Vascular Endothelial Growth Factor In Expression Of Renal Tubular Epithelial Mesenchymal Conversion Between Inhibitory Effect And Mechanism Of The Discussion

The Establishment of Human Kidney Cell Lines with Stable Over-expression of VEGF (HKC-SOEV cells)Background and Aim vascular endothelial growth factor (VEGF) is very important for maintenance survival and proliferation of vascular endothelial cells, and plays a vital part in regulating rebirth and reconstitution of blood vessel. As a general rule, VEGF of the kidney of fetus and the adult mainly comes from podocytes and renal tubular epithelial cells. Renal tubular epithelial cells can secrete VEGF121and VEGF165, which are the main source of VEGF in renal tubulointerstitial matrix. We demonstrated for several times that exogenous VEGF can suppress TGF-β1-induced epithelial-mesenchymal transition (EMT) previously,.but the mechanism is still unclear.The aim of this study is to establish a new HKC cell lines, HKC-SOEV cells, which may over-express VEGF stablely and secrete VEGF121and VEGF165,which modulate the in vivo enviroment, in order to clarify the mechanism of VEGF suppressing EMT.Methods We got total gene synthesis of VEGF121and VEGF165by biochemistry. VEGF121was made locate behind the CMV promoter of pBuDCE4.1carrier, and VEGF165locate behind the EF-1α promoter of pBuDCE4.1carrier. And the resistance of zeocin was inserted. By detection of gene sequence, it is recognized that Z7842-2is the correct plamid, and resistance is Zeocin.Z7842-2plamid was made get into HKC by electric conversion, and cells were screened by the pressurized method of zeocin step by step. The concentration of VEGF in culture medium was tested by ELASA.Results The concentration of VEGF in culture medium of normal HKCs was112.918pg/ml, but the concentration of VEGF in culture medium of HKC-SOEV cells that were transferred to Z7842-2plamid was1210.061pg/ml, which concentration was10.72times higher than that in normal HKCs(p<0.05).Conclusion Human kidney cell lines with stable over-expression of VEGF (HKC-SOEV cells) were successfully established. Suppression of EMT by VEGF Overexpresssion and Inhibition of Smad3ExpressionBackground and Aim:TGF-β1plays a biological role by Smad signaling pathway, and Smad proteins are the main signaling molecules behind receptors of TGF-β1, which mediates signal transduction from membrane to nucleus. Epithelial-mesenchymal transition (EMT) is an important way to renal interstitial fibrosis. Smad3is the key adjustment factor in the process of EMT that induced by TGF-β1,. We have demonstrated that VEGF can inhibit TGF-β1-induced EMT. Recently the human kidney cell lines with stable over-expression of VEGF (HKC-SOEV cells) were successfully established in our lab. Therefore, the aim of this study is to examine the effect of VEGF over-expression on EMT and Smad3expression using HKC-SOEV cells.Methods:The cultured cells were divided into following groups:A. normal HKC cells, B.normal HKC cells+TGF-β1(5μg/l) for24H and48H, C. HKC-SOEV cells (HKC stable over-expression of VEGF), D. HKC-SOEV cells+TGF-β1(5μg/l) for24H and48H.The protein expressions of Smad2and Smad3were detected with Western blot, and Smad3was also determined with real-time PCR and RT-PCR. E-cadherin and α-SMA expressions were measured with western blot and laser scanning confocal microscope(LSCM). The morphological changes of cells were observed with40optical microscope.Results:a-SMA expression in group B was significantly higher than that in group A (p<0.05), while E-cadherin expression in group B was significantly lower than that in group A. a-SMA in group D was lower than that in group B(p<0.05), and E-cadherin in group D was higher than that in group B (p<0.05). P-Smad3expression in group B was significantly higher than that in group A (p<0.05), while P-Smad3and Smad3in group D was significantly lower than that in group B(p<0.05). Smad3in group C was significantly lower than that in group A (p<0.05), P-Smad3/Smad3in group B was significantly higher than that in group A(p<0.05), while P-Smad3/Smad3in group D was significantly lower than that in group B(p<0.05). With40optical microscope, normal HKCs and HKC-SOEV cells showed a cobblestone-like, and normal HKCs stimulated with TGF-β1were spindle-shaped, while HKC-SOEV cells stimulated with TGF-β1showed no obvious change in shape.Conclusions The results indicated that over-expression of VEGF may reduce the expression and phosphorylation of Smad3of HKCs, which suppresses TGF-β1induced EMT Down-Regulation of Smad3Expreesion by VEGF Overexpression Depent on PI3K/Akt signal pathwayBackground and Aim:Epithelial-mesenchymal transition (EMT) is an important way to renal interstitial fibrosis, in which TGF-β1/Smad pathway plays an important biological role, and Smad3is the key adjustment factor in the process of EMT. We have demonstrated that VEGF may inhibit