Protective Role Of HIF-1 In Rats Myocardial Infarction And Ischemia-reperfusion Injury

IntroductionHypoxia Inducible Factor-1(HIF-1)is a heterodimericα,βtranscription factor that mediates tissue responses to hypoxia.HIF-1αpromotes dministrated of genes involved in oxygen homeostasis in response to diminished oxygen tension,including vascular endothelial growth factor(VEGF),inducible NO synthase(iNO),and heme oxygenase-1.Under hypoxic conditions,the activity of HIF-1αtransactivation domains increase.However,it is not known what can be enhanced if intramyocardial injection of HIF-1αbe taken in an acute myocardial infarction model in the rat.It is well known ischemic preconditioning can attenuate postischemic injury,but we don't know the role of protein kinase C(PKC)signaling in ischemic preconditioning to attenuate acute myocardial infarction.Dimethyloxalyglycine(DMOG)is prolyl hydroxylase inhibiter,it can increase the activity of HIF-1α.We don't know what effect wll be produced in myocardial ischemia-reperfusion injury if DMOG is dministrated.PartⅠProtective Effect of Hypoxia Inducible Factor-1 in Rats Acute Myocardial InfarctionIntroductionHypoxia Inducible Factor-1(HIF-1)is a heterodimericα,βtranscription factor that mediates tissue responses to hypoxia.We have known that ischemic preconditioning can attenuate postischemic injury and increase the expression of HIF-1α.However,it is not known what can be enhanced if intramyocardial injection of HIF-1αbe taken in an acute myocardial infarction model in the rat.So we evaluate the effect of hypoxia-inducible factor-1α(HIF-1α)to acute myocardial infarction of rat through Intrarnyocardial injection of HIF-1αMaterials and Methods.72 healthy male Wistar rats weighting 250-350g were divided into three groups.The first group with intramyocardial injection of HIF-1α(Ad-HIF-1αgroup); The second group with intramyocardial injection of Ad-null of HIF-1α(Ad-null group);The third group was the sham-operation group(control group).each group was divided into 3d,7dand 14d subgroups;There are 8 rats in each subgroup.Rats in Ad-HIF-1αgroup andAd-null group received ligation of the proximal left anterior descending coronary artery(LCA)to induce acute myocardial infarction.After the ligation of LCA,the total 50μg plasmid HIF-1αand Ad-null was injected into the infarct area at three locations in Ad-HIF-1αgroup and Ad-null group respectively.The control group don't take ligation of LCA.After 3days,7days and 14days of operation,rats were killed.The heart was excised and LV was cut to immersed into 4% paraformaldehyde in phosphate-pufferd saline for more than 24 hours.The infarct size,expressed as a percentage of the left ventricle,was calculated by dividing the circumference of the infarct by the total circumference of the left ventricle including the septum.The capillary density by immunohistochemistry was mesureed at 3d,7d and 14d after operation.The HIF-1αand VEGFmRNA gene expression was observed through Reverse transcription-polymerase chain reaction(RT-PCR).Results1.Infarct size:The infarct size in Ad-HIF-1αgroup and Ad-null group was (23.85±2.25)%,(38.74±3.12)%respectively,p＜0.01.2.capillary density:The capillary density at3d,7d and 14d in Ad-null group was 365±23/mm~2,435±68/mm~2,492±45/mm~2 respectively;The capillary density at 3d,7d and 14d in Ad—HIF-1αgroup was 376±25/ mm~2,826±44/mm~2,938±67/ mm~2 respectively.The capillary density at 7d and 14d in Ad—HIF-1αgroup was more than that in Ad-null group respectively.3.HIF-1αandVEGFmRNA gene expression:The HIF-1αand VEGFmRNA gene expression was observed at 3d and 7d in Ad—HIF-1αgroup and in Ad-null group through Reverse transcription-polymerase chain reaction(RT-PCR);The HIF-1αand VEGFmRNA gene expression increases at 3d and 7d in Ad—HIF-1αgroup more than that in Ad-null group.4.Plasma VEGF level:The plasma VEGF level at 3d,7d and 14d in Ad-null group was4.8±0.7pg/ml,3.3±0.9 pg/ml,1.04±0.4 pg/ml respectively;The plasma VEGF level at 3d,7d and 14d in Ad-HIF-1αgroup was7.44±0.3pg/ml,5.5±0.8 pg/ml, 1.24±0.5 pg/ml respectively;The plasma VEGF level at 7d and 14d in Ad—HIF-1αgroup was more than that in Ad-null group respectively.ConclusionIntramyocardial injection of HIF-1αcan decrease the infarct size of AMI rats and increase capillary density significantly;It also can increase HIF-1αand VEGFmRNA gene expression and increase plasma VEGF level.It has obvious protection to AMI in rats.PartⅡHypoxia Inducible Factor-1 Gene Expression in acute Myocardial infarction of Rat and The Role of PKCIntroductionIt is well known ischemic preconditioning can attenuate postischemic injury,but the mechanism is not clear,and we also don't know the role of protein kinase C(PKC) signaling in ischemic preconditioning to attenuate acute myocardial infarction.So we observed the role of PKC through ischemic preconditioning before AMI model in rats.Materials and Methods.32 healthy male Wistar rats weighting 250-350g were divided into four groups.The first group was ischemic preconditioning group(IP group);The second groupwas simple Myocardial infarction group(Migroup);The third group was ischemic preconditioning plus PKC inhibiter group(IP+I group);The forth group was the sham-operation group(control group).There are 8 rats in each group.IP group,MI group and IP+I group rats received ligation of the proximal left anterior descending coronary artery(LCA)to induce acute myocardial infarction.The control group don' t take ligation of LCA.In IP group,short-term