Inhibition Of TGF?1-Induced Epithelial-mesenchymal Transition (EMT) By VEGF Is Related To Down-regulations Of Jagged-1 And Notch1 In HK2 Cells

Background and Objective:It is well known that epithelial-mesenchymal transition (EMT) is a novel mechanism involved in renal interstitial fibrosis and Jagged-1/Notch signaling pathway plays a crucial role in transforming growth factor-?1(TGF-?1) induced EMT. Our previous work have indicated that vascular endothelial growth factor (VEGF) is a potential antifibrotic cytokine that partially blocks EMT induced by TGF-?1. However, the underlying mechanism remains largely unclear. Evidences have shown that Notch moleculars are regulated by VEGF in endothelial cells and crosstalk between VEGF, TGF-?1 and Notch is essential for angiogenesis. All of these indicates that VEGF may influence Notch moleculars expression induced by TGF-?1.This study is designed to establish the effect of VEGFI65 on Jagged-1, Notch1 expression and EMT induced by TGF-(31.Methods:To examine the effect of VEGF on expression of Jagged-1 and a-SMA at different time points, cultured HK2 cells were incubated with TGF?1(5?g/L) in the absence or presence of VEGF165(100?g/L) respectively. Expression of a-SMA and Jagged-1 mRNA were assessed with RT-PCR at 0,2,4,8,24 hours respectively after incubation; a-SMA and Jagged-1 protein expression were assessed with Western Blot at 0,2,4,8, 12,24,48 hours respectively after incubation.To test the influence of different concentrations of VEGF on expression of Jagged-1, Notchl and EMT induced by TGF-?1, cultured HK2 cells were divided into four groups:negative control, incubated with TGF-?1(5?g/L) along, with VEGF165(100?g/L) along, or incubated with TGF?1(5?g/L) and different concentrations of VEGF165(0.1,1, 10,100?g/L) for 24 hours. Double-stain immunocytochemistry was used to detect expression of?-SMA and E-cadherin. Confocal microscope was used to detect expression of?-SMA. Protein and mRNA expression of?-SMA, Jagged-1 and Notch1 were assessed with RT-PCR and Western Blot respectively.Results:Both mRNA and protein expression of?-SMA in HK2 cells incubated with TGF?1 alone(group T) increased dramatically at earlier stage than that co-treated with TGF?1 and VEGF165 (group T+V). Compared to group T, protein expression of?-SMA decreased significantly in group T+V at 24h and 48h. Expression of Jagged-1 mRNA increased biphaticly both in group T and group T+V, although was relatively lower in group T+V. Protein expression of Jagged-1 increased earlier in group T than group T+V, and decreased significantly in group T+V at 24h and 48h compared with group T.As revealed by double-stain immunocytochemistry, TGF-?1 inhibited E-cadherin protein expression and enhanced?-SMA expression in HK2 cells. VEGF165 reversed TGF-?1-induced inhibition of E-cadherin protein and promotion of?-SMA expression in a dose dependent matter. Confocal microscope also showed that VEGF165 inhibited?-SMA protein expression induced by TGF-?1. Revealed by RT-PCR and Western Blot, mRNA and protein expression of?-SMA, Jagged-1 and Notch1 significantly decreased in cells incubated with TGF-?1 and VEGF165 (100?g/L), compared with that treated with TGF-?1 alone (P<0.05). VEGF165 markedly repressed TGF?1 induced?-SMA, Jagged-1 and Notch1 expression in a dose dependent manner. There was no significant difference between negative control and the group treated with VEGF165 alone for mRNA and protein expression of Jagged-1 and Notch1 in HK2 cells.Conclusions:The results of the present study indicated that VEGF165 may partially inhibits TGF?1-induced EMT of HK2 cells in vitro, which is probably contributed to the repression of Jagged-1 and Notch1 expression. Further study still needs to be done. Background and Objective:It has been proved that epithelial-mesenchymal transition (EMT) plays an important role in renal interstitial fibrosis and is regulated by a lot of factors. For example, TGF-?1 promotes EMT through Notch signaling pathway and VEGF inhibits EMT. Sulodexide is isolated from porcine intestinal mucosa and comprised of four naturally glycosaminoglyan (GAG) components. It has been reported that sulodexide could decrease the expression of TGF-?1 in renal tissue of experimental diabetic rats, and also affect expression of several other cytokines involved in the EMT process. GAGs have been demonstrated inhibitory effect on experimental organ fibrosis, including liver and kidney. But different kind of GAG has not revealed the same consequences. The influence of sulodexide on renal interstitial fibrosis has not be studied yet.This study is aimed to determine the effect of therapy with sulodexide on renal interstitial fibrosis, EMT and Jagged-1, Notch1 expression in unilateral ureteral obstruction (UUO) mice, trying to reveal the likely mechanism underlying.Methods:78 mice were divided into five groups randomly:group A (sham) was subjected to surgical manipulation but without ureteral ligation; group B(UUO+NS) was subjected to ureteral obstruction and received saline subcutaneously (s.c.) once daily; group C(UUO+LS) was subjected to ureteral obstruction and received sulodexide(6mg/kg) s.c. once daily; group D(UUO+HS) was subjected to ureteral obstruction and received sulodexide(12mg/kg) s.c. once daily; group E (UUO+3dHS) was subjected to ureteral obstruction and received sulodexide(12mg/kg) s.c. once daily from the third day after operation. Mice from group A-D were sacrificed at day 3,7 and 14 after operation respectively and mice of group E at day 7. The severity of renal interstitial fibrosis were determined by pathologic techniques. The content of a-SMA, TGF-?1, VEGF, Jagged-1 and Notch1 were determined in the kidneys by immunohistochemical or biochemical techniques.Results: Morphology changes were distinguished after UUO operation:accumulation of extracellular matrix in tubular-interstitial areas, atrophy of tubules. Yet glomeruli almost remained untouched. The degree of interstitial fibrosis, expression of a-SMA, TGF-?1 and Jagged-1 increased significantly and expression of VEGF decreased in group B, compared to group A. Sulodexide treatment reduced interstitial fibrosis, expression of?-SMA, TGF-?1 and Jagged-1, but not affect the expression of VEGF when compared with group B. Expression of Notchl was not detectable in CD1 mice kidneys by RT-PCR and Western Blot.Conclusions:Sulodexide diminishes interstitial fibrosis in obstructed kidneys. Inhibition of EMT is most likely to be included, which may be related to downregulating the synthesis of TGF-?1 and Jagged-1. The mechanisms underlying these effects remain to be elucidated.