RTEF-1Prevents Senescence And Chlamydia Pneumonia Disrupts Lipid Metabolism In Human Umbilical Vein Endothelial Cells

Part ⅠEffects of RTEF-1on the H2O2-induced senescence in human umbilical vein endothelial cellsObjective To investigate the effects of Related transcriptional enhancer factor-1(RTEF-1) on the hydrogen peroxide (H2O2)-induced senescence in human umbilical vein endothelial cells.Methods The coding sequence of RTEF-1(NM003213) obtained from a pXJ40/RTEF-1construct. The Liposomal medium was used to transduce HUVECs, and stable transfectants were selected with G418(500μg/mL). Moreover, Small interfering RNA (siRNA) encoding human RTEF-1at a final concentration of50nM was transfected using Lipofectamine2000. The over-expression and RNA interfering efficiency were confirmed by real-time PCR (RT-PCR) and western blotting, respectively.HUVECs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium containing10%fetal bovine serum (FBS),100U/mL of penicillin, and100μg/mL of streptomycin at37℃in5%CO2. Then, they were randomly divided into7groups:(1) control group;(2) H2O2group:incubated the cells with100μmol/L H2O2for48h;(3) negative control group:incubated the control HUVECs with100μmol/L H2O2for48h;(4) RTEF-1o/e group:incubated the RTEF-1o/e HUVECs with100μmol/L H2O2for48h;(5) negative control siRNA group:negative control sequence transfected for6h, then incubated the cells with100μmol/L H2O2for48h;(6) RTEF-1siRNA1group:RTEF-1siRNAl sequence transfected for6h, then incubated the cells with100μmol/L H2O2for48h;(7) RTEF-1siRNA2group:RTEF-1siRNA2sequence transfected for6h, then incubated the cells with100μmol/L H2O2for48h. The number of senescence cells was observed using senescence associated-P-galactosidase (SA-β-gal) staining. The cell cycle analysis was detected by flow cytometry. The mRNA and protein expressions of RTEF-1were evaluated by RT-PCR and Western-blot, respectively.Results In the first part, compared with negative control, the over-expression of RTEF-1by gene stable transfection significantly increased RTEF-1mRNA and protein expressions. Compared with negative control siRNA, the RTEF-1siRNA sequences transfection inhibited mRNA and protein expressions of RTEF-1(p<0.05). In the second part, compared with control group, the number of SA-β-gal-positive cells increased in H2O2-treated HUVECs (p<0.05). Moreover, the proportion of HUVECs in the GO/G1phase increased and that in the S phase decreased, which indicated the presence of cell senescence. Compared with negative controls, increases in SA-β-gal-positive cells induced by H2O2were significantly attenuated in RTEF-1o/e HUVECs. However, RTEF-1siRNA-transfected HUVECs increased the sum of SA-β-gal-positive cells. In addition, the proportion of cells in the G0/G1phase was decreased in H2O2-treated RTEF-1o/e HUVECs, while the proportion of cells of RTEF-1siRNA-transfected HUVECs in the GO/G1phase was increased.Conclusion Incubated the HUVECs with100μmol/L H2O2for48h could induce cells senescence. Over-expressing RTEF-1in H2O2-treated HUVECs decreased SA-β-gal-positive cells and G0/G1cells population. However, specific siRNA of RTEF-1totally reversed the anti-aging effect of RTEF-1. It demonstrated that RTEF-1could protect cells from H2O2induced aging. Part ⅡEffects of RTEF-1on the expressions of key genes involved in cell cycle in H2O2-induced endothelial cell senescenceObjective To investigate the roles of key genes involved in cell cycle in RTEF-1-driven endothelial cell protection from H2O2induced aging.Methods HUVECs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium containing10%fetal bovine serum (FBS),100U/mL of penicillin, and100μ g/mL of streptomycin at37℃in5%CO2. Then, they were randomly divided into5groups:(1) negative control group:incubated the control HUVECs with100μmol/L H2O2for48h;(2) RTEF-1o/e group:incubated the RTEF-1o/e HUVECs with100μmol/L H2O2for48h;(3) negative control siRNA group:negative control