The Role Of HGF/VEGF-expressing Characters Of MSC In The Therapeutic Effects Of MSC On Lung Permeability And Lung Injury In Rat With LPS-induced ALI

Part One:Construction of MSC with low expression of VEGF/HGF mediated by RNA inteferenceObjective:To construct MSC with low expression of VEGF/HGF.Methods:The rat VEGF/HGF knockdown constructs expressing short hairpin RNA targeting endogenous VEGF(ShRNA VEGF)or HGF(ShRNA HGF)were encoded into a lentivirus-based ShRNA vector pGLV3/H1/GFP+Puro(LV3)driven by the H1 promoter containing GFP and Puromycin.LV3 containing non-specific ShRNA(LV3-GFP)was used as a negative control.The recombinant plasmids were then separately co-transfected with three packaging plasmids pGag/Pol,pRev,pVSV-G into 293T cells using RNAi-mate according to the manufacturer's instruction.The lentiviral particles were collected and stored in-80 ? for future use.Titer was obtained by GFP expression assay.MSC were seeded and cultured in 6-well plates for 24 hours.The lentiviral vectors(Carrying LV3-GFP or LV3-GFP-ShRNA HGF)were then added to the wells at a Multiplicity of Infection(MOI)value of 100:1 and cultured with MSC for 24 hours.After 24 h,the culture medium was changed,and puromycin was added at the minimal lethal concentration(1.5 ?g/ml)for transfected MSC.The puromycin-resistant cells were then collected.MSC-ShVEGF or MSC-ShHGF at 10th passage after lentiviral transduction was used in the following experiments.?Transfection efficiency of lentiviral vectors was evaluated by fluorescence microscopy.Total RNA and total cellular protein were isolated from MSC,MSC-GFP,MSC-ShVEGF or MSC-ShHGF.The culture medium from MSCs treated with LV3-GFP(MSC-GFP),LV3-GFP-ShVEGF(MSC-ShVEGF)or LV3-GFP-ShRNA HGF(MSC-ShHGF)was obtained respectively.Finally,the below index were detected:?The VEGF/HGF mRNA levels in MSC by q-PCR;?The protein expression of VEGF/HGF in MSC by western blot;?VEGF/HGF levels in MSC culture medium by ELISA;?MSC migration ability by Transwell assay and scratch assay.Results:The result showed that the transfection efficiency of Lentivirus-GFP,Lentivirus-ShHGF-GFP and Lentivirus-ShVEGF-GFP were 95.75%,93.55%and 95.23%respectively.No differences were found between each group;?VEGF/HGF mRNA expression was significantly lower in the MSC-ShVEGF/MSC-ShHGF group than that in the MSC(&p<0.05)and MSC-GFP group(#p<0.05).However,there was no significant difference between the MSC and MSC-GFP group(p>0.05)in the VEGF/HGF mRNA expression;?The result from western blotting analysis showed that the VEGF/HGF protein expression in the cytoplasm was decreased in the MSC-ShVEGF/MSC-ShHGF group compared with the MSC(&p<0.05)and MSC-GFP group(#p<0.05);?The VEGF/HGF protein levels in the cell culture medium of the MSC-ShVEGF/MSC-ShHGF group were also significantly lower than that in the MSC(&p<0.05)and MSC-GFP groups(#p<0.05).However,there was no significant difference in VEGF/HGF protein levels between the MSC and MSC-GFP group in either the cytoplasm or the cell culture medium;?The scratch assay showed that no significant difference was found in the wound width which reflected the horizontal migration ability of MSC between the MSC and MSC-GFP or MSC-ShVEGF/MSC-ShHGF groups.Moreover,Transwell migration assay showed that no statistical difference was found in the vertical migration ability between the MSC and MSC-GFP or MSC-ShVEGF/MSC-ShHGF groups.Conclusion:?The MSC-ShVEGF/MSC-ShHGF we constructed was efficient and stable.? VEGF/HGF gene knockdown in MSC had no impairment on its migration ability.Part Two:The role of VEGF-expressing character of MSC in the protective effect of MSC on lung permeability and lung injury in Rat with ALI induced by lipopolysaccharideObjective:To explore the role of VEGF-expressing character of MSC in the protective effect of MSC on lung permeability and lung injury in Rat with ALI induced by lipopolysaccharideMethods:Six to eight-week-old wild-type SD rats received an intratracheal instillation of LPS dissolved in 100 ?l phosphate buffered.PBS,MSC,MSC-GFP or MSC-ShVEGF(5×106 cells resuspended in 100 ?l PBS)were injected into the tail vein five hours after LPS challenge.Rats without LPS challenge were injected with PBS as a control.Rats were sacrificed at 1,6 and 24 hours after MSC injection,and the lung lobes were collected for the following analysis:?Lung permeability by lung wet weight to body weight(LWW/BW)ratios and Evans blue dye leakage assay;?VE-cadherin expression in the injured lung by immunofluorescence;?Lung vascular endothelial cells apoptosis by