Effect And Molecular Mechanism Research Of Visfatin On HUVECs In Oxidative Stress Condition

Objective:1. To explore a stable way of the culture and characterization of human umbilical vein endothelial cells (HUVECs) and compare the surface antigen expressions among primary HUVECs, HUVEC-CS and EA.hy926cell lines;2.①Hydrogen peroxide was used to induce the oxidative stress injury in HUVECs cultured in vitro;②To study the expression of visfatin in HUVECs under normal or oxidative stress injury situation.3. To study the effect of visfatin on the apoptosis of HUVECs and expression of various cytokines under normal and oxidative stress injury condition in vitro;4. To discuss the influencing factor of plasma visfatin and its correlation to coronary heart disease (CAD) and abnormal glucose metabolism. EZscan system and laboratory method were used to screen sugar metabolic abnormalities of patients with CAD to study the clinical application value and prospect of eZscan system.Methods:1. HUVECs were obtained from human umbilical vein digested with0.1%collagenase type Ⅱ and cultivated in M199complete medium containing20μg/mL ECGF. HUVEC-CS and EA.hy926were cultured in DMEM complete medium. Subculture was attained with trypsin/EDTA digestion when cells were90%confluent. Cell morphology was observed under inverted phsase contrast microscope, immunofluorescent staining and flow cytometry (FCM) were used to test the expression of VWF, CD31and CD34of these three cells;2.①EA.hy926cell lines subcultured in vitro were divided into two groups:model group and control group. Six concentration gradients (H2O2final concentration was100,200,400,600,800or100μmol/L) were tested to find out the optimal effect concentration in dose effect of intervention in model group. HUVECs were treated with the optimal concentration of H2O2for6hours,12hours or24hours to find out the optimal effect time in time effect of intervention. HUVECs were collected and tested:cells morphology change were observed under inverted phase contrast microscope, cell counting kit (CCK-8) was applied to test cell viability rate; DCFH-DA fluorescence probe and FCM was applied to test intracellular reactive oxygen levels, FCM TUNEL method was used to test cell apoptosis rate and proliferation rate;②EA.hy926cell line passages sbucultured in vitro was divided into two groups randomly:control group and H2O2treatment group (H2O2final concentration was50,100,200,400or1000μmol/L) and testing was conducted after1hour,2,4or24hours of cell culture. Visfatin protein and gene expression were tested via western blot and real time PCR.3. EA.hy926cell line subcultured in vitro were divided randomly and given corresponding treatment. Cells apoptosis rate and cell cycle were tested by flow cytometry, IL-6, MCP-1, VCAM, VEGF and iNOS concentration were tested quantitatively by ELISA.4. One hundred and sixty patients with CAD were recruited and divided into two groups:CAD combined sugar metabolic abnormalities group and CAD group. Fifty sugar metabolism disorder patients without any organic heart disease were recruited as sugar metabolism disorder group,50healthy persons were recruited as control group at the same time. Fasting laboratory index and plasma visfatin levels were tested and conducted with baPWN and ABI examination in all subjects. Then eZscan system (a system for early detection of diabetes and its complication risk) was applied to screen sugar metabolic abnormalities of established CAD patients via coronary angiography. The results were compared with that of laboratory examination to test the sensitivity and specificity of eZscan system.Results:1. The primary HUVECs were polygon with the characteristic "cobblestone" morphology, while HUVEC-CS and EA.hy926cell lines exhibited rounded or flat shape, also with "cobblestone" morphology. Primary HUVECs and EA.hy926were positive for von Willebrand factor (vWF) immunostaining, whereas HUVEC-CS as negative. The expression of CD31in primary HUVECs (77.93±0.25) or EA.hy926(78.23±0.40) was much higher than that of HUVEC-CS(4.70±0.46), while the expression of CD34in primary HUVECs (43.1±0.20) was much higher than that of HUVEC-CS or EA.hy926(1.20±0.40or0.97±0.40, respectively). The differences were statistically significant (P<0.001).2.①Our results indicated that cell morphology of HUVECs changed a lot in model group:cell outline was ambiguous, cell shape was irregular, cell volume and membrane shrinked, cell arrangement was confused, some cells floated and the characteristic "cobblestone" structure was destroyed to varying degree. Spindle cells reduced, sphere or oval cells appeared and these cells became more and more with the extension of action time and dose increase, which meant that H2O2had harmful effect on HUVECs. CCK-8results indicated the survival rate of HUVECs decreased gradually with the extension of dose increase and action time of H2O2. presenting a certain relationship of dose-effect and time-effect. Results of DCFH-DA fluorescence probe and flow cytometry indicated higher level of intracellular active oxygen. Flow cytometry technique TUNEL method results verified that H2O2did induce the apoptosis of HUVECs and the apoptosis rate increased distinctly with the increase of H2O2concentration;②HUVECs could express visfatin when culturing in vitro. At different H2O2concentration (50,100,200,400or1000μmol/L) for different period of time (1hour,2,4or24hours), significant increase of visfatin levels were detected when treated with low concentration of H2O2(50or100μmol/L), while significant decrease was detected if the concentration of H2O2was higher than200μmol/L with the extension of time (P<0.05); Visfatin levels increased when treating in short time of H2O2(1hour or2hours), while decreased when treating no less than4hours of H2O2with the increase of its concentration (P<0.05). Western blot gray analysis and real time PCR results showed no difference of variation trend of visfatin expression.3. After treating HUVECs with different concentration of visfatin (100,400or800ng/ml) for24/48hours in normal culture, the effect of visfatin on apoptosis rate and proliferation index of cells showed no difference between treating group and