Microvesicles Secreted By Mesenchymal Stem Cells Promote The Repair Of Pulmonary Microvascular Endothelial Cells By Transplanting Mitochondria

Objective: To study the role of mitochondria in MVs secreted by MSCs in the repair of PMVECs.Methods:(1)In order to screen out the optimal culture conditions of concentrated mitochondria,the MSCs were cultured under normoxia(21% O2)and hypoxia(1% O2)for 24,48,and 72 hours,respectively.The microvesicles in the supernatant were separated and extracted by differential centrifugation.The morphology and size of MV were observed under electron microscope.The surface markers of MV were analyzed by flow cytometry.The BCA protein quantitative method was used to determine the density of MV.JC-1 was used to judge apoptosis rate,and the expression of mitochondria-associated proteins(CYP1A1,CYP1A2)were detected by Western blot.(2)To observe the transfer of mitochondria from MV to PMVEC,mitochondria of MSC-MVs and PMVECs were stained with fluorescent dyes respectively.The movement of mitochondrial was observed under confocal fluorescence microscope.MSCs,MVs and PMVECs injured by lipopolysaccharide(LPS)were co-cultured,and were divided into EC group,EC+LPS group,EC+LPS+MSC group and EC+LPS+MV group.Western blot was used to detect the changes of mitochondria-associated protein in endothelial cells after co-culturing.(3)To assess the effect on repair of mitochondria,we used rhodamine-6G(rho)to inhibit mitochondrial respiratory chains and JC-1 and ROS kits were used to assess whether mitochondrial function was inhibited.Then MSCs,MVs,MVs-rho and lipopolysaccharide(LPS)induced PMVECs were co-cultured and divided into EC group,EC+LPS group,EC+LPS+MSC group,EC+LPS+MV group and EC+LPS+MV-rho group.Western blot was applied to assess the synthesis(iNOS,eNOS)and connexin protein(VE-cadherin)of endothelial cells;FITC-dextran assay was employed to detect the endothelial cell permeability;Early and late apoptosis rate of PMVECs were assessed by flow cytometry;JC-1 kit was used to detect mitochondrial membrane potential;Inflammatory factor levels were measured by ELISA;ROS kit was used to assess changes in reactive oxygen species.Results:(1)Electron microscopy showed that MVs was a circular/oval membrane structure with a diameter of 100 to 1000 nm.CD44 and CD29 were positively expressed,with the rate of 97.6% and 95.9%,respectively.CD34 was negative.Culturing for 24 h in normoxia conditions was the optimal culture conditions for concentration of mitochondria.(2)The mitochondria in MSC-MVs could be transferred to PMVECs under confocal microscopy.The results of Western blot showed that MSCs and MVs could significantly increase the expression of CYP1A1 and CYP1A2 in PMVECs.(3)After using rhodamine-6G,mitochondrial membrane potential and ROS were significantly decreased.Compared with LPS injury group,MSC and MV co-culture group could significantly improve endothelial cell synthesis(iNOS,eNOS)and increase anti-inflammatory cytokine(IL-10)level.The endothelial cell permeability,early apoptotic rate and reactive oxygen species levels were decreased.The expression of VE-cadherin was higher than that of in LPS group,but the difference was not statistically significant.Compared with MSC and MV group,the expression of endothelial cell synthesis protein and anti-inflammatory factor were significantly decreased.The permeability of endothelial cells,early apoptosis rate and reactive oxygen species levels were significantly increased.The expression of VE-cadherin showed a decreasing trend compared with the MSC and MV groups,but the difference was not statistically significant.Conclusion: MSCs can alleviate the damage of PMVECs through transferring mitochondria delivered by MVs.