sequence transfected for6h, then incubated the cells with100μmol/L H2O2for48h;(4) RTEF-1siRNAl group:RTEF-1siRNA1sequence transfected for6h, then incubated the cells with100μmol/L H2O2for48h;(5) RTEF-1siRNA2group:RTEF-1siRNA2sequence transfected for6h, then incubated the cells with100μmol/L H2O2for48h. The p53and p21mRNA and protein expressions were determined by RT-PCR and Western-blot, respectively.Results The p53and p21mRNA expression decreased in H2O2-treated RTEF-1o/e HUVECs and increased in RTEF-1knockdown HUVECs compared to their corresponding controls. The levels of p53and p21proteins decreased in H2O2-treated RTEF-1o/e HUVECs and increased in RTEF-1siRNA-transfected HUVECs compared to their corresponding controls.Conclusion RTEF-1could decrease the expressions of p53and p21in H2O2-treated HUVECs. These changes of p53and p21expressions might play a role in RTEF-1decreased G0/G1cells population in cell cycle analysis. It further illustrated that RTEF-1might participate in anti-aging process. Part ⅢThe involvement of Klotho activation in RTEF-1driven anti-aging roleObjective To investigate whether Klotho takes part in RTEF-1-driven endothelial cell anti-aging role.Methods Small interfering RNA (siRNA) encoding human Klotho at a final concentration of50nM was transfected using Lipofectamine2000. The RNA interfering efficiency was confirmed by real-time PCR (RT-PCR) and western blotting, respectively.HUVECs and Human embryonic kidney cell line (HEK293cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium containing10%fetal bovine serum (FBS),100U/mL of penicillin, and100μg/mL of streptomycin at37℃in5%CO2. Then, they were randomly divided into11groups:(1) negative control group:incubated the control HUVECs with100μmol/L H2O2for48h;(2) RTEF-1o/e group:incubated the RTEF-1o/e HUVECs with100μmol/L H2O2for48h;(3) negative control siRNA group:negative control sequence transfected for6h, then incubated the cells with100μmol/L H2O2for48h;(4) RTEF-1siRNA1group:RTEF-1siRNA1sequence transfected for6h, then incubated the cells with100μmol/L H2O2for48h;(5) RTEF-1siRNA2group:RTEF-1siRNA2sequence transfected for6h, then incubated the cells with100μmol/L H2O2for48h.(6) negative control siRNA-transfected control HUVECs group:negative control sequence transfected for6h, then incubated the control HUVECs with100μmol/L H2O2for48h;(7) Klotho siRNA1-transfected control HUVECs group:Klotho siRNA1sequence transfected for6h, then incubated the control HUVECs with100μmol/L H2O2for48h;(8) Klotho siRNA2-transfected control HUVECs group:Klotho siRNA2sequence transfected for6h, then incubated the control HUVECs with100μmol/L H2O2for48h;(9) negative control siRNA-transfected RTEF-1o/e HUVECs group:negative control sequence transfected for6h, then incubated the RTEF-1o/e HUVECs with100μmol/L H2O2for48h;(10) Klotho siRNA1-transfected RTEF-1o/e HUVECs group:Klotho siRNAl sequence transfected for6h, then incubated the RTEF-1o/e HUVECs with100μmol/L H2O2for48h;(11) Klotho siRNA2-transfected RTEF-1o/e HUVECs group:Klotho siRNA2sequence transfected for6h, then incubated the RTEF-1o/e HUVECs with100μmol/L H2O2for48h. The number of senescence cells was observed using senescence associated-β-galactosidase (SA-β-gal) staining. Promoter activity was determined using the dual luciferase assay system. The cell cycle analysis was detected by flow cytometry. The Klotho, p53and p21mRNA and protein expressions were determined by RT-PCR and Western-blot, respectively.Results In the first part, compared with negative control siRNA, the Klotho siRNA sequences transfection inhibited mRNA and protein expressions of Klotho (p<0.05). In the second part, Klotho mRNA and protein levels increased in H2O2-treated RTEF-1o/e HUVECs and decreased after using two specific sequences of RTEF-1siRNA for knockdown of RTEF-1in HUVECs, compared to their corresponding controls. The Klotho promoter activity exhibited an RTEF-1dose-dependent