Effects Of Hif-1? Mutant On Neovascular Permeability And Energy Metabolism In Acute Myocardial Infarction Rats

BackgroundThe incidence and mortality of coronary atherosclerotic heart disease are increasing year by year,it is one of the leading causes of death in developed countries and has become a major disease threatening the health of our people.Rational diet,lipid-regulating,antiplatelet,anticoagulation,stable plaque,etc.are the conventional treatment methods for coronary atherosclerotic heart disease;Percutaneous coronary intervention and coronary artery bypass grafting and other methods of revascularization have developed rapidly and have significant clinical effects,but there are certain requirements for patients'indications and there are problems such as postoperative restenosis;Therefore,it is necessary to find a new way to treat coronary atherosclerotic heart disease.In tissue and organ ischemia,the body will have a compensatory mechanism.The expression of pro-angiogenic factors and their corresponding receptors in the body will be upregulated,thereby promoting the formation of collateral circulation in ischemic local tissue and improving its ischemic and hypoxic conditions,but this compensatory effect is often very limited.Therefore,therapeutic angiogenesis,which is mainly supplemented with exogenous pro-angiogenic factors?recombinant protein or gene?,has become a new research hotspot.However,due to the large dose required,the short efficacy,the repeated use and other drawback limited the protein treatment.The gene transfection technology can be used in the localization of continuous expression,fewer side effects and other advantages make it a research hotspot in the field of angiogenesis in ischemic tissue.Angiogenesis is a complex biological process that requires the participation and coordination of multiple factors.The earliest studies that used to promote angiogenesis were VEGF and FGF.Studies have shown that it can increase ischemic myocardial perfusion,reduce infarct size,and have certain safety and feasibility.These results are encouraging.However,Phase II clinical trials have found that VEGF causes immature angiogenesis,high vascular permeability,and tissue edema,while FGF has a significant proteinuria in some experimental patients and other shortcomings restrict their application in the field of therapeutic angiogenesis.Therefore,it is necessary to find new angiogenic factors that are more suitable for clinical applications.Hypoxia-inducible factor-1?HIF-1?has attracted the attention of researchers because it is the upstream switch of master angiogenesis.HIF-1 is an important nuclear transcription factor that is widely present in human and mammalian cells under hypoxic conditions and participates in the regulation of various genes such as oxygen balance,angiogenesis,glucose metabolism,erythropoiesis,cell proliferation,cell death,and autophagy,etc.HIF-1 has been found to be involved in the regulation of angiogenesis in more than 30 genes?such as VEGF,PLGF,PDGF,etc?.HIF-1 is a heterodimer composed of an?-subunit and a?-subunit,HIF-1?is a structural subunit stably expressed in cells,and HIF-1?is a functional subunit regulated by oxygen concentration,which determines the activity of HIF-1.Under hypoxic conditions,the expression of HIF-1?is enhanced,but the half-life of HIF-1?under normoxia is very short,about 1²min,Under hypoxic conditions,the expression of HIF-1?is enhanced,but the half-life of HIF-1?under normoxia is very short,it can be completely degraded in 5minutes.HIF-1?has two important structural regions,oxygen-dependent degradation domain?ODDD?and transcription activation domain?TAD?,of which TAD includes N-TAD and C-TAD.It is currently known that C-TAD regulates most of the transcriptional activity by interacting with co-activators such as CBP/P300.Under normoxic conditions,prolyl hydroxylase acts on the two proline?Pro?residues at positions 402 and 564 of the ODDD region,causing it to undergo hydroxylation,resulting in the rapid degradation of HIF-1?Within minutes;Aspartate enzyme hydroxylation of asparagine?Asn?at position 803 in the C-TAD region of HIF-1?can prevent the binding of HIF-1?to some of the co-activating factors and reduce its transcriptional activity.Under hypoxic conditions,prolyl hydroxylase and asparaginase are inactivated,HIF-1?becomes stable,then binds to HIF-1?,and then through the combination of C-TAD and ancillary activating factors,thereby enhancing the expression of downstream pro-angiogenic factors.Based on the above,our group has previously combined mutations at three key sites of HIF-1?and used adenovirus as a vector,successfully constructed the recombinant adenovirus HIF-1?564/402/803?Ad-HIF-1?-Trip?,that improves the stability and transcription activity of HIF-1?on the basis of ensuring its integrity as much as possible,which makes it stable and efficient expression under normoxic conditions.Some studies have shown that the Ad-HIF-1?