The Implantation Of HTK1 Gene Modified Homogeneous EPCs Defers The Progression Of Monocrotaline Induced Pulmonary Arterial Hypertension In Rats

Objectives:To observe the effects of injection of human tissue kallik:rein 1(hTK1)gene modified homogenous endothelial progenitor cells(EPCs)on hemodynamics,nitric oxide(NO)level in serum and pathological changes in small pulmonary arterioles of monocrotaline(MCT)induced pulmonary arterial hypertension(PAH)in rats.Methods:1.ex vivo experiments:(1)Mononuclear cells were extracted from bone marrow of male Wistar rats using density gradient centrifugation and cultivated in specific EGM-2 complete medium.(2)Bone marrow derived mononuclear cells(BMMNCs)were identified as EPCs by morphological observation,Dil-acLDL and FITC-UEA-I double fluorescence staining.(3)Adenovirous-green fluorescence protein(Ad-GFP)was gradiently transfected into EPCs to determine the best multiplicity of infection(MOI)according to cell viability and fluorescence performance.(4)CCK8 proliferation assay,scratch healing test were conducted to investigate the effects of hTKl gene modification on the proliferative and migration ability of EPCs.(5)Nitric oxide(NO)concentration in cell lysate was detected to verify the the impact of hTKl gene transfection on the NO production released by EPCs.(6)Western blotting was carried out to demonstrate the levels of human tissue kallikrein(hTKl)and endothelial nitric oxide synthase(eNOS).2.in vivo experiments:(1)Thirty male Wistar rats aged 7-8 weeks were divided into four groups as follows:one was normal control group(NC)with six rats,the other three were models of MCT induced PAH,eight animals in each group with different treatments including intravenous injection of PBS(MCT+PBS),EPCs(MCT+EPCs)and Ad-hTK1-EPCs(MCT+Ad-hTK1-EPCs).About 106 cells in 0.5ml PBS were given twice three days after MCT intraperitoneal injection.The general condition was observed and body weight was measured every two weeks.(2)Ultrasonic cardiograph(UCG)was performed before the experiment as well as two and four weeks after modeling to monitor some hemodynamic parameters including right ventricular anterior wall thickness(RVAWT),pulmonary artery diameter(PAD),aortic diameter(AOD),PAD/AOD,pulmonary artery acceleration time(PRAT),pulmonary artery ejection time(PAET),PAAT/PAET,right ventricular end diastolic diameter(RVEDD),right ventricular end diastolic length(RVEDL),RVEDD/RVEDL,tricuspid annular plane systolic excursion(TAPSE)and left ventricular ejection fraction(LVEF).(3)Serum NO level of all groups was measured every two weeks after MCT injection.(4)After the last UCG examination,all rats were administrated with thoracotomy and right ventricular catheterization to measure pulmonary artery systolic and mean pressure(PASP,PAMP)and the peak increasing,decreasing rate of right ventricle pressure(namely dp/dtmax,dp/dtmin).(5)All rats were killed.Hearts and lungs were obtained to calculate right ventricle hypertrophy index(RVHI)and for hematoxylin-eosin(HE)staining of formalin-fixed paraffin-embedded lung tissues to study the changes irn vessel wall thickness and area index(WT%,WA%)of pulmonary arterioles with 100-200?m external diameter(ED).(6)Western blotting was done to detect the expression of hTKl,eNOS in lung tissues of all groups.(7)Two more rats were used for tracking of EPCs in vivo.The rats were administered with 1%MCT solution through intraperitoneal injection.Seven days later,Ad-GFP-EPCs were intravenously injected into the rats.Two days later,the rats were killed to obtain heart,liver,spleen,lung and kidney tissues.Green fluorescence was observed in frozen sections of the organs to show the distribution of Ad-GFP-EPCs.Results:1.ex vitro experiments:(1)Mononuclear cells isolated from bone marrow were successfully cultivated,expanded and induced to differentiate in specific EGM-2 culture medium.The primary cells mostly attached on the culture bottle four days after being seeded.Cell colonies started to appear from the 7th day and could be passaged after 2-3 weeks culture.The passage cells presenting small colonies or endothelium-like growth went down to next generation at intervals of 3-7 days.