The Protection Of Pericytes On Vascular Permeability In Hemorrhagic Shock And Its Relationship To Cx43

Hemorrhagic shock as a serious complication caused by war wound or trauma,is the important cause of multiple organ dysfunction.Increased vascular permeability is the key link to tissue hypoxia,capillary leakage syndrome and microcirculation disorders in shock patients[1],which is an important pathogenesis of organ dysfunction caused by hemorrhagic shock.Searching for effective measures to prevent and treat vascular barrier dysfunction caused by hemorrhagic shock is necessary to reduce the late mortality of patients with hemorrhagic shock.Pericytes(PCs)are a group of mural cell surrounded the outside of microvessel,and located on the basolateral side of endothelium[2].The synapses of PCs are radial,connected with multiple endothelial cells,and entangled in the capillary wall.The coverage of PCs plays a decisive role in vascular permeability[3].The connection between PCs and vascular endothelial cells(VECs)plays a vital role in the formation,stability and leakage of blood vessels.While,it is unknown whether PCs have a protective effect on the vascular barrier function following hemorrhagic shock.Gap junctions play an important role in intercellular communication by forming permeable intercellular channels.Gap junctions mediate the conduction of electrochemical signals between cells and participate in the regulation of vascular function[4].The gap junction proteins distributed in the cardiovascular system mainly include Cx46,Cx45,Cx43,Cx40 and Cx37.They participate in the rapid coordinated movement of cardiovascular system,and occurrence and development of a variety of cardiovascular diseases[5-6].As the main protein constituent of gap junction between cells,Cx43 plays a key role in atherosclerosis and hypertension[7-8].Does Cx43 between PCs and VECs affects the colonization of PCs on blood vessels and affects vascular permeability in hemorrhagic shock?In this study,hemorrhagic shock SD rats and vascular endothelial Cx43 knockout mice were used to observed whether Cx43 affects the colonization of PCs on blood vessels and the protective effect of PCs on vascular barrier function in hemorrhagic shock.[Research contents] Part I : Relationship of the changes of PCs to vascular permeability in hemorrhagic shock.Hemorrhagic shock rats were used to observe the relationship between the changes of amount of PCs and the changes of vascular permeability in hemorrhagic shock.Pulmonary and mesenteric blood vessels were used to observe the changes of vascular permeability after hemorrhagic shock,by detecting mesentery and lung tissue’s permeability to Evans Blue(EB)and FITC-BSA.The amount of PCs were observed by transmission electron microscopy(TEM).Part II : The protective effect of PCs on vascular permeability in hemorrhagic shock.Animal level Retinal PCs were extracted and cultured according to the literature[9],which were identified by immunofluorescence and flow cytometry.The cultured PCs(PC=106/rat)were used to treat hemorrhagic shock rats.The permeability of pulmonary and mesenteric microvasculature was observed by determining the permeability of mesentery and lung tissue to Evans Blue and FITC-BSA.The amount of PCs,the expression of connection proteins between PCs and VECs,and the structure between VECs and VECs were observed by TEM.Cellular level After PCs and VECs were co-cultured,the protective effect of PCs on the endothelial barrier of hypoxia was observed by transepithelial electrical resistance(TER)and the infiltration rate of FITC-BSA.Part III: The relationship of Cx43 to the protective effect of PCs on vascular permeability of hemorrhagic shock.(1)The effect of regulating the expression of Cx43 in PCs on vascular permeability.Animal level The cultured PCs(Cx43 sh RNA / over-expressed)were used to treat hemorrhagic shock rats.Pulmonary and mesenteric microvasculature were used to observe the effect of Cx43 on vascular permeability in hemorrhagic shock,by determining mesentery and lung tissue’s permeability to Evans Blue and FITC-BSA.The changes of the amount of PCs,the expression of connection proteins between PCs and VECs,and the structure between VECs and VECs were measured by TER and WB.Cellular level PCs and VECs were co-cultured.They were marked with adenovirus as green and red respectively.PCs(Cx43 sh RNA / over-expressed)were cultured on one side of transwell membrane,and VECs was cultured on the other side.The relationship of Cx43 to the protective effect of PCs on endothelial barrier function were observed by TER and the infiltration rate of FITC-BSA.