The Mechanisms Of Mesenchymal Stem Cell Protective Effects Against Pulmonary Microvascular Endothelial Cell Dysfuntction Via Paracrine Hepatocyte Growth Factor

Part One: The mechanism of m TOR/STAT3 activation mediates mesenchymal stem cell(MSC)-secreted hepatocyte growth factor(HGF)protective effects against pulmonary microvascular endothelial barrier dysfunctionObjective: To investigate the mechanism of mesenchymal stem cell(MSC)protective effects against pulmonary microvascular endothelial cell(PMVEC)dysfunction via paracrine hepatocyte growth factor(HGF).Methods: We introduced a co-cluture protocol of MSCs and PMVECs for 4h-72 h.Lipopolysaccharide(LPS)was added into PMVECs to stimulate LPS-induced PMVECs dysfunction.To investigate m TOR/STAT3 activation of HGF protective mechanisms,recombination HGF(20ng/ml)were introduced into PMVECs,and m TOR inhibitor rapamycin(100nmol/L)or STAT3 inhibitor S3I-201(100nmol/L)were also applied.These belove were tested :(1)Endothelial permeability analysis :paracellular and transcellular permeability were receptively tested by fluorescein isothiocyanate(FITC)-Dextran and FITC-albumin;(2)ET-1 and v WF concentration in PMVEC supernate : enzyme-linked immuno sorbent assay(ELISA);(3)Adherent junction protein in PMVECs: Occludin expression were detected by immunoflurescence;(4)PMVECs proliferation and apoptosis experiments: Cell proliferation and apoptosis were respectively tested by Cell Counting Kit-8(CCK8)and Annexin V-FITC/PI assay;(5)Associated signaling detection:p-Akt(Ser473),Akt,p-m TOR(Ser2448),m TOR,p-p70S6 Kinase(Thr389),p70S6 Kinase,p-STAT3(Ser727)and STAT3 were analysised by western blot(WB).Results:(1)LPS-induced pulmonary microvascular endothelial barrier dysfunction were established: Paracellular permeability showed that LPS at concentration of 100ng/ml raised PMVECs paracellular permeability compared with control groups,and were evaluated positive as dose-depentent(P<0.05).LPS-stimulated ET-1 and v WF production in endothelium were time-dependent positive at 4h,24 h and 72 h at concentration of 100ng/ml.(2)The effect of m TOR/STAT3 acttivation of MSC-secreted HGF in endothelial cells:LPS decreased adherent junction protein Occludin expression and use of recombinant HGF and MSC-PMVEC co-cultivate increased occludin expression examined by immunofluorescence staining.PMVECs pretreatment with recombinant HGF significantly induced LPS-induced endothelial paracellular and transcellular permeability at time-dependent(P<0.05).HGF stimulation lowered LPS-stimulated endothelium injury markers of ET-1 and v WF at time-dependent(P<0.05).WB analysis was employed to show that LPS-induced phosphorylation m TOR(Ser2448)and phosphorylation STAT3(Ser727)were evaluted with HGF treatment(20ng/ml).(3)The effect of m TOR signaling mediates protective effects of HGF on PMVECs barrier: Immunoflurescence experiments demonstrated that adherent protein Occludin in LPS-induced PMVECs were lowed.HGF increases occludin adherent junction protein expression in LPS-induced PMVECs and these protective effects were inhibited by rapamycin.And inhibition of m TOR decreased protective effect of HGF on LPS-induced endothelial paracellular and transcellular permeability.Rapamycin unregulated endothelium injury factors ET-1 and v WF in LPS-induced PMVECs with HGF treatment.WB analysis showed that phosphorylation Akt(Ser473),m TOR(Ser2448),p70S6Kinase(Thr 389)and STAT3(Ser727)were lowered with rapamycin compared with HGF treatment.