?-catenin Overexpression Mesenchymal Stem Cells Improve Seawater Inhalation Induced Acute Lung Injury

Background:Drowning accidents happen easily,and according to global statistics,the total number of resulting deaths reaches 140,000 annually.When seawater enters the lungs,destruction of the alveolar-capillary membrane barrier occurs,a physiological hallmark of acute respiratory distress syndrome(ARDS),thus releasing floods of proteinaceous exudates into alveolar spaces.This may impair gas exchange,leading to noncardiogenic pulmonary edema and ultimately,respiratory failure.At present,there is no effective treatment strategy for ARDS except for conservative symptomatic supportive treatment.However,recent studies have shown that the transplantation of mesenchymal stem cells(MSCs)could substantially decrease the mortality rate of ARDS.MSCs,with their properties of multipotency,have been reported to be able to differentiate into alveolar epithelial cells,promote re-epithelialization,alleviate inflammation,improve pathological impairment,and even reduce mortality in ARDS models.However,owing to the low engraftment and differentiation rates of MSCs in the lung tissue of ARDS models,the therapeutic effects remain limited.Therefore,clarifying the mechanisms that underlie MSCs function in epithelial repair may lead to improvement of cellular retention in injured lung tissue,differentiation of MSCs into alveolar epithelialcells,and consequently,MSCs-mediated therapeutic effects in ARDS.Several recent studies have demonstrated that Wnts and their downstream canonical signaling molecules have critical effects on both the self-renewal and differentiation of MSCs.A previous study revealed that activation of the canonical Wnt/?-catenin pathway promotes differentiation of mouse bone marrow-derived MSCs into type II alveolar epithelial cells,conferring resistance to oxidative stress,and promoting migration to injured lung tissue in vitro.However,the role of the Wnt/?-catenin pathway in the fate and therapeutic effect of MSCs in ARDS remains unexplored in vivo.Here,the more complicated environment and regulatory mechanisms differ from the specific and limited cultural conditions of in vitro differentiation,and may consequently affect the MSCs.Another study constructed a long-term,stable MSCs line modified with activated?-catenin via lentiviral vectors.The authors confirmed the ability to activate the Wnt/?-catenin pathway,which could regulate proliferation,migration,and differentiation of the MSCs,thus proving suitability of the cell line for in vivo investigations.Objective:The aim of our current study was to identify MSCs and build ?-catenin overexpression MSCs lines,and confirm the effects of ?-catenin overexpression on the repair of injured alveolar epithelia and its overall therapeutic effect in SWI-ALI in rats.Methods:Part ?MSCs isolation,culture,and identification: The bone marrows of 4-week-old,male Sprague Dawley rats were used to obtain MSCs.The cells were cultured in L-DMEM medium,and inpassaged by 0.02% EDTA + 0.02% trypsin digestion.MSCs were identified via flow cytometry,multi-directional differentiation capacity,and morphology.MSCs transfection: The EF1?-MCS-3FLAG-CMV-EGFP-T2A-Puromycin(over-expressing Ctnnb1)lentivector was constructed and packaged.MSCs transduced overexpression of the target genes,induced from the lentiviral vectors.The MSCs were cultured in normal culture medium for 10 passages after transduction to assay their long-term transfection efficiency.This was observed by fluorescence microscopy,westernblotting and quantitative real-time PCR.Part ?The rats were randomly divided into four groups: the normal control;the seawater(SW)+ PBS group;the SW + MSCs control group;and SW + MSCs-Ctnnb1 group.Samples were collected from each rat for hematoxylin and eosin staining.Lung edema was evaluated using the ratio of lung wet weight to dry weight(W/D).Cytokine and protein measurements in BALF were measured via murine cytokine-specific ELISA kits and BCA Protein Assay Kit.Occludin protein was