ischemic was taken,three times for each 5 minites prior ligation of LCA;In IP+I group,rats were injected with the PKC inhibiter (chelerythrine,5mg/kg,iv).After 1days of operation,rats were killed.The heart was excised and LV was cut to immersed into 4%paraformaldehyde in phosphate-pufferd saline for more than 24 hours.The infarct size,expressed as a percentage of the left ventricle,was calculated by dividing the circumference of the infarct by the total circumference of he left ventricle including the septum..The HIF-1αand VEGFmRNA gene expression was observed through Reverse transcription-polymerase chain reaction(RT-PCR)..The HIF-1αand VEGFmRNA protein expression was also observed through Westen blot.Results1.Infarct size:The infarct size in IP group,IP+I group and MI group was (24.18±2.25)%,(38.11±2.94)%and(37.73±3.32)%respectively,The infarct size in IP group decreased significantly vs IP+I group and MI group(P＜0.01).2.HIF-1αandVEGFmRNA gene expression:The HIF-1αand VEGFmRNA gene expression was observed at 1d through Reverse transcription-polymerase chain reaction(RT-PCR);The HIF-1αand VEGFmRNA gene expression increases in IP group more than that in MI group and IP+I group.3.HIF-1αandVEGFmRNA protein expression:It has the same change as HIF-1αand VEGFmRNA gene expression.4.Plasma VEGF level:The plasma VEGF level atld in IP group,MI group and IP+I group after operation was 6.3±0.6pg/ml,3.0±0.5 pg/ml,3.6±0.4 pg/ml respectively; The plasma VEGF level atld in IP group was more than that in MI group and IP+Igroup(P＜0.01).ConclusionIschemic preconditioning can attenuate postischemic injury,and HIF-1αplay an important protective role;HIF-1αandVEGF gene expression depend on protein kinase C(PKC)signaling transform in ischemic preconditioning to attenuate acute myocardial infarction.PartⅢProtective Role of Hypoxia Inducible Factor-1 in Rat myocardial ischemia-reperfusion injuryIntroductionThe re-establishment of blood flow after ischemia adds paradoxically to damage done by prolonged ischemia.This phenomenon is known as ischemia reperfusion injury(IRI).we have known Heme oxygenase 1(HO-1)has protective effect to IRI, HIF-1αgene can promotes transcription ofHO-1 gene.The role of hypoxia inducible factor-1 in rat myocardial ischemia-reperfusion injury is not clear today. Dimethyloxalyglycine(DMOG)is prolyl-hydroxylase inhibiter,it can increase the activity of HIF-1α.We don't know what effect wll be produced in myocardial ischemia-reperfusion injury if DMOG is dministrated.So we observed the role of hypoxia inducible factor-1 in rat myocardial ischemia-reperfusion injury and what effect of DMOG to IRI.Materials and Methods32 healthy male Wistar rats weighting 250-350g were divided into four groups.The first group take ischemic preconditioning before IR(IP group);The second group was simple myocardial ischemia-reperfusion injury(saline group);The third group plus DMOG(DMOG group);The forth group was the sham-operation group(saline group).There are 8 rats in each group.IP group,IR group and DMOG group rats received ligation of the proximal left anterior descending coronary artery (LCA)for 30 minites to induce acute myocardial infarction,and followed by reperfusion for 180 minites.The control group don't take ligation of LCA.In IP group,short-term ischemic was taken,three times for each 5 minites prior ligation of LCA;In DMOG group,rats were administered intraperitoneally 24h before myocardial ischemia-reperfusion injury.After 180 minites of operation,rats were killed.The heart was excised and LV was cut to immersed into 4%paraformaldehyde in phosphate-pufferd saline for more than 24 hours.The infarct size,expressed as a percentage of the left ventricle,was calculated by dividing the circumference of the infarct by the total circumference of he left ventricle including the septum..The HIF-1αand HO-1mRNA gene expression was observed through Reverse transcription-polymerase chain reaction(RT-PCR)..The HIF-1αand HO-1mRNA protein expression was also observed through Westen blot.The level of plasma IL8 and MPO were mesureed by ELLSA kit.Results1.Infarct size:The infarct size in IP group,DMOG group and MI group was (21.56±2.27)%,(23.56±2.12)%and(35.21±2.34)%respectively,The infarct size in IP group and DMOG group decreased significantly vs MI group(P＜0.01).2.HIF-1αand HO-1mRNA gene expression:The HIF-1αand HO-1mRNA gene expression was observed at 180 minites after IRI through Reverse transcription-polymerase chain reaction(RT-PCR);The HIF-1αand HO-1mRNA gene expression increases in IP group and DMOG group increase more than that in saline group and control group(P＜0.01).3.HIF-1αandHO-1 protein expression:It has the same change as HIF-1αandHO-1mRNA gene expression.4.Caspase-3 protein expression:caspase-3 protein expression in IP group and DMOG group significantly decreased vs that in saline group and control group(P＜0.01).5.Plasma IL8 and MPO level:The plasma IL8 and MPO level in IP group and DMOG group significantly decreased vs that in saline group and control group(P<＜0.01).ConclusionIschemic preconditioning can attenuate IRI injury,and HIF-1αplay an important protective role;HIF-1αand HO-1 gene expression increasesd significantly in IP group and DMOG group than that in saline group and control group;HIF-1αandHO-1 protein expression have the same change as HIF-1αand HO-1mRNA gene expression;The infarct size in IP group and DMOG group decreased significantly than that in MI group;DMOG has effect as ischemic preconditioning.