(4)The effect of STAT3 signaling mediates protective effects of HGF on PMVECs barrier: Immunoflurescence experiments showed that adherent protein Occludin in LPS-induced PMVECs were lowed and increased with HGF treatment.However,with STAT3 inhibitor S3I-201,it reversely decreased.HGF treatment to PMVECs decreased paracellular and transcelluar permeability compared with LPS stimulation.Inhibition of STAT3 dramatically increased paracellular and transcelluar permeability compared with HGF treatment(P<0.05).STAT3 inhibition also raised endothelium injury factors ET-1 and v WF in LPS-induced PMVECs which could decrease responding to HGF(P<0.05).WB analysis showed that phosphorylation STAT3(Ser727)was lowered with STAT3 inhibitor S3I-201 compared with HGF treatment.(5)The effect of m TOR/STAT3 signaling mediates protective effects of HGF apoptosis:The CCK8 reagent showed that HGF could improve cell proliferation induced by LPS(P<0.05).However,the improvement was declined by rapamycin and S3I-201.Annexin V/PI method revealed that HGF attenuated LPS-induced cell apoptosis.m TOR inhibitor rapamycin and STAT3 inhibitor S3I-201 reversely the results.Conclusions:(1)MSC-secrected HGF improves pulmonary microvascular endothelial barrier by decreasing paracellular and transcellular permeability,raising endothelial adherent protein Occludin expression,decreasing enthelial injury markers ET-1 and v WF,increasing endothelial proliferation and decreasing apoptosis.(2)Activation of m TOR/STAT3 pathway provides mechanistic insights into MSC-secreted HGF protective against LPS-induced pulmonary microvascular endothelial dysfunction.Part Two: Hepatocyte growth factors-expression character of mesenchymal stem cells activated m TORC1 ad m TORC2 signaling in recovery of pulmonary microvascular endothelial cell barrierObjective: The aim of this study was to determine whether m TORC1 and m TORC2 mediated protective effects of MSC-secreted HGF against LPS-induced pulmonary microvascular endothelial barrier dysfunction.Mentods: We introduced MSC-PMVEC co-culture transwell system for 4-72 h.Lipopolysaccharide(LPS,100ng/ml)was added into PMVECs to stimulate LPS-induced PMVECs dysfunction and recombinant murine HGF(20ng/ml)was applied on endothelial cell barrier dysfunction stimulated by LPS.To explored potential mechanisms of m TORC1 and m TORC2 signaling in HGF treatment,m TORC1(raptor)and m TORC2(rictor)gene knockdown modification(PMVEC-sh Raptor,PMVEC-sh Rictor)by lentivirus vector were made.Moreover,Akt inhibitor AZD5363(1 μM)was also applied.These belove were tested:(1)PMVEC permeability analysis:paracellular and transcellular permeability were receptively tested by FITC-Dextran and FITC-albumin;(2)Adherent junction protein in PMVECs: VE-cadherin expression were detected by flow analysis;(3)PMVECs apoptosis and proliferation experiments : Cell apoptosis and proliferation were respectively tested by Annexin V-PE/7-AAD and Cell Counting Kit-8(CCK8)assay;(4)Associated signaling detection: p-Akt(Ser473)、Akt、Raptor、Rictor、p-m TOR(Ser2448)、m TOR were analysised by western blot.Results:(1)Effects of MSC-secrected HGF on the adhesion junction protein VE-cadherin in PMVECs: The data of flow analysis showed that he expression of VE-cadherin in PMVECs could be increased in the co-culture group of MSCs-PMVECs group and recombinant HGF group compared with the control group(P<0.05).In the related studies of signal pathway,with treatment of recombinant HGF(4h-24h),the expressions of m TOR,m TORC1(raptor) and m TORC2(rictor)gradually increased,showing a time-dependent relationship(P<0.05).