The Mechanism Of Bone Marrow Derived Mesenchymal Stem Cells Overexpressing Cav1F92A Gene Alleviating Pulmonary Vascular Remodeling In Pulmonary Arterial Hypertension Rats

BackgroundPulmonary arterial hypertension(PAH)is an incurable and progressive cardiovascular disease with an unfavourable prognosis.The primary pathological change is pulmonary vascular remodeling,which leads to vascular lumen narrowing,increasing pulmonary arterial pressure,eventually right heart failure and death.The phenotypic switching of small pulmonary arterial smooth muscle cells(PASMCs)is considered as an important factor in vascular remodeling.Phenotypic switching in PASMCs from contractile to synthetic phenotype contributes to cell proliferation and migration,resulting in concentric medial thickening of small arterioles,neovascularization of non-muscular capillary-like vessels,and the progression of PAH.Numerous studies have shown the contribution of inflammation and oxidative stress to the progression of PAH.Despite improvement in our understanding of PAH etiology and pathogenesis,no treatment options that inhibit the vascular remodeling are available.Therefore,early intervention for vascular remodeling may delay disease progression and improve survival and quality of life.Stem-cell based therapies have received attention by more researchers.Mesenchymal stem cells(MSCs)therapies attenuate or reverse the remodeling of vascular structures and repair damaged tissues,which move toward pre-clinical and clinical studies progressively.Several studies have shown that MSCs have the function of the anti-inflammatory and anti-oxidative properties.Therefore,MSCs therapy has been considered as a promising therapeutic strategy for PAH.However,the very first challenge encountered in MSCs transplantation is the low survival rate of transplanted cells due to detachment-induced apoptosis,called anoikis,which limits the application of MSCs.NO has been suggested to enhance the anoikis resistance,however,the effect of NO on anti-anoikis of MSCs is yet to be elucidated.Endothelial injury and dysfunction contributes to pulmonary vascular remodeling,which reduces NO bioavailability in PAH.NO is distributed broadly in organisms,exerting as an important signal molecule,neurotransmitter and regulatory factor.NO has an impact on the regulation of vasodilation,blood pressure,inhibiting proliferation and migration of PASMCs.It has suggested that phenylalanine 92(F92)in Caveolinl(Cavl)scaffolding domain is critical for eNOS binding and inhibition.Substitution of alanine for phenylalanine achieved Cav1F92A,which disrupted the inhibitor action of Cavl toward eNOS,improved NO production and did not modulate Cavl biochemical properties.Genetic modification of stem cells may improve stem cell function.Numbers of gene therapeutic approaches have been used such as lentiviral vectors(Lv)and it has also been demonstrated the safety profile of Lv in recent clinical trials.Thus,Lv-Cav1F92A modulated MSCs may be benefical to PAH treatment.ObjectiveIn this study,Lv-Cav1F92A was constructed,then used to transduce rat bone marrow derived MSCs(rBMSCs/Cav1F92A).We will explore following aspects:1.The effect of Cav1F92A gene on the anoikis in rBMSCs.2.The effect of rBMSCs/Cav1F92A on the phenotypic switching in monocrotalin(MCT)-treated rat PASMCs(rPASMCs)by co-culture method.3.The effect of rBMSCs/Cav1F92A on vascular remodeling in MCT-induced PAH rats by intravenous delivery of modulated rBMSCs.Methods1.Part ? The mechanism of Cav1F92A gene suppressing rBMSCs anoikis1).The rBMSCs were isolated by density gradient centrifugation using Ficoll,and identified by morphological observation,MSC surface antigen,and the osteogenic,adipocyte and chondrogenic differentiation;2).Negatvie,Cavl or Cav1F92A lentivirus vector were deliveried to rBMSCs using Lipofectamine2000 Transfection Reagent,respectively;3).NO production,cell adhesion and migration in the modulated rBMSCs were monitored;4).After inducing anoikis,we dected the cell viability,the expression of cell adhesion-related protein Integrins a5 and Integrins ?1(ITG?5?1),FAK/ERK signal,survivin and activated-caspase-3 and the changes of DNA ladder and TUNEL positive cell.2.Part ? The mechanism of rBMSCs/F92A inhibiting MCT-induced phenotypic switching of rPASMCs1).The production of inflammatory cytokines and anti-oxidant in rBMSCs were monitored using flow cytometry or ELISA,respectively;2).rPASCMs was isolated and cultured using adherent method;3).rPASMCs was treated with MCT for 24h,and then co-cultured with the modulated rBMSCs for 72h;4).The activated NF-?B,TNF-a and TGF-?1 were measured in rPASCMs;5).The migration and proliferation of rPASMCs were detected using transwell plate and EdU.The SMCs-special