TGF-β1induced EMT, in which VEGF transmits signal mainly with PI3K/Akt pathway. Recently HKC-SOEV cells (the human kidney cell lines with stable over-expression of VEGF) were successfully established in our lab. The aim of present study is to investigate whether VEGF over-expression suppresses Smad3with PI3K/Akt pathway or not.Methods:The cultured cells were divided into following groups:A. normal HKC cells, B: normal HKC cells+TGF-β1(5μ/l) for24and48H, C. HKC-SOEV cells (HKC stable over-expression of VEGF), D. HKC-SOEV cells+TGF-β1(5μg/l)24and48H. E. HKC-SOEV cells+TGF-β1(5μg/l)+LY294002(20μmol/l) for24and48H. LY294002is a specific inhibitor for PI3K activity. The protein of P-PI3K, PI3K, P-Akt, Akt, P-Smad3and Smad3were detected with Western blot. The gene levels of Smad3were measured with real time PCR. E-cadherin and a-SMA were measured with western blot and laser scanning confocal microscope (LSCM). The morphological changes of cells were observed with40optical microscope.Results:PI3K and P55-PI3K in group C were higher than those in group A, respectively(p<0.05). P-PI3K in HKC-SOEV cells stimulated by TGF-β1for48H was significantly higher than that in HKC cells stimulated by TGF-β1for48H (p<0.05), and P-PI3K in group E was significantly lower than that in other groups(p<0.05). Akt in HKCs stimulated with TGF-β1for48H was significantly lower than that in other groups, and no changes in Akt levels were found in other groups. P-Akt in group D was higher than that in group B, P-Akt in group E was lower than that in group D.Smad3in group C(HKC-SOEV cells) was significantly lower than that in group A(p<0.05), and Smad3in group D significantly lower than that in group B (p<0.05); However, Smad3in group E was higher than that in group D(p<0.05). P-Smad3and P-Smad3/Smad3in group B was significantly higher than that in group A (p<0.05); P-Smad3and P-Smad3/Smad3in group D was lower than those in group B (p<0.05); P-Smad3and P-Smad3/Smad3in group E was higher than that in group D(p<0.05).a-SMA in group B was significantly higher than that in group A and D respectively (p<0.05), while α-SMA in HKC-SOEV cells stimulated by TGF-β1and LY294002was significantly higher than that in HKC-SOEV cells stimulated by TGF-β1at48H (p<0.05). E-cadherin in group A was significantly higher than that in group B (p<0.05), but significantly lower than that in in group C (p<0.05), E-cadherin in HKC-SOEV cells stimulated with TGF-β1was significantly higher than that in HKCs stimulated with TGF-β1at24H (p<0.05).With40optical microscope, normal HKCs and HKC-SOEV cells showed a cobblestone-like, and normal HKCs stimulated with TGF-β1were spindle-shaped, while HKC-SOEV cells stimulated with TGF-β1showed no obvious change in shape. However, HKC-SOEV cells stimulated with TGF-β1and LY294002were spindle-shaped.Conclusions:This study showed that inhibition of expression and phosphorylation of Smad3of HKCs by over-expression of VEGF is probably depent on PI3K/Akt pathway, which is very important to the effect of VEGF on TGF-β1induced EMT. VEGF overexpression inhibit miR192in EMT induced by TGF-β1Background and Aim:TGF-β1-induced epithelial-mesenchymal transition (EMT) is an important process of renal fibrosis. It is suggested that VEGF may suppress EMT, and VEGF transmits sigal mainly with PI3K/Akt pathway, while the mechanism still needs to be elucidated. Recently the changes in miR-192expression was suggested to be related to renal fibrosis. This study is undertaken to examine the effect of VEGF overexpression on miR-192in TGF-β1-induced EMT of HKC cells.Method:The cultured cells were divided into following groups:A. normal HKC cells, B:normal HKC cells+TGF-β1(5μg/l) for24H and48h, C. HKC-SOEV cells(HKC stable over-expression of VEGF), D. HKC-SOEV cells+TGF-β1(5μg/l) for24H and48H. E. HKC-SOEV cells+TGF-β1(5μg/1)+LY294002(20μmol/l) for24H and48H. The levels of gene expression of miR-192and Smad3were determined by real-time PCR.The correlation between miR-192and Smad3was analyzed with SPSS.Results:miR-192expression in HKCs stimulated by TGF-β1for48H was higher than that in group A (p<0.05), while miR-192expression in HKC-SOEV cells stimulated by TGF-β1for48H was lower than that in group B(p<0.05). miR-192in group E is higher than that in HKC-SOEV cells stimulated by TGF-β1for24H (p<0.05). Smad3in group B is significantly higher than that in group A(p<0.05), and Smad3in HKC-SOEV cells is significantly lower than that in group A(p<0.05). Smad3in group D is significantly lower than that in group B(p<0.05), but Smad3in group E is significantly higher than that in group D(p<0.05). The levels of miR-192were positively correlated with Smad3expression.Conclusion:The results demonstrated that TGF-β1may upregulate the expression of miR-192, while VEGF may downregulate expressions of miR-192and Smad3in HKC cells. The levels of miR-192were positively correlated with Smad3expression.