detected via Western immunoblot analysis.NIR815-labeled cells were directly instilled into the trachea of the SW + MSCs control rats and the SW + MSCs-Ctnnb1 rats.Ex vivo lungs from three subjects per group were imaged at two time points(12 h and 48 h post-instillation)using a Maestro In-Vivo Optical Imaging system.Results:Part?1.Identification of rats MSCs: Spindle cells comprised the main colonies,and were reminiscent of a whirlpool,all with the same directionality.Each passaging was performed after 4 or 5 days of growth.Cell morphology and growth rate did not significantly differ after 10 generations of stable and continuous passage.The results of flow cytometry revealed a positivity rate of CD90 expression of 99.3%,and of 96.1% for CD29.Thus,the positivity rate of CD45 was 0.087%,and that of CD34 was 0.145% in fourth generation rat MSCs.The MSCs were severally added into the adipocytes-inducing agent and osteoblast-inducing agent.The oil red O staining and Alizarin red staining was positive.2.Lentiviral vector transduction efficiency in MSCs: The efficiency of the lentiviral vector transduction of MSCs-Ctnnb1(overexpression of Ctnnb1)after 10 passages in MSCs was detected via fluorescence microscopy.The analysis revealed transduction efficiencies above 90%.Ctnnb1 m RNA expression in MSCs was verified via quantitative real-time PCR.The results revealed that Ctnnb1 mRNA expression was significantly higher in the MSCs-Ctnnb1 cells compared to that in the MSCs-GFP cells(P<0.001,n=9).Similar results for ?-catenin protein expression were also obtained via western blotting.These results suggest that the lentivirus-mediated transduction was efficient and stable.Part?1.Histopathological examination: Increased thickening of the alveolar wall,alveolar and interstitial inflammatory cell infiltration,hemorrhaging,alveolar exudates,and edema were observed in the lung tissue of rats after seawater-induced lung injury.However,these histopathological characteristics were alleviated at 4 h in the SW + MSCs control and SW+ MSCs-Ctnnb1 groups compared to that of the SW+PBS group(P<0.05,n=9).The effect was higher in the SW + MSCs-Ctnnb1 group than in the SW + MSCs control group(P<0.05,n=9).2.Lung epithelial permeability evaluation: The W/D and total protein concentrations was significantly reduced in the SW + MSCs-Ctnnb1 group and SW + MSCs control group compared to that of the SW + PBS group at 4 h(P<0.05,n=9).Total protein levels was more prominent in the SW + MSCs-Ctnnb1 group than in the SW + MSCs control group(P<0.05,n=9).Additionally,Occludin protein upregulation in the SW + MSCs control and SW + MSCs-Ctnnb1 groups compared to that of the SW + PBS group(P<0.05,n=9).The increase observed in the SW + MSCs-Ctnnb1 group was more significant than that in the SW + MSCs control group(P<0.05,n=9).3.Lung inflammation assessment: The levels of all three cytokines were significantly increased in the SW + PBS group compared to that in the normal control group at 4 h after seawater exposure(P<0.05,n=9).IL-1? and IL-6 levels were reduced in the SW + MSCs control and SW + MSCs-Ctnnb1 groups compared to that in the SW + PBS group(P<0.05,n=9),while IL-10 level was increased(P<0.05,n=9).Similarly,the decrease in IL-1?levels and increase in IL-10 levels observed in the SW + MSCs-Ctnnb1 group were more prominent than the changes observed in the SW + MSCs control group(P<0.05,n=9).4.Stem cells reservations: Color-coded fluorescence images indicated that the signals in the SW + MSCs-Ctnnb1 group were stronger compared to those in the SW + MSCs control group towards the end of 12 h and 48 h of exposure to seawater.Conclusion:Activated ?-catenin was used for improving stem cell proliferation,colonization,and differentiation in seawater inhalation induced acute lung injury via lentiviral vectors.It increased MSCs retention in the lung,further improved the lung epithelial permeability,and further attenuated acute pulmonary inflammation compared to that of control MSCs,thus contributing to an improved therapeutic effect of MSCs in ARDS.