Role Of Wnt/?-Catenin-p130/E2F4 On The Differentiation Of Mesenchymal Stem Cells Into Type ? Alveolar Epithelial Cells By Regulating Cell Cycle In Ards

Background Diffuse alveolar epithelial damage is the basic pathological change of acute respiratory distress syndrome(ARDS),and effective repair of damaged alveolar epithelial cells is important for the treatment of ARDS.It has been demonstrated that the canonical Wnt pathway can improve the differentiation of exogenous mesenchymal stem cells(MSC)into alveolar epithelial cells in the lungs of ARDS mice,but the specific mechanism is unclear.Cell cycle plays a key role in MSC differentiation p130/E2F4 is an important pathway regulating cell cycle,while the canonical Wnt pathway could regulate p130/E2F4.It is speculated that canonical Wnt signaling pathway could affect the differentiation of MSC into type II epithelial cells by regulating downstream p130/E2F4,and the mechanism of which may be related to the changes of cell cycle on MSC.The purpose of this study was to investigate the mechanism of Wnt/?-Catenin-p130/E2F4 regulating cell cycle on the differentiation of MSC into type II alveolar epithelial cells and the role of MSC overexpressing p130 or E2F4 on repairing lung injury in ARDS micePart I Stable overexpression of p130/E2F4 affects the multipotential differentiation abilities of bone-marrow-derived mesenchymal stem cellsObjective To evaluate the effects of p130/E2F4 overexpression on the multidifferentiation of MSC and its possible mechanismMethods Mouse MSCs(mMSCs)with p130 or E2F4 overexpression were constructed using lentiviral vectors.The transfection efficiency of mMSCs after was identified using fluorescence microscopy,and the percentage of GFP positive cells was determined by flow cytometry analysis(FCM).The mRNA and protein levels in MSC-p130(overexpressing p130)and MSC-E2F4(overexpressing E2F4)were detected by qRT-PCR and Western blot,respectively.For cell cycle analysis,the cells were stained with propidium iodide and detected by FCM.The effects of p130/E2F4 on the multipotential differentiation abilities of mMSCs were evaluated by osteoblastic,adipogenesis and chondrogenic differentiation kit.The expression of the OSX,Runx2,PPAR-y,C/EBPa,Sox9 and Col2alwere measured by qRT-PCR and Western blot respectively.The proliferation of mMSCs was analyzed by Cell Counting Kit-8 and the migration abilities were determined by scratch assay and Transwell assayResults Our data showed that the transduction efficiencies of p130 or E2F4 mediated by lentiviral vectors were 80.3-84.4%.p130 and E2F4 mRNA expression was significantly higher in MSC-p130 and MSC-E2F4 cells than in MSC-NC cells.Similar results were also observed for p130 and E2F4 protein expression.After osteogenic or adipogenic differentiation,the G1 phase was significantly delayed in the MSC-p130 and MSC-E2F4 groups compared to that in the MSC-NC group.However,the G1 phase in the MSC-p130 and MSC-E2F4 groups did the opposite after chondrogenic differentiation.Moreover,overexpressing p130 or E2F4 significantly improved osteogenic differentiation while inhibiting adipogenic and chondrogenic differentiation of mMSCs.Moreover,overexpressing p130 or E2F4 significantly improved migration but not proliferation of mMSCsConclusion Our data suggest that cell cycle regulation could be involved in p130/E2F4-mediated changes in the multipotential abilities of bone-marrow-derived mMSCsPart ? Role of Wnt/?-Catenin-p130/E2F4 on the differentiation of mesenchymal stem cells into type ? alveolar epithelial cellsObjective To evaluate the role of Wnt/?