The Role And Mechanism Of EPO Gene Modified Bone Marrow Mesenchymal Stem Cells In Airway Remodeling In Asthma

OBJECTIVE:There was no effective measures can be obtained at present to reverse or prevent airway remodeling.We investigated the therapeutic effect of Erythropoietin(EPO)gene modified mesenchymal stem cells(MSCs)on asthmatic airway remodeling and the possible underlied molecular mechanisms.METHODS:EPO gene was transfected into MSCs via lentivirus vector.The transfected cells(EPO-MSCs)were identified by the EPO secreting function was detected by PCR and western blot.MSCs or EPO-MSCs were administrated to albumin(OVA)-induced chronic asthmatic mouse model via tail veins.The asthmatic phenotype was analyzed.Number of cells in bronchoalveolar lavage fluid(BALF)was counted using a haemocytometer.Histological findings of airways were evaluated by microscopic examination.The concentrations of interleukin 4(IL-4),interleukin 5(IL-5)and interleukin13(IL-13)in lung homogenate were determined by ELISA.The activation state of transforming growth factor-?1(TGF-?1),Transforming growth factor beta-activated kinase 1(TAK1)and p38 Mitogen Activated Protein Kinase(p38MAPK)signaling was detected by Real-Time PCR and Western blotting.RESULTS:(1)Construction of the EPO-MSCs.EPO gene-modified MSCs were successfully constructed.And there was no changes in the function of EPO-MSCs occurred as a result of EPO gene-modification.After established EPO-MSCs,expression of the mRNA and Protein of EPO were increased significantly in EPO-MSCs than in MSCs(p<0.05).(2)Significantly reduced airway inflammation in EPO-MSCs administrated mice.We found that both MSCs and EPO-MSCs significantly reduced the amount of airway cellular infiltrates after challenge with OVA(MSCs,P<0.01;EPO-MSCs,P<0.01).Differential cell counting revealed a profound reduction of airway eosinophilia in both Asthma+MSCs and Asthma+EPO-MSCs groups(MSCs,P<0.01;EPO-MSCs,P<0.01).Notably,EPO-MSCs had a more potently suppressive effect on local airway inflammation than MSCs(total cell number,P<0.05;number of eosinophils,P<0.05).(3)Reduced tissue pathology in EPO-MSCs administrated mice.In EPO-MSCs group,yellow fluorescence was found in lung slices by fluorescence microscope,indicating that EPO-MSCs can migrate to lung tissue and differentiate into lung tissue cells.In asthmatic control mice,airway challenge leads to a dense peribronchiolar inflammatory infiltrate of lymphocytes,and to the production of mononuclear and polymorphonuclear cells with epithelial shedding and extended columnal cells.In Asthma+MSCs and Asthma+EPO-MSCs groups,however,tissue inflammation was greatly reduced,with significantly less peribronchial and perivascular cellular infiltration and mucin staining.Thus,both MSCs and EPO-MSCs can protect mice against a range of allergic airway inflammatory pathologies,including the infiltration of inflammatory cells.Furthermore,EPO-MSCs had a more potent inhibitory effect than MSCs.The changes of airway remodeling index were performed with pathological staining and computerized image analysis system.In asthma group,the numbers of goblet cells as well as the thicknesses of smooth muscle layer,collagen density,percentage of PCNA~+mesenchymal cells and vWF~+vessels were significantly higher than control group(P<0.01).In Asthma+MSCs group,the numbers of goblet cells,the thicknesses of smooth muscle layer,collagen density,percentage of PCNA~+mesenchymal cells and vWF~+vessels were significantly lower than the asthma group(collagen density and vWF~+vessels,P<0.05;others,P<0.01).Moreover,in Asthma+EPO-MSCs group,significant improvement occurred in all histological parameters compared with Asthma+MSCs group(collagen density,P<0.01;others,P<0.05).(4)Reduced cytokine in EPO-MSCs administrated mice.We measured local cytokine IL-4,IL-5 and IL-13 levels in lung homogenate.After airway challenge the levels of IL-4,IL-5 and IL-13 were elevated in the lung homogenate of asthmatic mice.However,IL-5was significantly diminished by MSCs and EPO-MSCs administrated(MSCs,P<0.01;EPO-MSCs,P<0.01).Reductions in the key agents in the mobilization and extravasation of eosinophils provided a mechanistic explanation for the dramatically reduced airway eosinophilia in MSCs and EPO-MSCs administrated mice.Similarly,the elevation of the IL-4,IL-13 level was also reversed by MSCs and EPO-MSCs administrated(MSCs,P<0.05;EPO-MSCs,P<0.01),which may explain the down-regulation of goblet cell number in MSCs and EPO-MSCs administrated mice.Notably,EPO-MSCs had a more potent inhibitory effect on the level of IL-4,IL-5 and IL-13 than MSCs(IL-4,IL-5,P<0.05;IL-13,P<0.01).(5)Reduce the activity of TGF-?1-TAK1-p38MAPK signal pathway.To investigate the possible molecular mechanisms,we further studied mRNA expression of TGF-?1,TAK1 and p38MAPK.Twenty-four hours after the final challenge,EPO-MSCs treated animals revealed a lower expression level of TGF-?1,TAK1 and p38MAPK mRNA in lung tissue than asthma group.TGF-?1 signal pathway-related proteins(TGF-?1,TAK1,p-TAK1,p38MAPK and p-p38MAPK)were also detected in lung tissue in each group.EPO-MSCs treatment group showed a reduction in TGF-?1 and the transcription regulators(TAK1,p-TAK1,p38MAPK and p-p38MAPK)(P<0.05 or P<0.01).According to the data,the ratios of p-p38/p38 and p-TAK1/TAK1 have the same trend.EPO-MSCs treated animals revealed a lower ratio of p-p38/p38 and p-TAK1/TAK1(P<0.05 or P<0.01).CONCLUSION:Our results indicate that EPO gene modified MSCs may more efficiently attenuate asthmatic airway remodeling,which maybe related with the downregulation of TGF-?1-TAK1-p38MAPK pathway activity.