| Background and Objective It is well known that epithelial-mesenchymal transition (EMT) is an important event in renal interstitial fibrosis. Transforming growth factor (TGF)-β1is believed to induce EMT. Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that promotes endothelial cell proliferation, differentiation and survival, mediates endothelium-dependent vasodilatation, induces microvascular hyperpermeability and participates in interstitial matrix remodeling. Several studies have shown that VEGF may relieve experimental renal diseases, including renal interstitial fibrosis. This study is to examine the effect of VEGF on TGF-β1induced EMT of HK-2cells.Methods The cultured HK-2cells were divided into the following groups:a, no treatment, b, treated with TGF-β1(5μg/L) as positive control, c, co-treated with TGF-β1(5μg/L) and VEGFi65(0.1,1,10,100μg/L) for48h. a-smooth muscle actin (a-SMA) and E-cadherin expressions of HK-2cells were assessed with double-stain immunocytochemistry method and cofocal microscopy. a-SMA, E-cadherin, Vimentin mRNA and protein expressions were assessed with RT-PCR and Western Blot, respectively. The culture supernatants were collected to examine the expressions of fibronectin (FN) and collagen I (COL I) using immunosorbent assay (ELISA) methods.Results By double-stain immunocytochemistry method, confocal microscopy, RT-PCR and Western blot, we showed that a-SMA expression significantly increased and E-cadherin expression significantly decreased in HK-2cells treated with TGF-β1(5μg/L) compared with no treatment. a-SMA expression decreased and E-cadherin expression increased in HK-2cells co-treated with TGF-β1(5μg/L) and VEGF165compared with TGF-β1(5μg/L) alone. VEGF165dramatically abrogated TGF-β1induced a-SMA expression and restored the E-cadherin expression in HK-2cells in a dose-dependent manner. At the concentrations of10,100μg/L, VEGF165almost completely blocked a-SMA and vimentin mRNA and protein expression induced by TGF-β1(5μg/L)(P<0.05), and the expression of FN and COL I in the supernatant of HK-2cells were downregulated concomitantly (P<0.05), but E-cadherin protein and mRNA expression was upregulated (P<0.05).Conclusions The results documented that TGF-β1may induce EMT in HK-2cells, and VEGF165may partially inhibit TGF-β1-induced EMT in HK-2cells in vitro. Background and Objective TGF-β1-induced EMT is mainly mediated through Smad and non-Smad signaling pathway, including Erkl/2pathway. In the process of EMT, SnoN is able to inhibit Smad-induced transcription activity as co-repressor. This study is to examine the relationship between the inhibitory effect of VEGF on EMT of HK2cells and changes in the expressions of SnoN and whether Smad and Erkl/2pathways are involved in the effect of VEGF mentioned above.Methods The cultured HK-2cells were divided into four groups:a, no treatment, b, treated with TGF-β1(5μg/L) as positive control, c, treated with VEGF165(100μg/L), d, co-treated with TGF-β1(5μg/L) and VEGF165(100μg/L). a-SMA, p-Smad2/3and Smad2/3, p-Erkl/2and Erkl/2, Smad7, SnoN expressions were assessed with Western blot and RT-PCR, respectively.Results By Western Blot and RT-PCR analysis, we showed that a-SMA expression significantly increased in HK-2cells treated with TGF-β1(5μg/L) compared with no treatment (P<0.05), and the ratio of p-Smad2/3and Smad2/3and the ratio of p-Erkl/2and Erkl/2significantly increased at30min and60min,too(P<0.05). These phosphorylation markers mentioned above decreased in HK-2cells co-treated with TGF-P1(5μg/L) and VEGF165(100μg/L) compared with positive controls, especially at30min (P<0.05). But Smad7expression was exactly opposite to that of these phosphorylation markers mentioned above. VEGF165dramatically abrogated TGF-β1induced a-SMA expression and phosphorylation of Smad2/3and Erk1/2and restored Smad7expression in HK-2cells (P<0.05). However, VEGFi65itself failed to induce phosphorylation of Smad2/3and the expression of Smad7, while induced phosphorylation of Erkl/2. SnoN protein expression significantly increased in HK-2cells treated with TGF-β1(5μg/L) compared with no treatment (P<0.05), but was not different in HK-2cells co-treated with TGF-β1(5μg/L) and VEGF,65(100μg/L) compared with positive controls(P>0.05). SnoN mRNA expression was not different in each group (P>0.05).Conclusions The