Experimental Investigation Of Biological Effects Of Endotoxin-induced Acute Lung Injury By Bone Marrow-derived Mesenchymal Stem Cells In Rat

Objectives Because the most common cause of acute lung injury(ALI)is sepsis, and current specific treatment strategies for ALI are lacking.Our objective was to study the engraftment of bone marrow-derived mesenchymal stem cells(MSC)in lung tissue and their biological effects in the rat model of lipopolysaccharide(LPS) induced ALI.That will provide experimental basis for using stem cell in the therapy of ALI.Methods and Results1.Culture and identification of MSCMSC were isolated from adult male rat bone marrow and purified by means of adherent culture.Flow cytometry analysis demonstrated that phenotypes of MSC were CD34^-/CD45^-/CD90⁺.Differentiation assays demonstrated that MSC retained their ability to form osteblasts and adipocytes at passages 4,showing a multipotent plasticity.The cells had a spindled,fibroblast appearance in culture that is consistent with MSC.2.Engraftment of male MSC in female rat lung tissueThe engraftment of male MSC in female rat lung tissue was monitored by fluorescence in situ hybridization(FISH)using Y specific gene probes for rat. Lungs from animals given LPS that did not receive MSC showed minimal background staining.At the 24 h time point,lungs from animals that received MSC but not LPS contained few positive cells,but lungs from animals receiving both LPS and MSC contained large numbers of these cells.By week 3 after LPS, the positive cells could be detected in lungs from rat that received LPS and MSC. However,overall engraftment levels were low(＜10%).3.Biological effect of LPS-induced ALl intravenously by MSC in rat65 female Wistar rats were randomly assigned to one of three experimental groups that received 1)saline solution(NS)plus MSC,2)LPS plus NS,3)LPS plus MSC.MSC(1×10⁶)or an equal volume of NS was delivered through the right tail vein of rats 2 h after 8 mg/kg LPS or an equal volume of NS administration through the left tail vein of rats.Subgroups of animals were killed at 6 h,24 h,4 d,lw and 3 weeks after MSC.At the 6 h time point,PaO₂ in LPS plus NS-group was 67.1±11.6 mmHg,significantly decreased compared with control(P＜0.01).At 4 d time point,animals treated with MSC had a significant improvement in the degree ofneutrophil infiltration as assessed by the tung injury score(P＜0.05).Neutrophil counts in BALF also were lower in the LPS plus MSC-group vs the LPS plus NS-group(P＜0.05).Myeloperoxidase(MPO) activity in lung tissue also were similarly decreased between the LPS plus MSC-group and the LPS plus NS-group(P＜0.05).Both at 24 h and 4 d,the levels of TNF-αin serum were significantly lower in the LPS plus MSC-group compared with LPS plus NS-group(P＜0.05).The levels of IL-1βin serum also were significantly lower at 4 d(P＜0.05).The levels of IL-10 in serum were not significantly increased at all time points.Kaplan-Meier survival assay showed that, although did not reach significance,the LPS plus MSC-group appeared predominance in improving survival rates of LPS-induced lung injury(p=0.22), compared with the LPS plus NS-group.The content of hydroxyproline(HYP)in lung tissue at 3w also were similarly decreased between the LPS plus MSC-group and the LPS plus NS-group(p=0.09).4.Biological effect of LPS-induced acute inflammatory lung injury intraperitoneaily by MSC in rat60 female Wistar rats were randomly assigned to one of three experimental groups that received 1)NS alone,2)LPS plus NS,3)LPS plus MSC.Rats were inoculated intraperitoneally with 2 mg/kg LPS or an equal volume ofNS.MSC (1×10⁶)were infused 1h after LPS administration through a tail vein.Subgroups of animals were killed at 6,24,48 h and 2 weeks after MSC.Administration of NS alone caused no detectable alterations in lung structure.Lungs from animals receiving LPS but not MSC showed vascular congestion and a general increase in cellularity predominantly due to the presence of neutrophils.These changes were present by 6 h and were more pronounced at 24 h time point when thickening of the alveolar