Protective Effect And Mechanism Of PEDF/PEDF-R On Ischemic Heart Disease

Part1 Effect of PEDF on Cardiac Function in rats with acute myocardial infarctionObjective: In this study we delivered lentivirus carrying PEDF or PEDF RNAi by using intramyocardial injections to overexpress or knockdown PEDF in a rat AMI model.We aimed to investigate whether PEDF could improve cardiac function in rats with AMI.Methods: Myocardial ischemia was induced by ligation of the left-anterior descending coronary artery(LAD)in anesthetized Sprague-Dawley male rats.PEDF-small interfering RNA(si RNA)-lentivirus(PEDF-RNAi-LV)or PEDF-LV was delivered into the myocardium along the infarct border immediately after surgery.Control animals received an equivalent volume of lentivirus vector.The animal models were randomly divided into four groups: Group A(normal);Group B(si PEDF),PEDF-RNAi-LV was transferred after surgery;Group C(PEDF),PEDF-LV was transferred after surgery;Group D(vector control),NC-RNA-LV was transferred into the ischemic myocardium and treated as a LV vector control.Four weeks after LAD artery ligation,echocardiography was employed to evaluate cardiac functional parameters,and then the hearts were removed for MIS analyses by the method of 2,3,5-triphenyltetrazolium(TTC)staining and Magnetic resonance imaging(MRI).Results: Adult Sprague-Dawley rat models of AMI were surgically established.PEDF protein expression in the ischemic heart was downregulated immediately after LAD ligation.In the PEDF group,we found that an overexpression of PEDF protein was evident,while PEDF protein expression in the si PEDF group was significantlyinhibited.Cardiac function was examined using echocardiography 4 weeks after AMI.Significant improvement in cardiac systolic function in the PEDF group was present 4 weeks after AMI and that the values of FS% and EF% in this group were significantly larger than those of the si PEDF and the other control groups.PEDF treatment significantly decreased myocardial infarct size(MIS)than vector control 4 weeks after AMI.However,MIS percentage was markedly increased in si PEDF-treated groups than in vector control rats.Conclusion: PEDF reduces myocardial infarct size and improves cardiac function in rats with AMI.Part2 A study on the effect and mechanism of PEDF on protecting cardiomyocyte against ischemia via PEDF-RObjective: To verify the hypothesis whether PEDF could protect cardiomyocyte against ischemia or hypoxia-induced cell apoptosis via PEDF-R both in vivo and in vitro.Methods: The animal models and groups were identical to the part1.Four weeks after LAD artery ligation,cardiomyocyte apoptosis in vivo was determined by double-labeling immunofluorescence staining of TUNEL.Western blot was conducted to determine the protein levels of caspase-8,-9,-3.H9c2 cells and rat primary cardiomyocytes were divided into groups(Normal,Hypoxia control,PEDF,PEDF+si PEDF-R,PEDF+GW9662).Hypoxia was achieved by culturing the cells in D-Hank’s liquid with serum deprivation in an tri-gas incubator saturated with 5% CO2/1% O2 at 37oC for the indicated time periods.Cell apoptotic rates were examined using double labeling with cleaved caspase3 immunofluorescence and TUNEL staining.Western blotting analysis was used to test cleaved caspase-3.The generation of intracellular ROS was measured by DCFH-DA method.T-SOD and MDA activities were measured using respective detection kits according to the manufacturers’ instructions.Results: The results of TUNEL staining showed that the amount of cardiomyocyte apoptosis was significantly increased in the si PEDF group 4 weeks after AMI,whereas the amount of cardiomyocyte apoptosis in the PEDF group was reduced when compared with vector control group.In addition,we further determined the expression of proteins related to apoptotic signal pathways.In the si PEDF group,increased expression of cleaved caspases-8,-9,and-3 was evident,however,in the PEDF group,cleaved caspases-8,-9,and-3 expression was significantly decreased.Compared with the vector control group,PEDF treatment decreased cardiomyocyte apoptosis and ROS level,increased the activity of i PLA2 and PPARγ in hypoxia-induced cell.Likewise,cultured cells pretreated with silenced PEDF-R(si PEDF-R)and/or PPARγ antagonist(GW9662)attenuates the beneficial effects of PEDF as determined by TUNEL assay and ROS staining.Conclusion: PEDF protectes cardiomyocyte against ischemia or hypoxia-induced cell apoptosis both in vivo and in vitro.PEDF activates i PLA2 attached on PEDF-R,promotes the activity of PPARγ and then decreases the generation of ROS to inhibit cardiomyocyte apoptosis.Part3 A study on the effect and mechanism of PEDF on inhibiting vascular permeability via PEDF-RObjective: To verify the hypothesis whether PEDF could inhibiting vascular permeability via PEDF-R both in vivo and in vitro.Methods: The animal models and groups were identical to the part1.To determine vascular permeability in the rat hearts,a Miles assay was employed based on the method Evans Blue injection four weeks after AMI.To further determine vascular permeability affected by PEDF expression,we examined the changes in the main functional proteins of AJs(VE-cadherin/β-catenin)and TJs(Occluding).RAECs were isolated and cultured,and then RAECs exposed to hypoxic conditions(94% N2–5% CO2–1% O2)in an anaerobic system at 37°C for 24 h,while the controls were left in normoxic conditions at 37°C for the equivalent periods.For this,RAECs were exposed to normoxia or hypoxia for 24 hours in the absence or presence of PEDF and measured for permeability.We also examined the changes in the main functional proteins of AJs(VE-cadherin/β-catenin)and TJs(Occluding and ZO-1)by western blot and co-immunoprecipitation(co-IP).Results: The normal heart was dye-free.The amount of dye that leaked out of the coronary artery and infiltrated into the myocardium in the PEDF group was significantly less than the amounts in the si PEDF and vector control groups.However,a large quantity of Evans Blue residing in the myocardium was detected in the si PEDF group.The level of VE-cadherin tyrosine protein phosphorylation was high in the si PEDF group and was low in the PEDF group.The results of co-IP showed that the level of VE-cadherin associated with β-catenin was significantly low in the si PEDF group.In contrast,a high level of VE-cadherin expression was found in the PEDF group.We also found that occludin protein expression was decreased in the siPEDF group and was increased in the PEDF group.Hypoxia-induced increases of endothelial permeability were abolished when PEDF treatment.When we added PPARγ inhibitor GW9662,PEDF’s effect on the suppression of the endothelial permeability was significantly lost.Western blots of RAECs lysates under hypoxia treated with PEDF showed significant up-regulation of TJs(Occludin and ZO-1)compared with the hypoxia group.However,the up-regulation of occludin and ZO-1 proteins retracted in the presence of PPARγ inhibitor GW9662.The level of VE-cadherin tyrosine phosphorylation was high in the hypoxia group but was low in the PEDF group.The results of co-IP showed that the level of VE-cadherin associated with β-catenin was low in the hypoxia group but was significantly high in the PEDF group.Under these conditions,when we added PPARγ inhibitor GW9662,PEDF’s effects on the AJs(VE-cadherin/β-catenin)were nearly unchanged.We also found PEDF could increase the expression of PPARγ in RAECs via PEDF-R.Conclusion: PEDF prevents vascular leakage in rats with AMI.PEDF inhibits RAECs permeability under hypoxia via enhancing the expression of TJs and AJs.PEDF-mediated PPARγ production via PEDF-R in hypoxic RAECs promotes up-regulation of TJs,leading to a sustained barrier function.The effects which PEDF up-regulates AJs may be via the other path.