TUNEL assay;? Lung injury by histopathology and lung injury scores;?IL-1 ? and IL-10 levels in the injured lung by ELISA;?VEGF levels in the injured lung by ELISA.Results:?The LWW/BW and Evans Blue Dye extravasation in the MSC-ShVEGF group,were significantly higher compared with the MSC or MSC-GFP group(p<0.05).? After treatment with MSC or MSC with VEGF gene knockdown,the VE-cadherin expression was increased in the injured lung,compared with the ALI group(p<0.05).However,the increase in VE-cadherin expression in the MSC-ShVEGF group was markedly lower than that in the MSC group(p<0.05);?The apoptosis index of the lung endothelium in the MSC-ShVEGF group was significantly higher than that in the MSC or the MSC-GFP group(p<0.05);?The lung injury score in the MSC-ShVEGF group was significantly higher than that in the MSC and MSC-GFP group(p<0.05);?The lung IL-1 ? levels in the MSC-ShVEGF group was higher than that of the MSC or MSC-GFP group at 24 hours(p<0.05).However,the lung IL-10 levels in the MSC-ShVEGF group was significantlylower than that of the MSC or MSC-GFP group at 24 hours(p<0.05);?The VEGF level in the injured lung decreased significantly in the MSC-ShVEGF group,compared with the MSC or MSC-GFP group at 24 hours(p<0.05).Conclusion:These data suggested that MSC restores lung vascular permeability,which might be associated with the protective effects of MSC on the adherens junction protein VE-cadherin and lung vascular endothelial cell apoptosis,reduces inflammation and attenuates lung injury in LPS-induced ALI in rats in part by maintaining the VEGF to a sufficient level in the injured lung.Part Three:The role of HGF-expressing character of MSC in the protective effect of MSC on lung permeability and lung injury in Rat with ALI induced by lipopolysaccharideObjective:To explore the role of HGF-expressing character of MSC in the protective effect of MSC on lung permeability and lung injury in Rat with ALI induced by lipopolysaccharideMethods:Six to eight-week-old wild-type SD rats received an intratracheal instillation of LPS dissolved in 100 ?l phosphate buffered.PBS,MSC,MSC-GFP or MSC-ShHGF(5×106 cells resuspended in 100 ?l PBS)were injected into the tail vein five hours after LPS challenge.Rats without LPS challenge were injected with PBS as a control.Rats were sacrificed at 1,6,24 and 48 hours after MSC injection,and the lung lobes were collected for the following analysis:?Lung permeability by lung wet weight to body weight(LWW/BW)ratios and Evans blue dye leakage assay;?VE-cadherin expression in the injured lung by immunofluorescence;? Lung vascular endothelial cells apoptosis by TUNEL assay;? Lung injury by histopathology and lung injury scores;?IL-1? and IL-10 levels in the injured lung by ELISA;?MSC retention in the injured lung by immunohistochemical assay;?HGF levels in the injured lung by ELISA.Results:?The LWW/BW and Evans Blue Dye extravasation in the MSC-ShHGF group,were significantly higher compared with the MSC or MSC-GFP group(p<0.05);? After treatment with MSC or MSC with HGF gene knockdown,the VE-cadherin expression was increased in the injured lung,compared with the ALI group(p<0.05).However,the increase in VE-cadherin expression in the MSC-ShHGF group was markedly lower than that in the MSC group(p<0.05);?The apoptosis index of the lung endothelium in the MSC-ShHGF group was significantly higher than that in the MSC or the MSC-GFP group(p<0.05);?The lung injury score in the MSC-ShHGF group was significantly higher than that in the MSC and MSC-GFP group(p<0.05);?The lung IL-1? levels in the MSC-ShHGF group was higher than that of the MSC or MSC-GFP group at 24 hours(p<0.05).However,the lung IL-10 levels in the MSC-ShHGF group was significantlylower than that of the MSC or MSC-GFP group at 24 hours(p<0.05);?No significant differences were found in the retention of MSC with GFP in the lung tissue between the MSC-GFP and MSC-ShHGF groups(p>0.05);? The HGF level in the injured lung decreased significantly in the MSC-ShHGF group,compared with the MSC or MSC-GFP group at 24 and 48 hours(p<0.05).Conclusion:These data suggested that MSC restores lung vascular permeability,which might be associated with the protective effects of MSC on the adherens junction protein VE-cadherin and lung vascular endothelial cell apoptosis,reduces inflammation and attenuates lung injury in LPS-induced ALI in rats in part by enhancing the HGF level in the injured lung to a beneficial level.Moreover,the HGF-expressing character plays an important part in the therapeutic effect of MSC on ALI.