septae was especially apparent.By 48 h,there were residual inflammatory cells and some alveolar wall thickening,but these changes were resolving.Lungs from animals receiving LPS and MSC were devoid of these changes and were histologically similar to lungs from control animals that did not receive LPS.The number of neutrophils in histological sections of lungs from animals receiving LPS and MSC were significantly decreased compared with the animals receiving LPS but not MSC at both 24 h and 48 h after MSC.The wet-dry weight ratios of lung and protein levels in BALF also were significantly decreased between the LPS plus MSC-group and the LPS plus NS-group(P＜0.05).LPS caused a consistent acute systemic inflammatory response reflected in increased serum concentrations of the proinflammatory mediators,IL-1β,MIP-1α,TNF-αand IL-10.This response peaked at 6 h and largely subsided by 24 h. Administration of MSC moderated the increase in each of these proinflammatory mediators(P＜0.01).LPS also caused an increase in serum concentrations of the anti-inflammatory cytokine IL-10,effects were not altered by MSC administration (P=0.21).5.Interactions of MSC and LPS-stimulated lung cells ex vivoFirstly,alveolar macrophages(AM),lung capillary endothelial cells(EC)and lung cells(LC)were respectively isolated from adult rat lung.Partâ… Coculture of LPS-stirnulated alveolar macrophages and MSCTo determine whether MSC are able to regulate production ofproinflammatory cytokines by LPS-stimulated AM by a cell-ceU contact-dependent or -independent mechanism,AM were cocultured with MSC in either a standard 24-well or in a Transwell(0.4-um pore size),and then stimulated with LPS.Control wells were prepared with only AM stimulated with LPS,and MSC stimulated with LPS alone. The cell culture supernatants were then collected after the 4-h incubation to assay for the levels of IL-1β,MIP-1α,TNF-αand IL-10 by ELISA.In the presence of MSC,concentrations of IL-1β,MIP-1αand TNF-αwere consistently lower than in the absence of MSC.In addition,the MSC effect on MIP-1αwas much greater in experiments where MSC could contact AM wells than in experiments in which contact between AM and MSC was not possible.In addition,IL-10 concentrations were only significantly increased in experiments where MSC could contact AM wells.Partâ…¡Migration of MSC toward the lung cells ex vivoTo determine whether MSC are able to migrate toward the lung cells ex vivo, additional wells were prepared in which a Transwell(3-um pore size)insert was used with AM,AM and EC,LC respectively cultured in the bottom compartment and MSC in the upper compartment.The cells in the bottom compartment were either treated with LPS or not.After 3 days in coculture,we examined the filters and counted numbers of cells as a measure of MSC migration toward the lung cells.Significantly higher numbers of cells were migrated by cells treated with LPS compared with control cells(P＜0.01).The migrating cells did not appear to change their morphology.In addition,we examined the migrating cells by means of immunohistochemistry withâ…§factor associated antigen.The results were negative.Conclusions 1.Isolation,purification and culture of MSC derived from rat bone marrow were finished successfully in vitro.2.MSC engraftment in injuried lung reveale lung homing,even though overall engraftment levels were low. Administration MSC by intravenous infusion have the ability to modulate both systemic and local acute inflammatory response,induced by LPS in vivo,attenuate the severity of lung injury.This therapeutic effect was related to the decrease of proinflammatory cytokines and the increase of anti-inflammatory cytokine,which would alter the milieu from pro- to anti-inflammatory.In addition,studies of interactions between lung cells and MSC in coculture define a bilateral conversation in which lung cells stimulate MSC to migrate,and MSC promote an anti-inflammatory cytokine milieu by producing soluble factors and by mechanisms requiring physical contact of MSC and lung cells,suggesting benefits to inflammation resolution and tissue repair.