Effect And Machanism Of Growth Differentiation Factor15on Human Aortic Endothelial Cells

In the atherosclerotic process,endothelial dysfunction is recognized as its initial step. Growth differentiation factor-15(GDF-15) is strongly upregulated in disease states such as tissue hypoxia, inflammation and oxidative stress. GDF-15can protect the vascular endothelial cells and inhibit its apoptosis, is a key factor of prevention and treatment of cardiovascular disease especially anti-atherosclerosis.Objective:1.To study the effect of GDF-15on proliferation of human aortic endothelial cells(HAECs) and its relation to the ERK1/2signaling pathway.2.To study the effect of GDF-15on apoptosis of HAECs which induced by H2O2and its relation to the PI3K/Akt signaling pathway.Methods:1.Cell proliferation experiment:HAECs were cultured in vitro and randomized into five groups of group1(blank control),group2(treated with GDF-155ng/ml),group3(treated with GDF-1550ng/ml),group4(preincubated with PD98059followed by GDF-155ng/ml) and group5(preincubated with PD98059followed by GDF-1550ng/ml). Cell proliferation was determined by CCK8cell proliferation assay kit,and p-ERK expression was detected by Western blot after24hours of culture.2. Cell apoptosis experiment:HAECs were cultured in vitro and randomized into six groups of group1(blank control),group2(induced by H2O2500μmol/L),group3(treated with GDF-155ng/ml plus H2O2500μmol/L),group4(treated with GDF-1550ng/ml plus H2O2500μmol/L),group5(preincubated with LY294002followed by GDF-155ng/ml plus H2O2500μmol/L) and group6(preincubated with LY294002 followed by GDF-1550ng/ml plus H2O2500μmol/L). Cell apoptosis was determined by Hoechst3258staining and flow cytometry,and p-Akt expression was detected by Western blot after12hours of culture.Results:1. Proliferation experiment:(1)Cell proliferation:It was determined by the CCK8cell proliferation assay kit,compared with control group, the proliferation in low concentration GDF-15group was significantly increased(P<0.05),which were significantly reversed by PD98059in low concentration GDF-15+PD98059group.However, the proliferation in high concentration GDF-15group had no change compared with control group and high concentration GDF-15+PD98059group.(2) The expression of p-ERK protein:It was detected by Western blot,compared with control group and low concentration GDF-15+PD98059group, the expression of p-ERK protein in low concentration GDF-15group was significantly increased(P<0.05). However, the expression of p-ERK protein in high concentration GDF-15group had no change compared with control group and high concentration GDF-15+PD98059group.2. Apoptosis experiment:(1)Cell apoptosis:It was observed by Hoechst3258fluorescence staining and determined by flow cytometry,compared with control group, the cell apoptosis rate in H2O2group was significantly increased(P<0.05). Compared with H2O2group,the cell apoptosis rate was decreased in a dose-dependent manner in low concentration GDF15+H2O2group and high concentration GDF15+H2O2group(P<0.05),which were significantly reversed by LY294002in low concentration GDF-15+H2O2+LY294002group and high concentration GDF-15+H2O2+LY294002group(P<0.05).(2)The expression of p-Akt protein:It was detected by Western blot,compared with control group and H2O2group, the expression of p-Akt protein was increased in a dose-dependent manner in low concentration GDF15+H2O2group and high concentration GDF15+H2O2group(P<0.05),which were significantly reversed by LY294002in low concentration GDF-15+H2O2+LY294002group and high concentration GDF-15+H2O2+LY294002group(P<0.05).Conclusion:1.Low concentration GDF-15can promote the proliferation of human aortic endothelial cells,which may be related with the upregulation of p-ERK expression.However, high concentration GDF-15has no this effect.2.GDF-15can supess H2O2-induced apoptosis of human aortic endothelial cells in a dose-dependent manner,which may be related with the upregulation of p-Akt expression.