: 1. Transfected with pcDNA3.1(-)-hSR-B1 plasmid or the SR-B1 siRNA-S2 for 48 hours respectively, then incubated with 30μg/ml HDL for 24 hours; 2. 30μg/ml apoA1 cultured ECV304 endothelial cells for 24 hours; 3. Respectively pre-treated with 25μmol/L PI3K specific inhibitor LY294002 or 50μmol/L eNOS specific inhibitor L-NAME for 3 hours, then 30μg/ml HDL incubated for 24 hours. Incubation with DMEM medium alone as a negative control group, 30μg/ml HDL group as a positive control. Subsequently, RT-PCR and (or) Western blot detected the PGIS expression, COX-2 expression and phosphorylation of CREB. Meanwhile, immunocytochemistry assay of COX-2 expression in endothelial cells and ELISA assay of 6-keto-PGF1αin medium were performed.Results: Both in transcription and post-transcription level, the overexpression of SR-B1 did not obviously up-regulate COX-2 expression induced by HDL, while siRNA silenced SR-B1 significantly reduced HDL induced COX-2 expression in endothelial cells, and immunocytochemistry could get the same result. 30μg/ml apoA1 treated alone for 24 hours appeared to slightly increase COX-2 expression also. However, HDL, SR-B1 siRNA and pcDNA(3.1)-hSR-B1 group all did not significantly affect the expression of PGIS. The PI3K or eNOS specific inhibitors treated alone did not change the PGI2 production, but inhibited PI3K or eNOS could significantly reduce the HDL induced PGI2 production, and the reduction of PGI2 was more significant in the group pre-treated 25μmol/L LY294002 (PI3K specific inhibitor). Interestingly, LY294002 or L-NAME pre-treatment turned out an inhibition on HDL induced COX-2 expression and phosphorylation of CREB in endothelial cells, which were same to the effect of such inhititors on HDL induced PGI2 production.CONCLUSIONS:1. HDL induced COX-2 expression and PGI2 release partly depends on SR-B1 receptor in endothelial cells.2. Inhibited PI3K or eNOS activity may down-regulate HDL induced CREB phosphorylation, COX-2 expression and PGI2 production in endothelial cells, and the role of PI3K special inhibitor were more significant.3. PGI2 induction by HDL depended on SR-B1 may be associated with SR-B1 mediated PI3K-Akt-eNOS pathway activation.