stimulating. Moreover, Klotho siRNA significantly increased the SA-β-gal-positive cells and G0/G1cells population both in RTEF-1o/e HUVECs and control HUVECs. Klotho siRNA up-regulated p53and p21expressions in control HUVECs but that was not observed in RTEF-1o/e HUVECs.Conclusion Klotho siRNA could significantly inhibit Klotho expression in control and RTEF-1o/e HUVECs. RTEF-1up-regulated Klotho gene expression and activated its promoter. Furthermore, Klotho siRNA significantly blocked RTEF-1-driven endothelial cell protection from H2O2-induced aging and increased p53and p21expressions. These results revealed that RTEF-1was a potential anti-aging gene and could prevent H2O2-induced endothelial cell senescence by activating Klotho. Part ⅠEffects of Chlamydia pneumoniae infection on lipid metabolism in human umbilical vein endothelial cellsObjective To investigate the effects of Chlamydia pneumoniae (C.pn) on lipid metabolism in human umbilical vein endothelial cells (HUVECs).Methods C.pn strain AR-39was propagated in HEp-2cells. HUVECs were seeded onto six-well plates at a density of1×106cells per well in Dulbecco’s modified Eagle’s medium (DMEM) medium containing10%fetal bovine serum (FBS) at37℃in5%CO2, then they were randomly divided into3groups:(1) negative control group:HUVECs were incubated with LDL (50mg/L) for48h;(2) C.pn infection group:in the presence of LDL (50mg/L), HUVECs were infected with C.pn (1×106IFU) for48h;(3) positive control group:HUVECs were incubated with oxidized LDL (50mg/L) for48h. C.pn infection on Hep-2cells and HUVECs were observed using the light microscope. The measurement of thiobarbituric acid reactive substances (TBARS) was used to to determine the extent of LDL oxidation. The contents of intracellular total cholesterol (TC) and cholesteryl esters (CE) were detected by enzymic chromatometry.Results Compared with uninfected HUVECs (negative control group), supernatants of C.pn-infected HUVECs but not cell lysates induced a significantly distinct LDL-oxidation (p<0.05). In addition, C.pn infection increased total cholesterol (TC) and cholesteryl esters (CE) in LDL-treated HUVECs, compared with negative controls (p<0.05). However, C.pn infection did not show significant difference in increasing TC and CE levels, compared with positive controls (p>0.05).Conclusion C.pn infection induced LDL-oxidation and lipid metabolism disturbance in endothelial cells. Part ⅡEffects of Chlamydia pneumoniae infection on the expressions of key genes involved in human umbilical vein endothelial cells lipid metabolismObjective To investigate the roles of scavenger receptor A (SR-A1), CD36, Acyl-CoA cholesterol acyltransferase-1(ACAT1) and ATP-binding cassette transporter Al/G1(ABCA1/G1) in C.pn-induced lipid metabolism disturbance in endothelial cells.Methods Human umbilical vein endothelial cells (HUVECs) were randomly divided into3groups:(1) negative control group:HUVECs were incubated with LDL (50mg/L) for48h;(2) C.pn infection group:in the presence of LDL (50mg/L), HUVECs were infected with C.pn (1×106IFU) for48h;(3) positive control group:HUVECs were incubated with oxidized LDL (50mg/L) for48h. The mRNA and protein expressions of SR-A1, CD36, ACAT1, ABCA1and ABCG1were evaluated by RT-PCR and Western-blot, respectively.Results Compared with negative controls, C.pn infection not only up-regulated the mRNA and protein expressions of SR-A1, CD36and ACAT1, but also down-regulated the mRNA and protein expressions of ABCA1and ABCG1in LDL-treated HUVECs (all p<0.05). However, C.pn infection did not show significant difference in the expressions of SR-A1, CD36, ACAT1, ABCA1and ABCG1at mRNA and protein levels, compared with positive controls (p>0.05).Conclusion C.pn infection might destroy the homeostasis of intracellular cholesterol metabolism by increasing SR-A1, CD36and ACAT1expressions, and inhibiting ABCA1and ABCG1expressions in LDL-treated HUVECs, which could lead to endothelial dysfunction.