-Trip is stably expressed in the ischemic hindlimbs of rabbits,and can promote the expression of downstream angiogenic factors,promote neovascularization,and improve local tissue ischemia and hypoxia without significantly increasing the permeability of neovasculari-zation.But it is unknown whether the Ad-HIF-1?-Trip has the same effect in ischemic myocardium.In addition,energy metabolism is the material basis of cardiac activity.ATP is the direct energy source for maintaining the normal life activities of the heart,and myocardial acute ischemia is often accompanied by changes in energy metabolism,but the exact relationship between the two is not yet clear.What is the effect of Ad-HIF-1?-Trip on the energy metabolism of ischemic myocardium has not been reported.Therefore,based on the previous study of our group,this study investigated the effects of Ad-HIF-1?-Trip on the neovascular permeability and energy metabolism of ischemic myocardium in rats with acute myocardial infarction.ObjectiveTo investigate the effect of HIF-1?mutant on the permeability of neovascularization and energy metabolism in ischemic myocardium of acute myocardial infarction rats.MethodsHIF-1?mutants Ad-HIF-1?-402/564 and Ad-HIF-1?-Trip were constructed and confirmed the transfection concentration of the virus by cell culture?preliminarily completed by our research group?.The model of myocardial infarction was established by ligation of the left anterior descending coronary artery on 250 male Sprague-Dawley rats,and then were randomly divided into five groups:Saline group?n=50?,Ad-Null group?n=50?,VEGF group?n=50?,Ad-HIF-1?-564/402 group?n=50?,and Ad-HIF-1?-Trip group?n=50?.The ischemic myocardium received saline,Ad-Null,VEGF,Ad-HIF-1?-564/402,or Ad-HIF-1?Trip 0.1ml?the virus titer is 2.0×10⁸ PFU?,respectively,by intramuscular injection.Five days after gene transfection,the rats were randomly selected from the gene transfection group for immunofluorescence examination to observe the effect of gene transfection.On the 7^th,14^th and 28^th days after gene transfection,compared the difference of body weight,body mass ratio,left ventricular mass to total heart mass in acute myocardial infarction rats in each group;the real-time PCR and Western-blot were used to assess the nucleic acid and protein expression levels of angiogenesis-related factor HIF-1?and its downstream genes VEGF,PLGF,and PDGF in ischemic myocardium;In the heart of rats with acute myocardial infarction,the heart general view,HE staining and immunohisto-chemical staining of vWF were performed to observe the angiogenesis;the evans blue staining was used to evaluate the permeability of neovascularization;the Na⁺-K⁺ATPase,Ca²⁺ATPase,total ATPase,and Succinate dehydrogenase?SDH?levels were detected by spectrophotometer to evaluate the energy metabolism of ischemic myocardium.Results1?The results of the difference of body weight,body mass ratio,left ventricular mass to total heart mass after gene transfection showed:Comparison between groups at each time point:There was no statistical difference in body weight after gene transfection?P>0.05?.The intra-group comparison of the weight difference shows:There was significant difference between 28 days and 7 days in Ad-Null group?P<0.05?,while there was no statistical difference between the rest time points?P>0.05?;In Saline group,VEGF group,Ad-HIF-1?-402/564 group and Ad-HIF-1?-Trip group,there was no significant difference between 14 days and 7 days?P>0.05?,but the differences between 28 days and 7 days,28days and 14 days were statistically significant?P<0.05?.The ratios of HW/BW and LHW/HW showed a certain downward trend from 7 days,14 days to 28 days,and the 28-day comparison between groups showed that the HW/BW and LHW/HW ratios of Ad-HIF-1?-402/564 group and Ad-HIF-1?-Trip group were lower than those of other groups,Ad-HIF-1?-Trip group was lower than Ad-HIF-1?-402/564 group,whlie whether the between groups or intra-group comparison was no statistical difference?P>0.05?.2?The heart general view and HE staining qualitative results of acute myocardial infarction rats showed:Myocardial infarct size range from large to small was Saline group/Ad-Null group>VEGF group>Ad-HIF-1?-402/564 group>Ad-HIF-1?-Trip group;And HE staining showed that the cardiac morphology and arrangement of myocardial fibers in Ad-HIF-1?-Trip group were relatively neat,inflammatory cells were relatively few,and vascular tissue was seen in the peri-infarct area.3?At 7 days after gene transfection,the expression level of HIF-1?mRNA in the myocardium from strong to weak were:Ad-HIF-1?-Trip group>Ad-HIF-1?-564/402group>VEGF group/Ad-Null/Saline group,the difference was statistically significant?P<0.05?.Over time,the expression of HIF-1?was obviously reduced in Ad-HIF-1?-Trip group and Ad-HIF-1?