TGF-β1 Signaling Induced Activation Of Hepatic Stellate Cells Promotes Angiogesis Of Vascular Endothelial Cells

PART ONE TGF-Β1 ACTIVATED HEPATIC STELLATE CELLS INDUCE ANGIOGENESIS OF VASCULAR ENDOTHELIAL CELLS IN VITROObjective: The aim of this part is to investigate the mechanism of transforming growth factor beta 1(TGF-β1)in the activation of mouse hepatic stellate cells(mHSCs),and promoting effect of active HSCs on the angiogenesis in vitro.Methods: After transfection with virus,mHSCs are divided into TGF-β1 overexpressing group,TGF-β1-RⅠ knocked-down group and their control groups;The mRNA levels of α-SMA,Smad2/3,VEGFA and TGF-β1-RⅠ were determined by Quantitative Real-time PCR;the protein levels of α-SMA,Smad2/3,VEGFA and TGF-β1-RⅠ were determined by Western blot;the proangiogenic effect of activated mouse hepatic stellate cells(mHSCs)on Human Umbilical Vein Endothelial Cells(HUVECs)was detected by Endothelial Cell Tube Formation Assay.Results:TGF-β1 can induce mouse hepatic stellate cells(mHSCs)high expression of α-SMA(2.057±0.114 vs.0.664±0.063,P > 0.0001)and the downstream signal molecules of classical TGFβ1/TGF-β1-RⅠ/Smad2/3 signaling pathways,TGF-β1-RⅠ(1.710±0.380 vs.0.278±0.069,P > 0.0001),Smad2/3(2.780±0.138 vs.0.259±0.012,P > 0.0001),while the HSCs get its proangiogenic potential through up-expressing VEGFA(mean length of newly formed tube control groups 174.4±1.9μm;TGF-β1 overexpressing groups 258.2±6.9μm;TGF-β1-RⅠ knockdown groups 98.5±1.1μm,P > 0.0001).Conclusion : the study showed that TGF-β1 signaling activated mHSCs through the classical Smad2/3 pathway,and the activated mHSCs have the function of promoting the angiogenesis of vascular endothelial cells.PART TWO TGF-Β1 ACTIVATED HEPATIC STELLATE CELLS INDUCE LIVER ANGIOGENESIS OF EXPERIMENTAL ANIMAL IN VIVOObjective: The aim of this part is to investigate the proangiogenic ability of stable TGF-β1-transfected mouse hepatic stellate cells(m HSCs)in liver regeneration of mice via cells transplantation.Methods: Through intrasplenic transplantation of TGF-β1-overexpressing m HSCs,TGF-β1-RⅠ-silenced m HSCs,GFP-m HSCs into acute liver injury mice models,m HSCs engrafted in livers;after cells transplantation for 14 d,livers from each groups were taken out and liver functions of each mice were also detected.The m RNA levels of α-SMA,VEGFA and CD34 were determined by Quantitative Real-time PCR;the newly formed vessels in mice livers from each groups were detected by immunohistochemical.Results:The TGF-β1 activated m HSCs promoted the expression of VEGFA(2.420±0.140 vs.0.147±0.053,P > 0.0001)in the liver of recipient mice under the condition of cell transplantation,which increased the formation of neovascularization in damaged liver during acute liver injury,thereby promoting the recovery of damaged liver function.Conclusion:The results indicate that m HSCs activated by TGF-β1 can promote hepatic angiogenesis in vivo by increasing VEGFA secretion under cell transplantation during mouse liver regeneration.