Prostaglandin E2/EP2 Mediates Mesenchymal Stem Cell Homing To Injured Lung Tissue Following Acute Lung Injury In A Mice Model

Part One:E-prostanoid 2 receptor overexpression promotes mesenchymal stem cell homing to injured lung tissue and attenuates lung injuryObjective:To ascertain whether E-prostanoid 2 receptor overexpression promotes mesenchymal stem cell homing to injured lung tissue and attenuates lung injury.Methods:(1)MSCs from passage 4-6 were transduced with recombinant lentiviral vectors.Transduction efficiency was evaluated by detecting GFP expression using a fluorescent microscope and through flow cytometry.MSC EP2 expression was assessed using Western blotting and PCR.(2)Transwell chambers were used to assess the migration ability of MSCs,MSCs-GFP and MSCs-EP2.(3)Mice model of lung injury was induced via the intratracheal administration of LPS from Escherichia coli,MSCs-GFP and MSCs-EP2 were injected into mice via the tail vein.(4)Near-infrared imaging and immunofluorescent staining were performed to evaluate the homing efficiency of the implanted MSCs to injured lung tissue.(5)Vascular endothelial permeability of the lung was assessed by measuring the lung wet weight/body weight ratio(LWW/BW)and Evans blue leakage.(6)Lung injury was scored according to lung histopathological assessment.The levels of proinflammatory cytokines(tumor necrosis factor-alpha[TNF-a]and interleukin-1 beta[IL-1β])and anti-inflammatory cytokines(interleukin-10[IL-10])in the lung homogenates were measured using ELISA kits.Results:(1)MSCs were successfully transduced with EP2 plasmid as evidenced by the results of Western blotting and PCR(2)Compared with the GFP-MSCs and the control MSCs,the EP2-MSCs migrated significantly more to PGE2 in vitro after 6 h;*P<0.05.(3)Near infrared imaging and immunofluorescence both shown that administration of MSC-EP2 significantly enhance MSC homing to injured lung tissue compared to that of MSC-GFP group;*P<0.05.(4)our in vivo experimental results suggested MSC-EP2 also further reduced LPS-induced pulmonaryvascular permeability as reflected by reducing levels of Evans blue in lung parenchymal homogenates compared to that of MSC-GFP group;*P<0.01.(5)Compared with the MSC-GFP group,the total severity scores of lung injury were significantly reduced in the MSC-EP2 group both at 24 h and 72 h;n=6,*P<0.05.(6)The MSC-EP2 treatment decreased the IL-1β and TNF-a level to a greater extent than did the MSC-GFP treatment at both 24 and 72 hours.Similarly,the MSC-EP2 treatment increased the expression of IL-10 to a greater extent than did the MSC-GFP treatment at both 24 hours and 72 hours.Conclusion:In conclusion,our results suggested that treatment with EP2-transduced MSCs significantly enhances MSC homing to injured lung tissue and leads to additional improvements in lung inflammation and permeability.In addition,our in vivo experimental results suggested that the administration of MSCs overexpressing the EP2 gene can greatly improve the histopathology of lung injury caused by LPS challenge.These results suggest that a combination of MSC and EP2 gene therapy could markedly facilitate MSC homing to sites of inflammation and represent a potential novel strategy for MSC therapy in inflammatory diseases.Part Two:PGE2/EP2 promotes the migration of Mesenchymal stem cells through the activation of FAK and ERK1/2 pathwayObjective:To ascertain whether EP2 prostanoid receptor promotes MSCs migration through activation of FAK and ERK1/2 pathways.Methods:(1)MSCs from passage 4-6 were transduced with recombinant lentiviral vectors.Transduction efficiency was evaluated by detecting GFP expression using a fluorescent microscope and through flow cytometry.MSC EP2 expression was assessed using Western blotting and PCR.(2)MSCs migratory ability was assessed by a 24-well transwell system inserting a 8 um pore size filter membrane.600 μl serum free medium in the presence or absence of PGE2 was added to the bottom chamber.(3)Migration was also evaluated by a wound healing migration assay.Subsequently,MSCs were incubated at 37℃ for 12 h in serum free medium with or without PGE2 in the presence or absence of PF573228 or PD98059.(4)The level of FAK and ERK1/2 was measured by Western blot.Each experiment was performed 3 separate times.Results:(1)To investigate the involvement of EP2 receptor in PGE2-induced MSCs migration.Transwell assay revealed that the number of MSCs-EP2 traversing the membrane was significant more than the control when stimulated with PGE2.(2)Immunoblotting revealed that the expression level of FAK is much higher in MSCs-EP2 treated with PGE2 compared to the MSCs stimulated with PGE2,indicating FAK signaling contributes to PGE2-mediated migration of MSCs.(3)Immunoblotting revealed that the expression level of ERK1/2 is much higher in MSCs-EP2 treated with PGE2 compared to the MSCs stimulated with PGE2,suggesting ERK1/2 pathways was involved in PGE2-induced MSCs migration.Conclusion:Our findings reveal EP2-mediated FAK and ERK1/2 activation was essential for MSCs migration induced by PGE2,indicating that activation of EP2 receptor and FAK/ERK pathways may be a promising strategy to accelerate homing efficiency of MSCs,which in turn enhances therapeutic potential of MSCs transplantation.