Effect And Mechanism Of Visfatin On Endothelin-1 Expression In Human Umbilical Vein Endothelial Cells

Objective:Recent studies have shown that adipose tissue is not only an energy storage organ,but also a real endocrine organ by releasing a variety of bioactive mediators named as adipocytokines.These adipocytokines may act on immune cells leading to local and generalized inflammation and may also affect directly or indirectly endothelial and vascular function.Visfatin,also known as pre-B-cell colony-enhancing factor?PBEF?or nicotinamide phosphoribosyltransferase?Nampt?,is a novel adipokine that appears to be preferentially produced by visceral adipose tissue and has insulin-mimetic actions.Plasma visfatin levels are increased in obesity and obesity-related vascular diseases.Visfatin also functions as a new proinflammatory adipocytokine which dose-dependently up-regulates the production of the pro-inflammatory cytokines interleukin?IL?-1?,IL-6and tumor necrosis factor?TNF?-?in human monocytes.Plasma visfatin concentrations are elevated in various inflammatory disorders.Moreover,it has been suggested that visfatin is a vascular inflammatory mediator that increases matrix metalloproteinase?MMP?-9 activity in THP-1 monocytes and TNF-?and IL-8 levels in peripheral blood mononuclear cells,affects the activation of gelatinases MMP-2 and-9 in endothelial cells.Visfatin may be involed in the development of atherosclerosis as the levels of plasma visfatin are increased in coronary heart disease,particularly in acute coronary syndrom.Visfatin may play a potential role in plaque destabilization based on its localization in macrophages within unstable atherosclerotic lesions.A recent clinical study reports that plasma visfatin levels are related to impaired endothelial function since the log10-transformed plasma visfatin concentration is negatively associated with vascular endothelial function evaluated by flow-mediated dilation in patients with type 2 diabetes mellitus.However,the underlying mechanisms by which visfatin may contribute to endothelial dysfunction remain undetermined.In spite of the experiment In vitro which has suggested that visfatin causes endothelial dysfunction by increasing inflammatory and adhesion molecule IL-6,IL-8,ICAM-1,VCAM-1 and E-selectin gene expression in endothelial cells.However,the role of visfatin in endothelial dysfunction has been largely unexplored.Endothelin?ET?-1 is one of the most potent vasoconstrictor peptide originally isolated from endothelial cells.ET-1 is involved in endothelial dysfunction in the pathogenesis of cardiovascular diseases,such as hypertension,hypercholesterolemia,atherosclerosis,diabetes,and coronary artery disease.Endothelial dysfunction is the initial step in the pathogenesis of atherosclerosis and vascular diseases in humans,and is regarded as an independent predictor of cardiovascular events.The preclinical and clinical studies propose that ET-1 can cause endothelial dysfunction.ET-1 may induce endothelial dysfunction by decreasing nitric oxide bioavailability either by decreasing its production or increasing its degradation.It has been reported that visfatin and endothelin-1 is significantly greater in adipose tissue from obesity women.Serum visfatin is significantly positive associated with endothelin-1 in African women by age-and obesity-adjusted univariate and multivariate analyses.However,there are no data directly linking visfatin to ET-1 expression in endothelial cells.Whether visfatin can induce ET-1 gene expression subsequently lead to endothelial dysfunction remains unknown.The aim of the present study is to examine the hypothesis that visfatin increases the expression of ET-1 in human umbilical vein endothelial cells?HUVECs?and determine the intracellular signal transduction pathways and transcription factor involved in this process.Methods:1.HUVECs?CRL-1730?was maintained in DMEM supplemented with 10%fetal calf serum with 5%CO₂ at 37?.HUVECs were serum starved overnight before treatment.2.ROS levels of the endothelial cells were determined by analyzing the fluorescent product DCF and fluorescence intensity was measured by laser-confocal microscopy.3.The levels of ET-1 protein secretion into the culture supernatant were assessed using a human ET-1 enzyme immunometric assay kit based on a double-antibody sandwich technique.4.The mRNA levels of ET-1 were measured by reverse transcription PCR and real-time quantitative PCR.5.The activity of ERK1/2 was analyzed by Western blotting using antibody directed against the phosphorylated form of ERK1/2.6.The activity of transcription factor AP-1 was assessed by EMSA with a specific probe for the AP-1 binding site.Results:1.HUVECs were subjected to various concentrations of visfatin for several time periods.Visfatin increased ET-1 mRNA levels which were observed as early as 4h but peaked at 6h and declined thereafter.At 6h,visfatin induced a dose-dependent increase in mRNA levels of ET-1.Consistently,a dose-and time-dependent increase of ET-1 protein expression was detected.Visfatin enhances ET-1 gene expression in a dose-and time-dependent manner in HUVECs.2.Visfatin significantly increased ROS generation in HUVECs,which was abolished in the presence of NAC.The pretreatment of HUVECs with NAC significantly suppressed visfatin-induced ET-1 mRNA levels and protein secretion.ROS mediate visfatin-induced ET-1gene expression in HUVECs.3.Visfatin induced phosphorylation of both p42 and p44 with a maximal response at 15 min and decreased thereafter.Both antioxidant NAC and the MEK inhibitor U0126 significantly inhibited visfatin-induced phosphorylation of ERK.Similarly,both NAC and U0126inhibited augmentation of ET-1 mRNA expression and protein secretion stimulated with visfatin.4.Visfatin stimulated the binding of nuclear extracts to the AP-1 consensus sequence in HUVECs,which was attenuated by specific competition.Pretreated cells with antioxidants NAC or MEK inhibitor U0126 attenuated visfatin-induced AP-1binding activity.Conclusions:1.Visfatin enhances ET-1 gene expression in HUVECs in a dose-dependent and time-dependent mannar.Visfatin is likely to promote endothelial dysfunction via increase in ET-1 gene expression in endothelial cells.2.Visfatin increases ROS generation in HUVECs.NAC suppresses visfatin-induced ET-1 mRNA levels and protein secretion and visfatin-induced ROS accumulation.ROS may play a role in visfatin-induced ET-1 gene expression in vascular endothelial cells.3.Visfatin activates ERK1/2 pathway in endothelial cells.Both NAC and U0126 inhibit visfatin-induced phosphorylation of ERK and the expression of ET-1 mRNA and protein secretion.The visfatin-activated ERK is a redox-sensitive signaling pathway,and ERK activation is necessary for visfatin-induced ET-1 gene expression.4.Visfatin promotes the activity of transcription factor AP-1 in HUVECs.Both NAC and U0126 attenuates visfatin-induced AP-1 binding activity.The AP-1 binding element is essential for the induction of the ET-1 gene by visfatin.ROS mediates AP-1 activation via ROS-dependent mechanism,and ERK pathway is involved in the visfatin-induced AP-1activation in HUVECs.5.Visfatin upregulates ET-1 gene expression at least in part through ROS-ERK-mediated AP-1-dependent pathway in HUVECs.