The Effects Of Apelin On The Expression Of Adhesion Molecules And Chemokines In HUVECs And The Cellular Signal Transduction Mechanism

Objective:To observe mRNA and protein expression of ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (Vascular cell adhesion molecule 1) and MCP-1 (Monocyte chemotactic protein 1) by human umbilical vein endothelial cells in vitro after the intervention of apelin with different concentrations or at various points in time.Methods:Primary human umbilical vein endothelial cells (HUVECs) were separated, cultivated and identifited. After the intervention of apelin with various concentrations in 0,10-10,10-9and 10-8M for 12h, the total RNA of HUVECs were extracted. The mRNA expression of ICAM-1, VCAM-1 and MCP-1 in HUVECs was detected by Real-Time PCR. Then HUVECs were intervened in time course at the points of 0,2,4,8,12 and 24h. Cells and culture supernatants were collected. To examine the ICAM-1,VCAM-1 and MCP-1 mRNA expression using Real-Time PCR, cell protein expression using Western Blot and secretory protein concentration in culture supernatants using ELISA.Results:There are markedly increases in the mRNA expression of ICAM-1, VCAM-1 and MCP-1 in HUVECs as the result of apelin intervention. The most efficient concentration seems to be 10-8M. The time course expression curve of ICAM-1 is like a bell and the highest level is at 4h. The expression levels of VCAM-1 and MCP-1 are increased with the extending of the time and reach the peak at 12h and 24h respectively.Conclusion:Apelin accelerates ICAM-1, VCAM-1 and MCP-1 expression in concentration-and time-dependant manners. The inflammatory reaction in HUVECs caused by apelin suggests that apelin may be an atherogenic factor. Objective:To observe the effect of APJ gene silencing by RNA interference on the expression of adhesion molecules and chemokines in human umbilical vein endothelial cells(HUVECs) in vitro induced by apelin and to state the possible mechanism of apelin-APJ system regulating atherosclerosis in endothelial cell level.Methods:ShRNA(small hairpin RNA) expression cassettes were designed based on human APJ cDNA encoding sequence, plasmids were constructed with pGenesil-1 vector and negative control vector was made also. Plasmids were transfected stablely into HUVECs mediated by Liposomes. Silencing effect of APJ was tested by mRNA and protein expression of APJ. After the intervention of apelin, mRNA and protein expression of ICAM-1, VCAM-1 and MCP-1 by HUVECs from untransfected group, negative transfected group and APJ silencing group were all detected.Results:The increase of apelin-induced ICAM-1, VCAM-1 and MCP-1 expression in HUVECs was inhibited markedly in APJ silencing group(ICAM-1 decreased for more than 90%) compared to other two groups.Conclusion:APJ, as apelin receptor in HUVECs, is indispensable in the positive regulation of apelin on adhesion molecules and chemokines. Endothelial inflammatory reaction caused by apelin-APJ system provides cytological evidence for APJ gene knockout alleviating mice atherosclerosis model. Objective:To clarify whether and how JNK (c-Jun N-terminal Kinase) and NF-κB (nuclear factor-kappa B) signal molecules participate the regulation mechanism of apelin induced endothelial inflammatory reaction.Methods:After HUVECs were intervented by apelin in a time course, total protein was collected in a phosphorylation protection circumstance to detect p-JNK expression using specific phospho-antibody by Western Blot. Nuclear protein and cytoplasmic protein were collected respectively to examine NF-κB-p65 and IκBαexpression. Pretreated with JNk inhibitor SP600125 or NF-κB inhibitor SN50 followed by apelin intervention, total RNA was extracted for ICAM-1, VCAM-1 and MCP-1 mRNA detection.Results:Apelin triggered a JNK phosphorylation peak at 60min and at the same time nuclear p65 protein was detected with a strongest signal. However, IκBαin cytoplasm was in the bottom of the Valley-type curve. SP600125 and SN50 both could inhibit apelin-induced ICAM-1, VCAM-land MCP-1 expression in HUVECs.Conclusion:Apelin accelerates adhesion molecules and chemokines expression in HUVECs through JNK phosphorylation and NF-κB nuclear translocation. The inflammatory reaction due to increased adhesion molecules and chemokines would promote the research in the mechanism of apelin-APJ related atherosclerosis to a further extent.