The Effects Of SPLA2-â…¡A In Human Umbilical Vein Endothelial Cells

Background:Coronary heart disease is a major cardiovascular disease in the world with higher morbidity and mortality. Atherosclerosis is a basic pathological change in cardiovascular disease. Inflammatory reactions and cytokines have been recognized as the most important factors to the pathogenesis of atherosclerosis. Recently, more and more evidences demonstrate that secretory phospholipase A2group IIA (sPLA2-IIA), as a new inflammatory cytokine appears to be an important inflammatory mediator of cardiovascular disease and may play a pivotal role in the pathophysiology of AS. Currently, studies of sPLA2-IIA are focused on the effect of macrophages and the lipid metabolism. There is no research revealed the function of sPLA2-IIA on endothelial cells. As the endothelial dysfunction is the initial genesis for atherosclerosis, to understand whether the affect of sPLA2-IIA on proinflammantory effects exert in vascular endothelial cells may help to comprehend the mechanism induced by the sPLA2-IIA involved in AS and may also contribute to the target management to prevent the development of AS.Objectives:1. To investigate the effect of different concentrations of sPLA2-IIA on Human Umbilical Vein Endothelial Cells (HUVEC), we measure the expression of nitric oxide(NO), endothelin-1(ET-1), nitric oxide synthase (eNOS), intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesionmolecule-1(VCAM-1) after the stimulation of sPLA2-IIA at different concentration in vitro.2. In order to understand whether the hydrolysis effect of sPLA2-IIA is involved in the induction of endothelial cells, we explore the mechanism of sPLA2-IIA which may lead to the dysfunction of endothelial cells through the observation of NO, ET-1, eNOS, ICAM-1and VCAM-1in HUVEC after stimulation of the cultured cells with4-Bromophenacyl bromide(4-BPB) which inhibits sPLA2-IIA enzymatic activity.Methods:1. Endothelial cells were isolated from human umbilicalcord vein and incubate in ECM complete culture medium in vitro.2. Endothelial cells were serially passaged (1:2ratio) into Costar tissue-culture flasks (75-cm2vessels) coated with1%gelatin. The3rd to5th generations were used to carry out the study.3. Confluent HUVEC cells were cultured in serum-free medium for6h before treatments for24h with three different concentrations groups(0.01、0.1、1ug/ml) of sPLA2-IIA and10ng/ml TNF-α,10nmol/L4-BPB alone or combined. Endothelial cell nitric oxide concentration in the supernatant was detected by kit. The mRNA expression levels of ET-1, eNOS, ICAM-1, VCAM-1were determined by Real Time-PCR.4. Stimulated HUVEC were lysed and protein concentrations were measured. Equal amounts of cell lysate protein were analyzed by12%SDS/PAGE gels. The ICAM-1protein expression was detected by Western blot method.5. All statistical analyses were performed using the SPSS statistics program, version17.0. Data are presented as means±SD. A p-value of p<0:05was considered as significant. One-way ANOVA were used to evaluate differences between2groups.Results:1. sPLA2-IIA of0.1ug/ml and1ug/ml significantly up-regulated the mRNA expression of ICAM-1and VCAM-1in a concentration dependent way (p<0.01). The effect of sPLA2-IIA on protein level of ICAM-1and VCAM-1were highly consistent with mRNA level.2.4-BPB abolished the over expression of mRNA of ICAM-1and VCAM-1induced by the sPLA2-IIA in1ug/ml (ICAM-1:0.65±0.12vs0.35±0.08, p<0.01; VCAM-1:0.85±0.14vs0.60±0.21, p<0.05) and the TNF-a in10ng/ml (ICAM-1:0.72±0.03vs0.47±0.03, p<0.05; VCAM-1;1.24±0.04vs0.82±0.09, p<0.05).4-BPB repressed the protein level of ICAM-1and VCAM-1up-regulated by sPLA2-IIA or TNF-a.3. sPLA2-IIA increased the mRNA expression of ET-1in a concentration dependent way (p<0.05);1ug/ml of sPLA2-IIA dramatically decreased the mRNA expression of eNOS (p<0.01), there were no significant difference by the stimulation of sPLA2-IIA in lower concentration (p>0.05); sPLA2-IIA increased the level of NO in0. lug/ml and1ug/ml respectively (P＜0.05, p<0.01).4.4-BPB abolished the up-regulated mRNA of ET-1、eNOS induced by sPLA2-IIA in1ug/ml (ET-1:1.52±0.06vs1.13±0.06, p<0.01; eNOS:-0.38±0.01vs-0.15±0.02, p<0.05) and TNF-a in10ng/ml (ET-1:1.89±0.00vs1.38±0.05, p<0.01; eNOS:-1.52±0.12vs-0.96±0.04, p<0.01).4-BPB was failed to reduce the over-expression of NO induced by the stimulation of sPLA2-IIA or TNF-a. Conclusions:1. In vitro, the normal function of endothelial cells may impair by the stimulation of sPLA2-IIA as it induced the over expression of cytokines including ICAM-1, VCAM-1and ET-1in a concentration dependent manner or the repression of eNOS and NO.2. Hydrolysis of sPLA2-IIA exactly take a part to regulate the expression of ICAM-1、 VCAM-1、ET-1and eNOS in ECs, but there were also other non-hydrolysis pathway to regulate the function of endothelial cells.3. The activation of sPLA2-IIA may involved in the over expression of ICAM-1, VCAM-1,ET-1and eNOS induced by TNF-a.