(2)Bone marrow derived mononuclear cells(BMMNCs)were identified as EPCs by morphological observation and double positive staining of Dil-acLDL and FITC-UEA-I.(3)Ad-GFP was transfected into EPCs with a step-increased MOI equal to 0,25,50,100,150,200 and 250.The fluorescence appeared to be the most and cell viability was't impaired as MOI=150,so it was chosen as the best MOI.(4)The results of CCK8 proliferation assay and scratch healing test showed that the absorbance values A450 and scratch healing areas of hTKl gene modified EPCs(Ad-hTK1-EPCs)were higher and larger than that of EPCs and Ad-GFP-EPCs(P<0.05),which indicated that hTK1 gene modification enhanced proliferation and migration ability of EPCs.(5)Ad-hTKl-EPCs,released more NO in cell lysate than EPCs and Ad-GFP-EPCs(P<0.05).(6)Western blotting demonstrated that hTK1 was stably expressed in Ad-hTK1-EPCs and higher eNOS level was detected compared with EPCs and Ad-GFP-EPCs(P<0.05).2.In vivo experiments:(1)In comparison with NC,PAH models were in poorer general conditions with lower survival rate.Weight increase measured at the second week in MCT+PBS and MCT+EPCs was less than that of NC and MCT+Ad-hTK1-EPCs(P<0.05).(2)The comparison analysis of echocardiographic parameters among the four animal groups showed that as time went on,the values of RVAWT,PAD/AOD,RVEDD/RVEDL increased while PAAT,PAAT/PAET,TAPSE decreased in the model animals especially in MCT+PBS and MCT+EPCs groups.The difference in terms of PAD/AOD,PAAT,PAAT/PAET,RVEDD/RVEDL between MCT+PBS and NC or MCT+Ad-hTK1-EPCs was statistically significant(P<0.05).There was statistical difference in PAAT,PAAT/PAET between MCT+EPCs and NC or MCT+Ad-hTK1-EPCs(P<0.05).(3)Serum NO content in MCT+Ad-hTK1-EPCs group two and four weeks after modeling was higher than the other three groups(P<0.05),while NO content of MCT+PBS was the least at either the second week compared to the other three groups(P<0.05)or the fourth week in contrast to NC or MCT+Ad-hTK1-EPCs(P<0.05).Serum NO level of MCT+EPCs increased at the second week,more than NC and MCT+PBS,but was less than MCT+Ad-hTK1-EPCs(P<0.05)and decreased in the fourth week,less than NC or MCT+Ad-hTK1-EPCs(P<0.05).(4)Right ventricular catheterization displayed that PASP,PAMP and dp/dtmax,dp/dtmin of models were elevated.There was statistic discrepancy in all the indicators between MCT+PBS and NC or MCT+Ad-hTK1-EPCs group(P<0.05).Statistic difference lay in terms of PASP and dp/dtmax between MCT+EPCs and NC group(P<0.05).(5)The comparison of RVHI among the four groups revealved increased RVHI in MCT+PBS compared with NC or MCT+Ad-hTK1-EPCs(P<0.05).HE staining exhibited that WT%,WA%of small pulmonary arterioles in MCT+PBS were enlarged compared to NC or MCT+Ad-hTK1-EPCs(P<0.05 or P?0.05).(6)The results of Western blotting showed that hTK1 and eNOS protein were expressed in lung tissues of MCT+Ad-hTK1-EPCs,while no obvious expression was observed in the other three groups.(7)Green fluorescence of Ad-GFP-EPCs could be seen in heart(relatively little),liver,spleen,lung and kidney,mainly located in the sites of vessel wall,blood sinus.Conclusion:hTKl gene transfection enhanced proliferation and migration ability of EPCs.hTKl gene modified EPCs could stably express hTKl and eNOS protein.Ad-hTK1-EPCs intravenous implantation attenuated MCT:induced PAH by alleviating hemodynamics and pathological changes,which may be attributed to increase of NO content in serum resulted from preventable intervention at early stage of PAH progression.