(2)Effect of Cx43 knocked out in VECs on the colonization and protective effect of PCs on vascular permeability in hemorrhagic shock mice.Cx43 knockout mice(Tie2-Cre;Cx43flox/+)were used.The hemorrhagic shock(30mm Hg,3h)model was established in both wild-type(WT)and Cx43 knockout mice(Tie2-Cre;Cx43flox/+)mice.The colonization of transplanted PCs in the vascular,and the protective effect on vascular permeability in hemorrhagic shock mice were observed.[Results] Part I: Relationship of the changes of PCs to vascular permeability in hemorrhagic shock rats.Following hemorrhagic shock,the amount of PCs was decreased,while the pulmonary and mesenteric microvasculature permeability were significantly increased.There was a negative correlation between the amount of PCs and vascular permeability.TME showed that the VECs were swelled,the tight junctions between VECs were destroyed.WB showed that the protein expression of VE-cadherin and ZO-1 was decreased after hemorrhagic shock.Part II: The protective effect of PCs on vascular permeability following hemorrhagic shock.It was found that the transplanted PCs could colonize on the pulmonary vasculature,and most of them were colonized at 12 hours after infusion.Transplanted PCs could significantly decrease the permeability of pulmonary and mesenteric microvasculature in hemorrhagic shock rats.TME showed that the tight junction structure of VECs was significantly improved,WB showed that the expression of VE-cadherin and ZO-1 was significantly increased after PCs treatment.At the cellular level,the results showed that the TER of VECs was decreased,the infiltration rate of FITC-BSA of VECs was increased,and the expression of ZO-1 and VE-cadherin was decreased after hypoxia.PCs could significantly increase the TER,reduce the infiltration rate of FITC-BSA,and up-regulate the expression of ZO-1 and VE-cadherin in VECs.Part III: The relationship of Cx43 to the protective effect of PCs on vascular permeability of hemorrhagic shock.(1)The effect of regulating the expression of Cx43 in PCs on the vascular permeability in hemorrhagic shock rats.After Cx43 sh RNA in PCs,the colonization of PCs in the pulmonary vascular was significantly decreased,the improving effect of PCs on pulmonary and mesenteric vascular permeability was weakened,and the expression of ZO-1 and VE-cadherin in blood vessels was decreased in hemorrhagic shock rats.Overexpression of Cx43 in PCs could increase their colonization and improve the permeability of pulmonary and mesenteric microvasculature,improve the tight junction structure of VECs,and increase the expression of ZO-1 and VE-cadherin in hemorrhagic shock rats.Cell experiments showed that,after Cx43 sh RNA in PCs,the proportion and area of direct contact between PCs and VECs were decreased,the expression of ZO-1 and VE-cadherin in VECs was decreased,the TER of VECs was decreased,and the permeability of VECs to FITC-BSA was increased.While overexpression of Cx43 in PCs could increase the proportion and area of contact of PCs and VECs,increase the expression of ZO-1 and VE-cadherin in VECs,decrease the permeability of VECs to FITC-BSA and increase the TER of VECs.(2)Effect of endothelial Cx43 knocked out on the protective effect of PCs on vascular permeability in hemorrhagic shock.After conditional knock out of Cx43 in VECs,colonization rate of PCs was significantly decreased,the protective effect of PCs on pulmonary and mesenteric microvasculature permeability was significantly weakened,and the expression of ZO-1 and VE-cadherin in blood vessels was significantly decreased following hemorrhagic shock.[Conclusion] 1.After hemorrhagic shock,the amount of PCs in vasculature was significantly decreased,the coverage of PCs on VECs was significantly decreased,and the permeability of pulmonary and mesenteric microvasculature was significantly increased.There was a negative correlation between the number of PCs and vascular permeability after hemorrhagic shock.2.Exogenous infusion of PCs could protect pulmonary and mesenteric microvasculature permeability.It was suggested that PCs played an important role in the regulation of vascular permeability in hemorrhagic shock.3.Cx43 could affect the colonization of PCs in blood vessels,and then affect its protective effect on vascular permeability.The mechanism might be that Cx43 affects the formation of direct connection between PCs and VECs,and affects the expression of adhesion junction proteins.