(2)Identification of low expression of m TORC1(raptor)and m TORC2(rictor)by lentivirus: First,lentivirus vector-mediated m TORC1(raptor)and m TORC2(rictor)knockdown in PMVECs(sh Raptor and sh Rictor as knockdown group,sh RNA-control as negative control)were conducted.The transduction efficacy detected by fluorescence microscopy showed that infected PMVECs had good green fluorescence signal.PCR results demonstrated that lower raptor and rictor m RNA expression in PMVEC-sh Raptor/PMVEC-sh Rictor than sh RNA-control(P<0.05).WB analysis showed that lower raptor and rictor protein were expressed in PMVEC-sh Raptor/PMVEC-sh Rictor than sh RNA-control.(3)The effect of m TORC1(raptor)and m TORC2(rictor)to endothelial adhesion protein VE-cadherin expression: Flow analysis demonstrated that the expression level of endothelial adhesion protein VE-cadherin increased after exogenous HGF application compared with LPS group.After sh Raptor and sh Rictor were given respectively to down-regulate raptor and rictor expression in PMVECs,the expression level of VE-cadherin were significantly reduced(P< 0.05).(4)The effect of m TORC1(raptor)and m TORC2(rictor)on endothelial cell barrier: Flow cytology analysis showed that the paracellular and transcellular permeability of the endothelial cells in PMVEC-sh Raptor and PMVEC-sh Rictor groups with administration of exogenous HGF increased compared with PMVEC-sh Raptor-control and PMVEC-sh Rictor-control(P<0.05).Moreover,WB results suggested that HGF increased raptor and phosphorylated p-p70s6k(Thr389)protein,as well as rictor and phosphorylated p-Akt(Ser473)in LPS-stimulated PMVECs.After gene silencing of m TORC1(raptor)and m TORC2(rictor)in PMVECs,the expression levels of raptor,rictor and phosphorylated p-p70s6k(Thr389)and p-Akt(Ser473)were decreased.(5)The effects of m TORC2/Akt on endothelial cell barrier: Flow cytology results showed that after blocking Rictor,the expression of VE-cadherin decreased in HGF-induced PMVECs,and decreased after following treatment with Akt inhibitor AZD5363(1 M)(P<0.05).Compared with LPS+HGF co-stimulation group,sh Rictor(+)could up-regulated the permeability of endothelial cells and trans-endothelial cells(P<0.05),and Akt inhibitor AZD5363 could followly up-regulated paracellular cells and transcellular permeability(P<0.05).(6)Effects of m TORC1(raptor)and m TORC2(rictor)on the apoptosis of endothelial cells: Apoptosis and proliferation assay showed that exogenous recombinant HGF could reduce the apoptosis of LPS injured endothelial cells and promote the proliferation of endothelial cells(P<0.05).However,after gene silencing of sh Raptor and sh Rictor,endothelial cell proliferation was significantly inhibited and apoptosis was increased(P<0.05).WB test of the apoptotic protein caspase-3 level tests revealed that gene silencing sh Raptor and sh Rictor increased the expression of the apoptosis-related protein cleaved-caspase-3,compared with the LPS+HGF costimulation group.Conclusions:(1)Activation of m TORC1 and m TORC2 signaling pathways are involved in recovery of LPS-induced PMVEC dysfunction via MSC-secrected HGF.(2)m TORC2 /Akt are involved in recovery of LPS-induced PMVEC dysfunction via MSC-secrected HGF.Part Three: Alleviate effects of hepatocyte growth factor in mitochondria-dependent apoptosis of lipopolysaccharide-induced pulmonary microvascular endothelial cell via m TOR/STAT3 signalingObjective: To investigate the effect of m TOR/STAT3 signaling pathway on paracrine HGF of MSC in alleviating mitochondrial dependent apoptosis of lung microvascular endothelial cells(PMVEC)induced by LPS.Methods: LPS(100ng/ml)was added into PMVEC to construct LPS-induced injured endothelial cells.Exogenous recombinant mouse HGF(20ng/ml)was added into PMVEC,and m TOR inhibitor rapamycin(100nmol/L)or S3I-201 inhibitor(100nm/L)were applied to detect the role of m TOR/STAT3 signaling pathway in reducing mitochondria-dependent apoptosis of PMVEC induced by LPS.Following were tested:(1)Intracellular calcium ion concentration detection: Flow analysis of intracellular calcium ion level by Fluo-4 fluorescence change;(2)Reactive oxygen species(ROS)test: DCFH-DA detection of ROS level was methoded by flow cytometry.