| 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 intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), endothial nitric oxide synthase (eNOS), nitric oxide(NO) and endothelin-1(ET-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.3. In order to understand whether the MAPKinase pathway is engaged 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 with PD98059(ERK inhibitor), SP600125(JNK inhibitor), B203580(p38inhibitor).4. In order to understand whether the NF-kB is engaged in the induction of endothelial cells, we observed NO, ET-1, eNOS, ICAM-1and VCAM-1in HUVEC after stimulation of the cultured cells with Bayl1-7085(NF-kB inhibitor).Methods:1. Endothelial cells were isolated from human umbilicalcord vein and incubate in ECM complete culture medium in vitro.2. HUVEC cells were cultured in three different concentrations groups (0.01,0.1,1μg/ml) of SPLA2-IIA. 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. The protein expression level of eNOS, ICAM-1, VCAM-1were measured by Western Blot. The protein expression level of ET-1was measured by ELISA.3. HUVEC cells were cultured with1μg/ml sPLA2-IIA and lOng/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. The protein expression level of eNOS, ICAM-1, VCAM-1were measured by Western Blot. The protein expression level of ET-1was measured by ELISA.4. HUVEC cells were cultured with1u g/ml sPLA2-IIA and1μmol/L PD98059or SP600125or SB203580alone 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. The protein expression level of eNOS, ICAM-1, VCAM-1were measured by Western Blot. The protein expression level of ET-1was measured by ELISA.5. HUVEC cells were cultured with1μg/ml sPLA2-IIA and1μmol/L Bayl1-7085alone 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. The protein expression level of eNOS, ICAM-1, VCAM-1were measured by Western Blot. The protein expression level of ET-1was measured by ELISA.6. All statistical analyses were performed using the SPSS statistics program, version 13.O. Data are presented as means±SD. A p-value of p<0:05was considered as significant. One-way ANOVA and independent sample t test were used to evaluate differences between2groups.Results:1. sPLA2-IIA of0.1μg/ml and1μg/ml significantly up-regulated the mRNA and protein expression of ICAM-1and VCAM-1in a concentration dependent way (p<0.05).2. sPLA2-IIA increased the mRNA and protein expression of ET-1in a concentration dependent way (p<0.05).1μg/ml of sPLA2-IIA dramatically decreased the mRNA expression of eNOS (p<0.01), but there were no significant difference by the stimulation of sPLA2-IIA in lower concentration (p>0.05). sPLA2-IIA decreased the level of NO in a concentration dependent way (p<0.05).3.4-BPB abolished the over expression of mRNA of ICAM-1and VCAM-1induced by the sPLA2-IIA in1μg/ml (p<0.01, p<0.05).4-BPB also repressed the protein level of ICAM-1and VCAM-1up-regulated by sPLA2-IIA.4-BPB abolished the up-regulated mRNA and protein level of ET-1induced by sPLA2-IIA in1μg/ml (p<0.01) and mRNA of eNOS induced by sPLA2-IIA in1μg/ml (p<0.05).4-BPB reduced the down-regulation of NO induced by the stimulation of sPLA2-IIA (p<0.01).4.4-BPB abolished the over expression of mRNA of ICAM-1and VCAM-1induced by the sPLA2-IIA in1μg/ml (p<0.01, p<0.05).4-BPB also repressed the protein level of ICAM-1and VCAM-1up-regulated by sPLA2-IIA.4-BPB abolished the up-regulated mRNA and protein level of ET-1induced by sPLA2-IIA in1μg/ml (p<0.01) and mRNA of eNOS induced by sPLA2-IIA in1μg/ml (p<0.05).4-BPB reduced the down-regulation of NO induced by the stimulation of sPLA2-IIA (p<0.01).5. PD98059abolished the over expression of ICAM-1, VCAM-1and ET-1induced by the sPLA2-IIA in1μg/ml (p<0.01). PD98059also reduced the down-regulation of NO induced by the stimulation of sPLA2-IIA (p<0.01).6. SP600125abolished the over expression of ET-1induced by the sPLA2-IIA in1μg/ml (p<0.01). SP600125also reduced the down-regulation of NO induced by the stimulation of sPLA2-IIA (p<0.01). However, SP600125had little effect on the over expression of ICAM-1and VCAM-1induced by the sPLA2-IIA in1μg/ml (p>0.05).7. SB203580had little effect on the over expression of ICAM-1, VCAM-1and ET-1induced by the sPLA2-IIA in1μg/ml (p>0.05). SB203580did not reduced the down-regulation of NO induced by the stimulation of sPLA2-IIA (p>0.05).8. Bayl1-7085abolished the over expression of ICAM-1, VCAM-1and ET-1induced by the sPLA2-IIA in1μg/ml (ICAM-1:p<0.01; VCAM-1:p<0.05; ET-1:p<0.01). However, Bay11-7085did not reduced the down-regulation of NO induced by the stimulation of sPLA2-IIA (p>0.05).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 took a part in regulating the expression of ICAM-1、 VCAM-1、ET-1and eNOS in endothelial cells.3. sPLA2-IIA up-regulated the adhesion molecules through MAPKinase ERK and NF-kB pathway.4. sPLA2-IIA up-regulated the expression of ET-1and down-regulated the NO production through MAPKinase ERK and JNK pathway. NF-kB was also involoved in the sPLA2-IIA induced ET-1expression. |
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