Visfatin Causes Apoptosis Of Endothelial Progenitor Cells By Induction Of Proinflammatory Mediators Through The NF-κB

Endothelial progenitor cells(EPCs), firstly identified by Asahara in 1997, are progenitor cells which originate from bone marrow and migrate to the peripheral circulation. EPCs have the capacity of proliferation and repair compentence. In the case of vessel impairment or tissue ischemia, EPCs can mobilize to peripheral blood circulation, specifically home in to damaged or ischemic tissue and differentiate further into mature endothelial cells to paticipate the restoration and regeneration of endothelial cells. Also EPCs could endocrine some factor to activate the regeneration of endothelial cells, which could promote the restoration of vascular. Nowadays with the change of people lifestyle, diabetes has become an increasing disease. And cardiovascular complications are the leading cause of death among diabetic patients. Many studies have demonstrated that a decreased circulating EPCs level and impairment of EPCs has been shown to independently predict atherosclerotic progression and poor prognosis in patients with coronary heart disease. EPCs are influenced by many factors, such as physical activity and smoking. Many diseases can cause the decrease in the quantity of EPCs. Hyperglycemia, obesity, hyperinsulinemia and hyperlipidemia can all lead to the impairment of EPCs.Visfatin, since identified as an adipokine which was specifically highly expression in adipose tissue, was correlated with type 2 diabetes mellitus and metabolic syndrome, especially atherosclerosis. Visfatin can cause oxidative stress and inflammation in endothelial cells, which subsequently damages the vascular endothelial function, leading to atherosclerosis. Because of its role in inflammation, visfatin has been implicated in the pathogenesis of various metabolic disorders.Emerging evidence has established a potential link between adipokines and vascular dysfunction.Studies have shown that EPCs dysfunction in diabetic patients and endothelial function is correlated with visfatin. And our previous studies have shown that EPCs dysfunction in obese rats is correlated with serum visfatin. However, the specific mechanism underlying this link remains unclear. Here we explore whether visfatin plays a role in promoting EPCs apoptosis and its potential mechanism.Objective: This study explored how visfatin affects apoptosis in endothelial progenitor cells(EPCs) and the mechanisms underlying such effects.Methods: Umbilical vein blood mononuclear cells(MNCs) of healthy pregnant woman donors were isolated by Ficoll density gradient centrifugation. After washing with PBS, MNCs were resuspended in EGM-2MV medium, supplemented with 20% fetal bovine serum, 1% Pen/Strep, 0.04% hydrocortisone, 0.1% heparin and 0.1% ascorbic acid in presence of IGF-1(50ng/ml), EGF(10ng/ml), FGF(50ng/ml), VEGF(50ng/ml) and fibronectin(10μg/ml). Tissue culture plates precoated with fiber protien and cells were seeded at a density of 2x106/ml and cultured in a 5% CO2, 95% humidity incubator at 37°C. Cells were observed daily under inverted microscopy. After cultured for 7days, immunophenotyping of cultured cells was detected by flow cytometry. And EPCs were characterized by cellular uptake of Di I-labeled acetylated LDL(Di I-Ac-LDL) and binding of fluorescein isothiocy- anate-conjugated lectin from Ulex europaeus agglutinin(FITC-Lectin-UEA-1), which were detected by laser scanning confocal microscope. After serum starvation, cultured EPCs were incubated with visfatin at different concentrations(0ng/ml, 50ng/ml, 100ng/ml, 150ng/ml) for 48 hours. In some experiments, EPCs were pretreated with visfatin inhibitor(FK866, 10μM), nuclear factor kappa B(NF-κB) inhibitor(BAY11-7085, 5μM) 1 hour before incubation with visfatin. Then, EPCs were treated with varying concentrations of visfatin for 48 hours. Apoptosis was evaluated by flow cytometry. The m RNA expression of apoptosis related proteins(caspase 3, Bax and Bcl-2) and NF-κB were measured by quantitative real-time PCR. And protein levels of apoptosis related proteins and proinflammatory mediators(intercellular adhesion molecule(ICAM)-1 and inter-leukin(IL)-6) and NF-κB were evaluated by Western blot analysis.Results:1 Newly isolated umbilical vein blood mononuclear cells were round and suspended in medium. After 48 hours of plating, part of the cells attached. The adherent cells gradually elongated with spindle shape. After 10 days of plating adherent cells grew as colonies. EPCs were detected by flow cytometry. CD309+CD34+ double-positive cells were determined as EPCs. By uptake of Dil-Ac-LDL and binding of FITC-Lectin-UEA-1, EPCs which were double-stained cells as orange were observed and counted under laser scanning confocal microscopy.2 After treated with various concentrations of visfatin(0ng/ml, 50ng/ml, 100ng/ml, 150ng/ml) for 48 hours, apoptosis of EPCs was increased in a dose-dependent manner; the expression of Bax and caspase 3 was up-regulated at both m RNA and protein levels; but the expression of Bcl-2 was decreased(P>0.05). Compared with control group, significant increases in apoptosis of EPCs along with increased m RNA and protein expression of Bax and caspase 3 were detected at a visfatin concentration of 150ng/ml(P<0.05), which was suppressed by pretreatment with FK866(P<0.05).3 Various concentrations of visfatin resulted in a dose-dependent increase of IL-6 and ICAM1 protein expression of EPCs, which was significant at a concentration of 150ng/ml(P<0.05), and this could be suppressed by FK866(P<0.05).4 Visfatin up-regulated the expression of NF-κB at both m RNA and proteins levels. Compared with control group, significant increases were detected at a visfatin concentration of 150ng/ml(P<0.05). EPCs were preincubated with BAY11-7085 for 1 hour, and then treated with visfatin(150ng/ml) for 48 hours. Compared with 150ng/ml visfatin treated group, the visfatin-induced apoptosis, the increased expression of caspase 3, Bax, ICAM1 and IL-6, and the decreased expression of Bcl-2 were all abolished in BAY11 treated group(P<0.05).Conclusions:1 The isolated umbilical vein blood mononuclear cells, which were cultured in the medium of EMG-2MV, could differentiate into EPCs. Cells were determined as CD34+CD309+ double positive.2 Visfatin upregulated the apoptosis as well as apoptosis related proteins of EPCs in a dose-dependent manner.3 Our findings suggest that visfatin causes EPCs apoptosis by increasing expression of proinflammatory mediators at least partially through up-regulation of NF-κB expression.