TGR5 Activation Attenuates Endothelial Progenitor Cells' Ability Of Angiogenesis In Obstructive Jaundice

?Objective?There are a variety of pathophysiological changes in the obstructive jaundice patients,and We found vascular endothelial cell damage and delayed wound repair in obstructive jaundice animals.Althought angiogenesis is an important part of wound repair and wound repair after surgery,if biliary obstruction directly inhibits the wound healing is unreported;and what factors play a role in vascular endothelial injury is not clear.Past research reported that endothelial progenitor cell is the key factor in wound healing and angiogenesis,yet mechanisms in these processes are related to the VEGF signaling pathway.The purpose of this study is to reveal the role of obstructve jaundice,bile acid and its membrane receptor in the endothelial progenitor cell-inducing ischemic wound healing.?Methods?(1)To identify the effect of obstructive jaundice on skin punch wound healing and observing the angiogensis in the wound by immunofluorescence.(2)Femoral artery ligation is the classic model of ischemic injury and was used to analyze the effect of obstructive jaundice on ischemic injury healing.(3)The effects of bile acid and bilirubin on the repair of ischemic injury after femoral artery ligation were observed.(4)EPCs(Endothelial progenitor cells)were isolated and then identified by flow cytometry and immunofluorescence.(5)Bile acid receptors were knockdown by small interfering RNA and validated by western blotting;EPCs were incubated with bile acid after bile acid reeptors knockdown and then to analyze its migration and tube formation abilities.(6)Proangiogenesis facotrs were detected by PCR after bile acid incubation.(7)EPCs was infected by lentivirus to knockdown and incubated with TGR5(also known as GPBAR1,protein-coupled bile acid receptor 1)specific angonist,after that EPCs were transinplantation into femoral artery ligation nude animal model to validate the role of TGR5 in EPC-inducing angiogenesis.?Results?(1)The obstructive jaundice inhibits skin punch wound healing,one week after injured,the positive density of CD31,Ki67,and ILB4 of the obstructive jaundice group(BDL group)all lower than the sham operation group(Sham group);suggesting that the obstructive jaundice has effect on inhibiting the wound healing,and it may be functioning by inhibiting angiogenesis;(2)In classical femoral artery ligation model,we do observe the obstructive jaundice inhibiting the subsequent angiogenesis of ischemic injury: 3 days,7 days and 14 days after the operation of the BDL group,at operated side,the recovering of blood perfusion and the density of vascular endothelial cell at the 14 th day of limb muscles(ILB4 immunofluorescence)all lower than the Sham group;(3)To identified which factors of the obstructive jaundice inhibit the subsequent angiogenesis of ischemic injury,we respectively research the bile acid and bilirubin.We found that,in femoral artery ligation models of mice,the recovering of blood perfusion can be inhibited after intraperitoneal injection of bile acid;moreover,at the 14 th day,the density of vascular endothelial cell of limb muscles(ILB4 immunofluorescence)show a significant low-level to the control group.However,when we apply bilirubin in the same way,it didn't show the same results above.These results identified the main function induced the obstructive jaundice inhibiting skin punch wound healing was by the bile acid.(4)Endothelial progenitor cells were isolated and culture in vitro,then identified the purity of cells was above 90% by multiple methods.(5)After EPCs incubated in bile acid,we found that,activated bile acid membrane receptor TGR5 could inhibit EPCs' migration and tube formation abilities;however,after specific interfering bile acid membrane receptor TGR5,this inhibiting effect was faded out.The interfered bile acid nuclear receptor FXR did not show the same effect above.(6)Being incubated in bile acid,several proangiogenesis factors of EPCs,such as VEGF-A,PDGF-B,FGF,CD31,IL-8,the expression of these all reduced.(7)The inhibitional effect of EPCs to the repairing of ischemic injury can be suppressed by specific knocking down the bile acid membrane receptor TGR5.Transplantate the TGR5-knockdown EPCs to mice,the time of blood perfusion recovering at operated side was faster than the control group notably,the density of vascular endothelial cell of limb muscles shown a higher-level as well.?Conclusion? We found the number of EPCs was negatively related with the level of bile acid in obstractive jaundice for the first time.The abilities of EPCs in migration,tube formation and angiogenesis factor expression were attenuated after bile acid incubation.TGR5 spectific activation attenuates the abilities of EPCs in migration,tube formation,angiogenesis and wound healing.Opposite outcomes were obtained when TGR5 was knockdown.All resultes hintes that TGR5 playes an important role in angiogenesis.