(3)Respiratory chain Complex I(Complex I)expression: WB ananlysis was used to detect the expression of Complex I core protein NDUFB8.(4)Apoptosis and proliferation detection: Annexin V-FITC/PI and CCK8 method were respectively used to detect the endothelial apoptosis and proliferation.(5)Apoptosis protein Caspase3 level: Western blot was applied.(6)Antiapoptotic genes of Bcl-2 and the Bcl-XL m RNA detection: Reverse transcription Real-time policy polymerase chain reaction(RT-q PCR)was used.Results:(1)The effect of m TOR/STAT3 signaling on the concentration of calcium ions in endothelial cells: Fluo-4 fluorescence test showed that LPS stimulation increased the intracellular calcium ion flow,and HGF could reduce the calcium flow compared with control.The calcium flow increased after the administration of m TOR inhibitor rapamycin and STAT3 inhibitor S3I-201(P<0.05).(2)The effect of m TOR/STAT3 signaling on ROS from endothelial cell: Flow DCFH-DA assay showed that the production of ROS decreased with HGF treatment(30-60-90-120min)(P<0.05).Moreover,HGF could reduce the ROS production induced by LPS in 30 min.However,the reduction of ROS production by HGF was inhibited under the inhibition of m TOR inhibitor rapamycin and STAT3 inhibitor S3I-201(P<0.05).(3)The effect of m TOR/STAT3 signaling on Complex I expression: WB analysis showed that LPS damaged Complex I(NDUFB8),and HGF reversely reduced LPS-induced Complex I(NDUFB8)expression.Complex I(NDUFB8)expression was also decreased after the administration of m TOR inhibitor rapamycin and STAT3 inhibitor s3I-201(P<0.05).It also showed that HGF increased Complex I activity compared with LPS group,which could be inhibited by rapamycin or S3I-201(P<0.05).(4)The effect of HGF on LPS-induced endothelial apoptosis and proliferation: The proportion of early apoptosis decreased with HGF treatment(1h,2h,4h)tested by flow cytometry of Annexin V-PE/7-AAD apoptosis assay.The proliferation test of endothelial cells showed that LPS could reduce the optical density(OD)value of endothelial cells,and the OD value of endothelial cells increased after the treatment of HGF for 4h-12h-24h(P<0.05).(5)The effect of m TOR/STAT3 signaling on endothelial cell apoptosis protein: WB results showed that LPS promoted the increase of active cleaved-caspase-3,and HGF reversely reduced the expression of cleaved-caspase-3.m TOR inhibitor rapamycin and STAT3 inhibitor S3I-201 inhibited the expression of apoptotic cleaved-caspase-3 protein(P<0.05).(6)The effect of HGF on antiapoptotic genes Bcl-2 and Bcl-XL m RNA expression: RT-q PCR showed that HGF increased the expression of Bcl-2 and Bcl-XL m RNA compared with LPS group,and m TOR inhibitor rapamycin and STAT3 inhibitor S3I-201 could inhibit the expression of Bcl-2 and Bcl-XL m RNA(P< 0.05).After gene silencing of m TORC1(raptor)and m TORC2(rictor),the expression of anti-apoptotic gene Bcl-2 and Bcl-XL m RNA were inhibited(P<0.05).(7)The effect of HGF on endotheial junction VE-cadherin and Occludin expression: WB analysis demonstrated that HGF could increase VE-cadherin and Occludin of injured endothelial cells;however,the expression levels of VE-cadherin and Occludin decreased after m TOR inhibitor rapamycin and STAT3 inhibitor S3I-201 were given.Conclusion: The activation of m TOR/STAT3 signaling pathway is involved in MSC-secretd HGF to reduce the mitochondria-dependent apoptosis of PMVEC induced by LPS.