| Part One:Ang Ⅱ-AT2R Increases Mesenchymal Stem Cell Migration by signaling through the FAK and RhoA/Cdc42 Pathways in vitroObjective:Mesenchymal stem cells(MSCs)migrate via the bloodstream to sites of injury and are possibly attracted by inflammatory factors.As a pro-inflammatory mediator,Angiotensin Ⅱ(Ang Ⅱ)reportedly enhances the migration of various cell types by signaling via the angiotensin Ⅱ receptor in vitro.However,few studies have focused on the effects of Ang Ⅱ on MSC migration and the underlying mechanisms.Methods:Human bone marrow MSC migration was measured using wound healing and Boyden chamber migration assays after treatments with different concentrations of Ang Ⅱ,an AT1R antagonist(Losartan)and/or an AT2R antagonist(PD-123319).To exclude the effect of proliferation on MSC migration,we measured MSC proliferation after stimulation with the same concentration of Ang Ⅱ.Additionally,we employed the focal adhesion kinase(FAK)inhibitor PF-573228,RhoA inhibitor C3 transferase,Rac1 inhibitor NSC23766 or Cdc42 inhibitor ML141 to investigate the role of cell adhesion proteins and the Rho-GTPase protein family(RhoA,Rac1 and Cdc42)in Ang Ⅱ-mediated MSC migration.Cell adhesion proteins(FAKdetected by western blot.The Rho-GTPase family protein activities were assessed by,Talin and Vinculin)were G-LISA and F-actin levels,which reflect actin cytoskeletal organization,were detected by using immunofluorescence.Results:Human bone marrow MSCs constitutively expressed AT1R and AT2R.Additionally,Ang Ⅱ increased MSC migration in an AT2R-dependent manner.Notably,Ang Ⅱ-enhanced migration was not mediated by Ang Ⅱ-mediated cell proliferation.Interestingly,Ang Ⅱ-enhanced migration was mediated by FAK activation,which was critical for the formation of focal contacts,as evidenced by increased Talin and Vinculin expression.Moreover,RhoA and Cdc42 were activated by FAK to increase cytoskeletal organization,thus promoting cell contraction.Furthermore,FAK,Talin and Vinculin activation and F-actin reorganization in response to Ang Ⅱ were prevented by PD-123319 but not Losartan,indicating that FAK activation and F-actin reorganization were downstream of AT2R.Conclusions:These data indicate that Ang Ⅱ-AT2R regulates human bone marrow MSC migration by signaling through the FAK and RhoA/Cdc42 pathways.This study provides insights into the mechanisms by which MSCs home to injury sites and will enable the rational design of targeted therapies to improve MSC engraftment.Part Two:Genetic Modification of Mesenchymal Stem Cells Overexpressing Angiotensin Ⅱ Type 2 Receptor Increase Lung Engraftment in LPS-induced Acute Lung Injury MiceObjective:The objective of our study was to determine whether overexpression of AT2R in MSCs augments their cell migration and engraftment after systemic injection in ALI mice.Methods:A human AT2R expressing lentiviral vector was constructed and introduced into human bone marrow MSCs.We also down-regulated AT2R mRNA expression,using a lentivirus vector carrying AT2R shRNA to transfect MSCs.The effect of AT2R expression on migration of MSCs was examined in vitro.A mouse model of lipopolysaccharide induce ALI was used to investigate the engraftment of AT2R overexpression MSCs and the therapeutic potential in vivo.Results:(1)Lentivirus-mediated high expression and interference with AT2R MSCs,transfection efficiency was up to 90%by fluorescence microscopy.Compared with untransfected MSCs,the AT2R mRNA level of AT2R overexpressed MSC(MSC-AT2R)was about 3 times that of untransfected cells(P<0.05),while AT2R-interfering MSC(MSC-ShAT2R)showed less than 80%that of untransfected cells(P<0.05).The effect of Ang Ⅱ on the migration of human bone marrow MSCs in MSC-AT2R group was significantly higher than that in control group(P<0.05).MSC-ShAT2R inhibited the migration induced by Ang Ⅱ(P<0.05)compared with MSC-Shcontrol group.(3)Compared with MSC-GFP,the fluorescence signal of lung tissue in MSC-AT2R group was significantly increased at 24h and 72h after transplantation.Compared with MSC-Shcontrol,MSC-ShAT2R group(P<0.05),and the expression of green fluorescent protein and Alu sequence in the lung tissue of MSC-AT2R group was significantly increased(P<0.05),while the fluorescence intensity of the lung tissue was significantly decreased in the lung tissue fluorescence microscopy and the Alu level.These date indicated that the MSC retention of lung tissue in MSC-AT2R group were significantly increased after transplantation,while the MSC of retention lung tissue in MSC-ShAT2R group were significantly reduced.(P<0.05).The lung wet weight/body weight ratio was reduced and the permeability of pulmonary microvascular endothelium was decreased(P<0.05).While the MSC-ShAT2R group reduced the lung wet weight/body weight ratio and decreased the permeability of pulmonary microvascular endothelium(P<0.05).(5)MSC-AT2R treatment can significantly reduce the pathological damage of lung tissue,reduce lung injury score(P<0.05),reduce the total number of cells and neutrophils in BALF(P<0.05),reduce IL-1β,IL-6 levels(P<0.05),and increase IL-10 level(P<0.05).In contrast,MSC-ShAT2R transplantation could aggravate lung injury in LPS-induced lung injury and increase lung injury score(P<0.05),increase the number of cells and neutrophils in BALF(P<0.05),IL-1β,IL-6 levels(P<0.05),and decrease the level of IL-10(P<0.05).Conclusions:Our results demonstrate that overexpression of AT2R enhance the migration and engraftment of MSCs and may provide a new therapeutic strategy for the injured lung. |
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