results documented that VEGF165may partially inhibit TGF-β1-induced EMT in HK-2cells in vitro, and this effect is related to suppress phosphorylation of Smad2/3directly and Erk1/2indirectly, and upregulate Smad7expression. However, the effect of VEGF165on EMT is not related to SnoN expression. Background and Objective Many studies demonstrated that PI3K/Akt was one of the major intra-cellular signaling molecules of VEGF. Connective tissue growth factor (CTGF) seemed to be an important downstream mediator of at least part of the profibrotic activity of TGF-β1. CTGF could promote EMT in vitro both directly and as a downstream mediator of TGF-β1. The aim of this study is to examine whether PI3K signal pathway is involved in the effect of VEGF on EMT and the changes in the expressions of Smad signal pathway and CTGF. Methods The cultured HK-2cells were divided into eight groups:a, no treatment, b, treated with TGF-(31(5μg/L) as positive control, c, treated with VEGF165(100μg/L), d, co-treated with TGF-β1(5μg/L) and VEGF165(100μg/L),e, LY294002(25μmol/L), f, TGF-β1(5μg/L) and LY294002(25μmol/L), g, VEGF165(100μg/L) and LY294002(25μmol/L),h, co-treated with TGF-β1(5μg/L) and VEGF165(100μg/L) and LY294002(25μmol/L). a-SMA and E-cadherin expressions of HK-2cells were assessed with cofocal microscopy. a-SMA, p-Smad2/3and Smad2/3, Smad7, and CTGF expressions were assessed with Western blot and RT-PCR, respectively. The expressions of FN and COL I in the supernatant of HK-2cells were assessed with ELISA.Results a-SMA, CTGF expressions, the ratio of p-Smad2/3and Smad2/3, and FN and COL I in the supernatant significantly increased in HK-2cells treated with TGF-β1(5μg/L) compared with no treatment (P<0.05), and those markers decreased in HK-2cells co-treated with TGF-β1(5μg/L) and VEGF165(100μg/L) compared with TGF-β1treatment alone (P<0.05). When LY294002was added to TGF-β1and VEGF165co-treated cells, the expressions of these markers mentioned above were markedly upregulated, and was not statistically significant when compared with that TGF-β1treatment alone. But E-cadherin and Smad7expression was exactly opposite to those markers mentioned above.Conclusions The results documented that VEGF165may partially inhibit TGF-β1-induced EMT in HK-2cells in vitro, and this effect is related to suppress phosphorylation of Smad2/3, upregulate Smad7expression, and inhibit CTGF expression and FN and COL I production, which may be partly dependent on PI3K pathway. Background and Objective Bone morphogenetic protein-7(BMP-7) can maintain the phenotype of epithelial cells and reverse EMT. BMP-7functioned mainly through Smad1/5/8signaling pathway. The objective of this study is to examine the relationship between the inhibitory effect of VEGF on EMT of HK2cells and changes in the expression of BMP-7and its Smad1/5/8signal pathway.Methods The cultured HK-2cells were divided into the following groups:a, no treatment, b, treated with TGF-β1(5μg/L) as positive control, c, co-treated with TGF-β1(5μg/L) and VEGF165(0.1-100μg/L) for different times. The p-Smadl/5/8, Smad1/5/8and BMP-7expressions were assessed with Western blot.Results BMP-7expression significantly decreased in HK-2cells treated with TGF-β1(5μg/L) compared with no treatment, however, increased in HK-2cells co-treated with TGF-β1(5μg/L) and VEGF165compared with TGF-β1(5μg/L) alone. VEGF165dramatically restored TGF-β1-inhibited BMP-7expression in HK-2cells in a dose-dependent manner. By Western Blot analysis, we showed that the ratio of p-Smad1/5/8and Smad1/5/8significantly increased in HK-2cells treated with TGF-β1(5μg/L) and VEGF165(100μg/L), and reached the peak at30min, then weaken with time. At30min, VEGF165induced phosphorylation of Smad1/5/8in concentration-dependent manners. TGF-β1alone significantly failed to induce phosphorylation of Smadl/5/8.Conclusions The results documented that the effect of VEGF165on TGF-β1-induced EMT in HK-2cells in vitro is related to induce BMP-7directly and phosphorylation of Smad1/5/8indirectly. Background and Objective Inhibitor of DNA binding/differentiation (Id) is the helix-loop-helix (HLH) protein family that comprises four members (Idl through4) They mostly act as negative transcriptional regulators in many biological processes such as cell differentiation, cell senescence, neurogenesis, apoptosis