marker protein was also tested by western blot assay;6).NO level was monitored using DAF-FM DA.The expression of sGC and cGMP mRAN were measured using RT-PCR.3.Part ? The effect of rBMSCs/Cav1F92A on pulmonary vascular remodeling treatment in PAH rats1).Rats received subcutaneous injections of MCT(60 mg/kg)for the construction of the PAH model;2).Rats were randomly assigned to six groups:Control group(Normal rats),Model group(PAH rats treated with saline),Negative group(PAH rats treated with rBMSC/Negatvie),Cavl group(PAH rats treated with rBMSC/Cavl),Cav1F92A group(PAH rats treated with rBMSC/Cav1F92A),and Cav1F92A+L-NAME group(PAH rats treated with rBMSC/Cav1F92A+L-NAME).14 days after MCT injection,approximately 1 × 106 cells in saline were slowly injected into rats via the tail vein for cell transplantation;3).The fluorescence of freezing slice in the lung tissues was observed 24h and 2 weeks after cell treatment;4)Twenty eight days after MCT injection,changes in pulmonary haemodynamics,right ventricular hypertrophy index(RV/(LV+S))were evaluated;5).The media thickness,percentage of media thickness with outer diameter(WT%),the ratio of vessel lumen cross-section area/total arterial cross-section area(WA%),and the neointima hyperplasia(neintima/medial area)and the collagen fibers production were measured in the small pulmonary artery of lung sections that stained with H&E or Masson staining;6).Ultrastructure changes and autophagosome in lung tissues were observed by transmission electron microscopy;7).The production of Nrf2,iNOS,3-NT,NOX4 and ROS were dectected;8).The BH4 level in serum,and NO production,eNOS dimerization,the expression of Cav1,sGC,cGMP and PKG-1 were measured in lung tissue;9).Mstl and autophagy-related proteins and pathway were also monitored;Results1.Part ?1).The expression of Cav1F92A increased in rBMSCs/Cav1F92A after transduction;2).Cav1F92A enhanced NO production,cell adhesion and migration in rBMSCs;3).In model of inducing anoikis,Cav1F92A groups showed an increase of cell viability,activated ITGa5?1/FAK/ERK siganl,an upregulation expression of survivin protein,a downregulation expression of activated-caspae-3 protein and the change of DNA ladder and TUNEL positive cell rate;4).Treated with L-NAME,an eNOS inhibitor,reactivated anoikisi in rBMSCs/Cav1F92A groups.2.Part ?1).Cav1F92A significantly increased anti-inflammatory cytokines interleukin-4(IL-4)and IL-10 production in rBMSCs(p all<0.01),and similar results were obtained in antioxidant GPx,GSH and SOD production(p all<0.01),which were reversed by L-NAME;2).Positive rate of the cultured rPASCMs was more than 80%;3).Co-cultured with rBMSC/Cav1F92A,the expression of NF-?B,TNF-a and TGF-?1 mRNA were inhibited in MCT-treated rPASCMs;4).rBMSC/Cav1F92A improved NO level and reactivated NO pathway in MCT-treated rPASMCs(p both<0.01).Moreover,rBMSC/Cav1F92A suppressed cell migration and proliferation,down-regulated the expression of synthetic phenotype markers OPN,tropoelastin and up-regulated the expression of contractile phenotype markers expression SM22a,calponin(p all<0.05)in MCT-treated rPASMCs.However,these changes were all reversed by an endothelial nitric oxide synthase inhibitor called NG-nitroarginine methyl ester hydrochloride(L-NAME).3.Part ?1).The positive number of engrafted cells to the lung in Cav1F92A groups is more than those in other groups;2).Twenty eight days after MCT injection,the mean pulmonary artery pressure increased,as well as the pulmonary vascular remodeling were observed in PAH group;3).Administration of rBMSCs/Cav1F92A decreased the mean pulmonary artery pressure and right ventricular hypertrophy index(p<0.01),reduced media thicken,MT%and neointima hyperplasia,increased WA%(p all<0.01),as well as ameliorated the ultrastructure changes in PAH rats;4).rBMSCs/Cav1F92A inhibited iNOS,3-NT,NOX4 and ROS production in MCT-induced rats via activating Mst1/Nrf2 signal(p all<0.05);5).rBMSCs/Cav1F92A enhanced BH4 and NO production,prevented eNOS uncoupling,and increased Cavl,sGC,cGMP and PKG-1 expression(p all<0.05);6).We also found the inactivated autophagy in MCT-induced PAH rats treated with rBMSCs/Cav1F92A(p all<0.05).Conclusion1.Transduction of rBMSCs with Cav1F92A lentivirus could prevent cell anoikis via activating ITGc?5?1/FAK/ERK signal induced by improving NO production;2.Co-cultured with rBMSCs/Cav1F92A,the phenotypic switching from a contractile phenotype to a synthetic phenotype was suppressed in MCT-treated rPASMCs via mediating NO/NF?B signal and reactivating NO pathway;3.rBMSC/Cav-1F92A alleviated the vascular remodeling in MCT-induced PAH rats by reactivated eNOS/NO/sGC/cGMP/PKG-1 pathway and inhibiting oxidative stress and autophagy via upregualtiing Mstl expression.