| Objective: Atherosclerosis is the main pathophysiological basis of heart head blood-vessel disease. In order to effectively prevent the damage caused by atherosclerosis, people have been investigating its mechanism for hundred years. Today, the inflammation has been prominently and widely accepted in the mechanism of atherosclerosis. Endothelial cell injury occurs in the early stage of atherosclerosis, playing a key role in the development of atherosclerosis. In the cardiovascular disease with the pathological basis of atherosclerosis, endothelial dysfunction, rennin-angiotensin system (RAS) activation and the inflammatory mechanisms play an important role.RAS over-activation engaged in a variety of diseases, such as hypertension, atherosclerosis, myocardial hypertrophy, apoptosis, heart failure, renal dysfunction and so on. In the classical RAS, the angiotensinⅡ(AngⅡ) is considered as the most important effective molecules, which can directly make smooth muscle contract, increaseing endothelin, and promote the production of superoxide to inactivate NO. Through the activation and recruitment inflammatory cells reaching the surface of damaged blood vessels, AngⅡcan regulate local inflammation. AngⅡcan regulate a variety of cell adhesion molecules, growth factors and chemotactic factor. AngⅡalso can act on some nuclear transcription factors, indirectly control inflammation, and accelerate atherosclerosis process.In clinic, the application of angiotensin-converting enzyme inhibitor (ACEI) and angiotensinⅡreceptor blocker (ARB) reduced the generation of AngⅡand inhibited AngⅡreceptors and showed some effects of atherosclerosis prevention. But the results of clinical trials realised that these effects are not enough to prevent the damage of atherosclerosis. Some research showed that latest RAS blockers—renin inhibitor (Aliskiren) can’t block the activation by receptor-mediated signaling pathway and stop the damages on target organ by activated transcription factors, failed to meet the expected ideal effect of RAS blockade.Recently, it has been found that (pro)renin receptor [(P)RR] was the common functional receptor of renin and (pro)renin. After renin and (pro)renin bind to the (P) RR active site, molecular structure was changed, enzyme activity increased, and activated signal transduction pathways. So renin and (pro)renin can play a biological role independent traditional RAS activation. (P)RR system composed by rennin, (pro)renin and (P)RR, was considered as a new member of RAS, and attract some focus.Mitogen-activated protein kinases (MAPKs) are serien/ threonine -specific protein kinases that respond to extracellular stimuli and regulate various cellular activities, such as cell proliferation, differentiation, transformation and apoptosis. Extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPKs are two of MAPKs which play an important role in inflammation and oxidative stress. It was not clear whether inflammation and oxidative stress induced by (P)RR were relatived to signaling pathway ERK1/2 and p38 MAPKs.Recently, our research group has confirmed the existence of renin receptor in human umbilical vein endothelial cells (HUVECs) by immunohistochemistry. Whether (P)RR system induced endothelial cell dysfunction and involved in atherosclerosis, there were no relevant reports.PI3K/AKT signaling pathway can directly phosphorylate a variety of cytokines, such as NFκB, Bcl-2, TGF-β, which is related with vascular wall cell phenotype conversion, proliferation, migration and hypertrophy. Thus, PI3K/AKT signaling pathway maybe involved in the machenism of (pro)renin and its receptor, we need to research wheter activation of (P)RR is able to influence PI3K/AKT signaling pathway.(Pro)renin propeptide (HRP) is a synthetic (pro)rennin propeptide containing 10 amino acids, which is presumed to affect the binding of (pro)renin and (P) RR, thus HRP is considered as the (P) RR blocker. But whether HRP can block (P) RR, there were not consistent related results.The purpose of our study are to investigate whether (pro)renin can activate the (P)RR leading to the phosphorylation of ERK and p38 MAPK singaling pathway and increase the expression of VCAM-1 and reduce the activity of SOD; and to study whether prorenin could activate PI3K, AKT, NFκB, IL-6 in HUVECs through (P)RR independent of angiotensinⅡ(AngⅡ); and study whether HRP exists effect on (P)RR blocking. Thus we want to study if (P)RR activistion can led to endothelial damage, and to explain the relationship between (P)RR and atherosclerosis.Methods:1.HUVECs were cultured in vitro, indentified by CD34 Flow Cytometry .2.HUVECs were incubated with (P)RR siRNA respectively 24, 48 and 72 hours, the mRNA of (P)RR expression was detected by RT-PCR.3.To determine the (pro)renin stimulation time on HUVECs, ERK1/2 and p38 pathway protein phosphorylation were evaluated by western blot when 5, 10, 20, 35, 60, 90 minutes after treatment with (pro)renin on HUVECs. Ang-Ⅱreceptor AT1 and AT2 were blocked by Olmesartan and PD123319.4.Ang-Ⅱreceptor AT1 and AT2 were blocked by Olmesartan and PD123319. Western blot was used to detected whether signaling pathway ERK1/2 and p38 were phosphorylated, ELISA was used to checked whether vascular cell adhesion molecule VCAM-1 expression was increased and Colorimetry was used to detected antioxidant SOD activity, and whether all these reactions were inhibited after (P)RR was interferenced.5.AT1/AT2 receptors of AngⅡwere blocked by Olmesartan(10-5M) and PD123319(10-4M) respectively to observe the prorenin(2×10-9M)’s effect on the expression of PI3K, p-AKT and NFκB-P65 in HUVECs, as well as IL-6 in cultural medium.6.(P)RR in HUVEC was treated by RNA interference with (P)RR - siRNA, and HRP(1×10-6M, 5×10-6M, 10×10-6M) respectively, the changes of p-AKT, P65, IL-6 were observed.7.PI3K/AKT signal transduction pathway was blocked with wortmannin(0.5×10-6 M), the expression of p-AKT, P65, IL-6 were observed.8.PI3K,p-AKT,p65 were evaluated by western blot.9. The concentration of IL-6 in cultural medium was evaluated by ELISA kit.Results:1. 98.35% HUVECs were identificated by CD34 Flow Cytometry.2.The weakest expression of (P)RR mRNA was at the point of 72 hours after interfered with (P)RR-specific siRNA.3.In HUVECs, ERK1/2 and p38 MAPKs protein were significantly phosphorylated after stimulated with (pro)renin, which were time dependent. 4.ERK and p38 MAPKs singaling pathways were actived and the expression of VCAM-1 was increased and the activity of SOD was decreased by (pro)renin, while interfered (P)RR with siRNA could inhibite these reactions.5.Prorenin up-regulated the expression of PI3K, p-AKT, NFκB-P65, and IL-6. The activation of p-AKT, NFκB-P65 and IL-6 was inhibited by (P)RR-RNA interference.6.The activation of P65 and IL-6 was not inhibited by wortmannin.7.All of the three doses of HRP did not affect the activation of AKT, NFκB-P65 and IL-6 caused by renin/prorenin/(P)RR.Conclusions:1.(P)RR expresses on HUVECs.2.Prorenin and renin can activate the (P)RR, lead to oxidative stress and increase phosphorylation of ERK and p38 MAPKs protein and increase the expression of VCAM-1 by independent AngⅡway.3.Prorenin can up-regulate the expression of PI3K, p-AKT, P65 and IL-6 independent of Ang II generation.4.The activation of AKT, NFκB-P65, IL-6 by (pro)renin is through (P)RR.5.The over expression of P65 and IL-6 by stimulating with prorenin may not be regulated through PI3K/AKT signal transduction pathway.6.HRP does not have effect on (P)RR blocking. |
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