-564/402 group,to 28 days after gene transfection,the expression of HIF-1?in the 5 groups has no significant difference.At 7 days after gene transfection,the expression level of HIF-1?downstream gene VEGF mRNA from strong to weak were:Ad-HIF-1?-Trip group/VEGF group>Ad-HIF-1?-564/402 group>Ad-Null group/Saline group,the difference was statistically significant?P<0.05?.At 14 days after gene transfection,the expression levels of VEGF in Ad-HIF-1?-Trip group and VEGF group were obviously reduced,the expression of VEGF in the 5 groups has no significant difference at 28 days.At 7 days after gene transfection,the expression level of PLGF and PDGF mRNA in each group showed Ad-HIF-1?-Trip group>Ad-HIF-1?-564/402group>VEGF group/Ad-Null group/Saline group,the difference was statistically significant?P<0.05?.At 14 days after gene transfection,the expression of PLGF and PDGF in the Ad-HIF-1?-Trip group and Ad-HIF-1?-564/402 group were obviously reduced,and there was no significant difference between the 5 groups at 28 days.4?At 7 days after gene transfection,the results of Western-blot analysis of ischemic myocardium in rats showed:Among the groups,the expression levels of HIF-1?,VEGF,PLGF and PDGF proteins were highest in the Ad-HIF-1?-Trip group,and the difference was statistically significant?P<0.05?.5?Immunohistochemistry results showed that the number of vWF positive:Ad-HIF-1?-Trip group>Ad-HIF-1?-402/564 group>VEGF group>Saline group/Ad-Null group.Microvessel density?MVD?statistics showed that the comparison between groups at each time point was:Ad-HIF-1?-Trip group>Ad-HIF-1?-402/564 gro-up/VEGF group>Saline group/Ad-Null group,the difference was statistically significant?P<0.05?.The comparison results in each intra-group showed that:The vascular density was the most at7D,reaching equilibrium around 14D,and tending to be stable at 28D;The number of MVD7D>14D>28D,the difference between 7D and 14D,7D and 28D was statistically significant?P<0.05?,but there was no significant difference between 14D and 28D?P>0.05?.6?The Evans blue perfusion experiment in acute myocardial infarction rats showed:The heart general view showed a comparison between the groups:blue staining in the ischemic myocardium,Ad-HIF-1?-Trip group<Saline group/Ad-Null group<Ad-HIF1?-402/564group<VEGF group;Intra-group comparison showed the content of Evans blue in the ischemic myocardium was 28D<14D<7D.The content of Evans blue in ischemic myocardium homogenate was detected,the results of comparison between groups were as follows:Ad-HIF-1?-Trip group<Saline group at 7D?P<0.05?;VEGF group>Saline group?P<0.05?and Ad-HIF-1?-Trip group<VEGF group/Ad-HIF-1?-402/564 group?P<0.05?at14D;Ad-HIF-1?-Trip group<Ad-HIF-1?-402/564 group?P<0.05?,VEGF group/Ad-HIF-1?-402/564 group>Saline group?P<0.05?and Ad-HIF1?-402/564 group>Ad-Null group?P<0.05?at 28D;there was no statistical difference between the other groups?P>0.05?.The content of Evans blue in ischemic myocardium homogenate intra-group comparison showed:In saline group,28D<14D<7D?P<0.05?;In Ad-Null group and VEGF group were28D<14D/7D?P<0.05?;In Ad-HIF-1?-Trip group 28D<7D?P<0.05?;There was no statisti-cal difference among the other intra-groups?P>0.05?.It can be seen that over time,the content of Evans blue in myocardium is gradually reduced.7?The results of detection of energy metabolism in ischemic myocardium of acute myocardial infarction rats showed,the comparison between groups at each time point:Na⁺-K⁺ATPase and Total ATPase in Ad-HIF-1?-Trip group were relatively high,but there was no significant difference among groups?P>0.05?;In 7D,Ca²⁺-ATPase showed Ad-HIF-1?-Trip group/Ad-HIF-1?-402/564<Saline group/Ad-Null group/VEGF group?P<0.05?,SDH was the highest in the Ad-HIF-1?-Trip group,and there was a statistically significant difference between the groups?P<0.05?;In 14D,Ca²⁺-ATPase showed Ad-HIF-1?-Trip group>Ad-HIF-1?-402/564>VEGF group>Saline group/AdNull group?P<0.05?,SDH was the highest in the Ad-HIF-1?-Trip group,and there was no statistically significant difference between the groups?P>0.05?;In 28D,Ca²⁺-ATPase showed Ad-HIF-1?-Trip group/Ad-HIF-1?-402/564 group>Saline group/Ad-Null group/VEGF group?P<0.05?,SDH showed Ad-HIF-1?-Trip group/Ad-HIF-1?-402/564group>Saline group/Ad-Null group?P<0.05?and Ad-HIF-1?-Trip group>VEGF group?P<0.05?;There was no statistically significant difference between the other groups.The comparison results in each intra-group showed:The Na⁺-K⁺ATPase and Total ATPase showed an increasing trend in7D,14D,28D?P<0.05?;Ca²⁺-ATPase and SDH showed a trend of increasing first?14D?and then decreasing?28D??P<0.05?.Conclusion1?HIF-1?-Trip can promote the neovascularization of ischemic myocardium in acute myocardial infarction rats,and the neovascular permeability does not increase significantly.2?HIF-1?-Trip can improve the energy metabolism of ischemic myocardium in rats with acute myocardial infarction.