control group, while the expression of IL-6, MCP-1, VCAM, VEGF and iNOS showed significantly difference between each group (visfatin100,400,800ng/ml and normal control).After treating HUVECs in oxidative stress with100,400or800ng/ml visfatin for24/48hours, cells apoptosis rate decreased significantly compared with H2O2group, while its apoptosis rate gradually was upregulated with the increasing concentration of visfatin. Any two of the groups showed significantly difference by pairwise comparison (P<0.05). After treating cells with visfatin for24hours, proliferation index of HUVECs in oxidative stress went up than H2O2group, different groups showed significant differences. Between different groups showed no statistical difference (P>0.05) after comparing one to the other, except for statistical difference between visfatin400ng/ml plus H2O2800μmol/L for24hours group and visfatin800ng/ml plus H2O2800μmol/L for24hours group (P>0.05). When the concentration of visfatin rose from100ng/ml to400or800ng/ml, cells proliferation index decreased gradually (P<0.05). After treating cells with visfatin100,400or800ng/ml for48hours, there was statistical difference between any two treatment groups. IL-6, MCP-1, VCAM, VEGF and iNOS expression all showed statistical differences between4treatment groups after treating cells with visfatin for24hours. For VCAM, each group showed statistical differences except between visfatin100ng/ml group and oxidative stress group. IL-6, MCP-1, VCAM, VEGF and iNOS expression all showed statistical differences between any2of4treatment groups after comparing one to the other when treating cells with visfatin for48hours (P<0.05).When treating cells with SB203580(p38MAPK inhibitor) and LY29004(PI3K inhibitor), apoptosis rate and proliferation index of different groups showed statistical differences (P<0.001):H2O2800μmol/L for24h, visfatin400ng/ml+H2O2800μmol/L for24h, SB20358020μmol/L90min+Visfatin400ng/ml for24h+H2O2800μmol/L for24h, LY29400220μmol/L90min+Visfatin400ng/ml for24h+H2O2800μmol/L for24h, any2groups also showed statistical differences (P<0.01) by pairwise comparison. IL-6, MCP-1, VCAM, VEGF and iNOS expression all showed statistical differences between4treatment groups. For MCP-1, each group showed statistical differences (P<0.05) except between SB20358090min treatment group and LY2900490min treatment group. For IL-6, VCAM, VEGF and iNOS, each group showed statistical difference (P<0.05).4. Plasma visfatin level was CAD group, sugar metabolic abnormalities group and control group in turns from high to low. Subgroup analysis indicated that plasma visfatin levels increased progressively among different clinical types of CAD (chronic stable angina pectoris, unstable angina and acute myocardial infarction). The visfatin level of CAD combined sugar metabolic abnormalities group was obviously higher than that of simple CAD patients (P<0.05). Linear dependence analysis results indicated that visfatin was positively correlated to BMI, SBP, TC, LDL-C, FPG, Fins or HOMA-IR (P<0.05). Multiple linear regression analysis results indicated that independence variable BMI and HOMA-IR had significantly effect on plasma visfatin levels (P<0.05). The sensitivity and specificity of eZscan system screening sugar metabolic abnormalities were88.89%and64.95%, respectively. The accuracy of eZscan system screening sugar metabolic abnormalities was expressed by receiver operating characteristic (ROC) curve, with statistical significance (area under the curve was0.769, P=0.000). With the aggravation of coronary stenosis, baPWV showed an increasing trend and ABI was negatively correlated to the degree of coronary stenosis (P<0.05)Conclusion:1. Human umbilical vein digested with collagenase type Ⅱ and cultured in M199complete medium can quickly obtain highly purified endothelial cells. EA.Hy926cell line can be characterized by more amounts of cells with simple culture method and high survival rate. Both of them are good sources for tissue engineering and related basic research;2.①The oxidative stress model in human umbilical vein endothelial cells could be established by coculturing cells with culture medium containing800μmol/L hydrogen peroxide;②Our findings suggest that H2O2had dose-effect and time-effect in promoting the expression of visfatin. H2O2could promote the expression of visfatin in short time or low concentration, while suppress the visfatin expression in long time or high concentration;3.①Our results indicated that apoptosis and proliferation of HUVECs in normal culture in vitro were not affected when treating cells with100,400or800ng/ml visfatin. The expression of IL-6, MCP-1, VCAM, VEGF and iNOS increased with the increasing concentration of visfatin and extension of action time;②Apoptosis rate of HUVECs in oxidative stress injury decreased significantly while its proliferation index rose up when treating cells with100,400or800ng/ml visfatin for24/48hours, compared to H2O2control group. But its effect didn’t increase with the increasing concentration of visfatin. The expression of IL-6, MCP-1, VCAM, VEGF and iNOS increased remarkably with the increasing concentration of visfatin and extension of its action tim;③Pretreating HUVECs with SB203580(p38MAPK inhibitor) and LY29004(PI3K inhibitor) could partially block the effect of visfatin on cell apoptosis, proliferation and expression of inflammatory cytokines, which indicated that the effect of visfatin on apoptosis, proliferation and expression of inflammatory cytokines of HUVECs may be related to p38MAPK signal pathway and PI3K signal pathway, especially the p38MAPK signal pathway;4. Our findings suggest that visfatin may have some effects on the process of development of sugar metabolic abnormalities and atherosclerosis. BMI, HOMA-IR had significantly effect on the level of plasma visfatin. EZscan system was simple, time-saving and noninvasively safe, not subject to fasting restrictions in early screening of sugar metabolic abnormalities, with higher sensitivity and moderate specificity. BaPWV and ABI test was noninvasive, simple, cost-effective and repeatable, playing a role in early screening of high-risk CAD patients and assessing coronary lesions.