and angiogenesis. Our previous study indicated that VEGF165may partially inhibit TGF-β1-induced EMT in HK-2cells in vitro, and this effect was related to up-regulated expression of Id2, but Id3was not proven to be involved. The objective of this study is further to examine whether VEGF165opposes TGF-β1-induced EMT in HK-2cells through Id2.Methods The cultured HK-2cells were divided into the following groups: non-silencing siRNA and Id2siRNA, then treated with TGF-β1(5μg/L) and/or VEGF165(100μg/L), or no treatment for48h, respectively. The162, a-SMA, and E-cadherin expressions were assessed with Western blot and RT-PCR, respectively.Results Id2expression significantly decreased in HK-2cells of non-silencing siRNA treated with TGF-β1(5μg/L) compared with no treatment, however, increased in HK-2cells of non-silencing siRNA co-treated with TGF-β1(5μg/L) and VEGF165compared with TGF-β1(5μg/L) alone(P<0.05). Knockdown of162with162siRNA was found to further enhance TGF-β1-induced α-SMA and decrease E-cadherin expression, compared with non-silencing siRNA(P>0.05). Knockdown of Id2before TGF-β1and VEGF165added, was found to significantly increase TGF-β1-induced α-SMA and decrease E-cadherin expression, compared with non-silencing siRNA (P>0.05).Conclusions The results document that VEGF165opposes TGF-β1-induced EMT in HK-2cells through Id2. Background and Objective:It is believed that EMT is an important event in interstitial fibrosis. Tubular EMT induced by TGF-β1is especially important during progressive renal interstitial fibrosis. CTGF seemed to be an important downstream mediator of at least part of the profibrotic activity of TGF-β1and promoted EMT in vitro both directly and as a downstream mediator of TGF-β1. BMP-7can maintain the phenotype of epithelial and reverse EMT in vitro, and attenuated renal fibrosis in vivo. We have shown that the inhibitory effect of VEGF165on TGF-β1induced EMT of HK-2cells in vitro is related to down-regulated expression of CTGF and up-regulated expression of BMP-7. This study is designed to determine the effect of VEGF on renal interstitial fibrosis and EMT in unilateral ureteral obstruction (UUO) mice and the relationship with the expressions of TGF-β1, CTGF and BMP-7.Methods:36male mice(21-24g) were divided into the following groups randomly: group A (sham group) was subjected to surgical manipulation but without ureteral ligation; group B (UUO group) was subjected to ureteral obstruction; group C (UUO+VEGF group) was subjected to ureteral obstruction and received VEGF121(50μg/kg, subcutaneously, twice daily) from the first day after operation. The VEGF121isoform was chosen because it is the only isoform of VEGF that has no heparin-binding ability and therefore results in a therapeutically effective plasma level when administered subcutaneously. Mice were sacrificed at day3,7and14after operation, respectively. The degree of renal interstitial fibrosis was determined by pathologic techniques. The content of α-SMA, E-cadherin, TGF-β1, CTGF and BMP-7in the kidneys were determined by immunohistochemical and biochemical techniques.Results:Morphology changes were distinguished after UUO operation:accumulation of extracellular matrix in tubular-interstitial areas, atrophy of tubules. Glomerulars almost remained untouched. Renal interstitial fibrosis in UUO mice was distinct compared with that in sham mice, and significantly aggravated as time went by. The degree of interstitial fibrosis, the expressions of α-SMA, TGF-β1, and CTGF increased significantly in UUO group, while E-cadherin and BMP-7expressions decreased significantly in UUO group, compared with sham group at day3,7and14, respectively. VEGF treatment reduced interstitial fibrosis, expressions of α-SMA, TGF-β1and CTGF, but increased the expressions of E-cadherin and BMP-7when compared with UUO group at day3and7. No differences in the expressions of these markers were found between the VEGF-treated and UUO group at day14except for CTGF.Conclusions:VEGF administration may ameliorate renal interstitial fibrosis and EMT at the early stage in UUO mice. This effect of VEGF may be probably related with down-regulation of TGF-β1and CTGF and the up-regulation of BMP-7in vivo. The exact mechanism underlying these effects remains to be elucidated.
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