-Catenin-p130/E2F4 on regulating the differentiation of mMSCs into type ? alveolar epithelial cells(AT ?)and its possible mechanlismMethods mMSCs with p130 or E2F4 overexpression were constructed using lentiviral vectors as previously described in our work.And a modified co-culture system with murine lung epithelial-12 cells and small airway growth media for 7-14d was used to efficiently drive mMSCs differentiation into AT ?.The differentiation eff-iciency was detected for surfactant protein C(SP-C)by immunofluorescence and Western blot assay as well as typical lamellar body-like structures by transmission electron microscopy.To detect the relationship between canonical Wit pathway and p130/E2F4,4 mmol/L LiCl or 200 ng/mL DKK-1 was also added in the co-culture system as previously described in our work.And the protein level of p-p130,p130 and E2F4 were also detected by Western blot assay.Cell cycle of mMSCs after differentiation was evaluate by flow cytometryResults The SP-C protein expression was significantly higher in MSC-p130(overexpression of p130)and MSC-E2F4(overexpression of E2F4)groups after induced differentiation into AT ?(p<0.05).Similar results for SP-C protein expression and lamellar body-like structures were also observed in immunofluorescence analysis and electron microscopy.The levels of p-p130,p130 and E2F4 were also increased in mMSCs when LiCl was added to the co-culture system to activate Wnt/?-catenin signaling pathway(p<0.05),while depressed to some extent by inhibiting the pathway with addition of DKK-1(p<0.05).As for cell cycle detection,G1+S phases were significantly increased when activating Wnt pathway(p<0.0001)while decreased when inhibiting it(p<0.0001).However,G1 phase was significantly delayed after differentiation of mMSCs overexpressing p130 or E2F4(p<0.0001)Conclusion Canonical Wnt signaling pathway can affect the differentiation of MSCs into AT ? by regulating downstream p130/E2F4,and the mechanism of which could be related to G1 phase extension in cell cycle of MSCsPart III Overexpressing p130/E2F4 in mesenchymal stem cells facilitates the repair of injured alveolar epithelial cells in LPS-induced ARDS miceObjective To evaluate the effects of overexpressing p130 or E2F4 in mMSCs on improving re-epithelization in lipopolysaccharide(LPS)-induced ARDS miceMethods mMSCs stably transfected with p130 and E2F4 were transplanted intratracheally into LPS-induced ARDS mice.After 7 and 14 d,the mice were sacrificed,and the histopathology of the lungs was assessed by hematoxylin-eosin staining and lung injury scoring.Mortality of the mice was assessey until 14d after LPS challenge.Homing and differentiation of mMSCs were analysed by labelling and tracking mMSCs with NIR815 dye and immunofluorescent staining.Surfactant proteins A and C and occludin in the lungs were assessed by Western blot.Permeability was evaluated by the ration between lung wet weight and body weight and by analysing the protein concentration of bronchoalveolar lavage fluid(BALF)using enzyme-linked immunosorbent assay.Alveolar fluid clearance was assessed by absorbance measurements of BALE.Lung fibrosis was assessed by Masson's trichrome staining and Ashcroft scoringResults The engraftment of mMSCs overexpressing p130 or E2F4 led to attenuated histopathological impairment of the lung tissue,and the lung injury scores of the LPS+MSC-p130 and LPS+MSC-E2F4 groups were also decreased(p<0.05).Although there was no significant difference of mortality at 14d in mice of LPS+MSC-p130 and LPS+MSC-E2F4 groups(p>0.05),a downward trend could also be seen in these two groups compared with LPS+MSC-NC group.Overexpression of p130 or E2F4 also increased the retention of MSCs in the lung(p<0.05),increased differentiation into type? alveolar epithelial cells(p<0.05),and improved alveolar epithelial permeability(p<0.05).Additionally,mMSCs overexpressing p130 or E2F4 inhibited lung fibrosis according to the deposition of collagen and the fibrosis score in the lungs(p<0.05)Conclusion Overexpressing p130 or E2F4 in mMSCs could further improve the injured structure and function of epithelial cells in the lungs of ARDS mice as a result of improved differentiation of mMSCs into epithelial cells.