HIF-1α-mutant On The Angiogenesis And Heart Function Of Ischemic Heart Disease

Background Coronary atherosclerosis sex heart disease is one of the leading causes of death in the developed countries in recent years,the morbidity and mortality in the developing countries also showed a trend of increased significantly.Anticoagulation and antiplatelet aggregation,lipid and stable plaques is in recent years,according to conventional drug treatment of ischemic heart disease,percutaneous intervention in blood vessel revascularization has been developing rapidly and coronary artery bypass grafting is an important treatment in recent years,but at the same time there are stent grafts restenosis and part of the diffuse is not suitable for interventional therapy for patients with problems,affect the prognosis of patients.In vascular stenosis or occlusion causes tissue organs such as ischemia,and factors related to promote angiogenesis in body expression level,promote the formation of local collateral circulation ischemia,but its compensatory effect often incomplete or not in time.Added exogenous angiogenic factors(recombinant protein or gene)can promote the formation of new blood vessels in theory,so as to improve the blood supply of the ischemic tissue,called the therapeutic angiogenesis.To ischemic experiment animals inrecent years to carry out the research of therapeutic angiogenesis of including protein therapy and gene therapy,protein therapy dose is large,relatively short time,and often need to use repeatedly,is now rarely used.Gene therapy can be expressed in local continuous work,may only need a single treatment without the need for repeated applications.So in gene transfection technique to promote angiogenesis is a current research hot spot.With the further study of mechanisms of angiogenesis,has found a variety of factors with the ability to promote angiogenesis.The earliest used in animal and clinical studies for VEGF and FGF.VEGF and FGF passed the phase I clinical trials,but the results of phase II clinical trial are not so good[1],the formation of VEGF can cause premature vascular greaten,vascular permeability and tissue edema in[2],FGF in promoting new blood vessels form at the same time,some experimental patients with proteinuria.So need to find new angiogenesis factors.Angiogenesis is a complex biological process,and need a variety of factors involved,rely on a single angiogenic factors is difficult to complete.In recent years,research shows that: HIF-1 is a very important nuclear transcription factor that can directly or indirectly control more than 100 kinds of downstream target genes[3,4],the downstream target genes in blood vessel growth,blood vessels,tension,sugar metabolism,erythropoiesis,stem cell differentiation,and other fields play an important role in[5].HIF-1 is composed of two subunits alpha and beta dimers[6],in the form of different source sex dimers.HIF-1 alpha is single-minded HIF-1 subunits by O2 adjustment,decided to the activity of HIF-1.In the field of angiogenesis gene therapy,research is focused on HIF-1 alpha.So far,has been found out by HIF-1 alpha directly regulated genes involved in angiogenesis of more than 30,is the core upstream of angiogenesis regulation factor[7-10].HIF-1 alpha Domain structure depend on the Degradation area containing Oxygen,Oxygen Dependent Degradation Domain,ODDD),Pro402 ODDD area and whether two proline residues of Pro564 hydroxylation decided the protein stability[11].HIF-1 alpha C terminal contains independent transcription activation zone(C-Transactivation Domains,C-TAD).C-whether TAD Asn803 hydroxylation decided its protein transcription activity[12].Oxygen,often Pro402,Pro564 and Asn803 hydroxyl,alpha HIF-1 is not stable,rapid degradation in 3 to 5 minutes,transcription activity decline.Low oxygen,the process is restrained[13,14].Hence,our study team had successfully constructed the recombinant adenovirusmediated hypoxia inducible factor-1α of triple mutant by point mutations of Pro402,Pro564 and Asn803.The enhanced stability of HIF-lα and transcriptional activity.So as to achieve a satisfactory effect on promoting angiogenesis effec.Our previous in vitro studies have shown that the key functional areas of ODDD Pro402,Pro564 a nd TAD Asn803 combined mutation construct Ad-HIF-1α-Trip under constant oxyge n can be stable,Thus further promote to the downstream promoting angiogenesis factor regulation.This study to establish a rat model of acute myocardial infarction,adopt the way of muscle injection with adenovirus as a carrier of HIF-1 alpha mutant Ad-H IF-1 alpha Trip transfection in regional myocardial ischemia of rats research Ad-HI F-1 alpha Trip to its effect on promoting angiogenesis for myocardial ischemia of rats and some security.Methods1.In the first stage of our experiment,Ad-HIF-1α-Trip 、Ad-HIF-1α-402/564 and Ad-Null were amplified in HEK293 A cells and purified by ultracentrifugation in Cs CI concentration gradient solutions.Virus drops is 1.0×1011plate forming unit(This topic first build).2.150 establish acute myocardial infarction model in rats,randomly divided into 5groups: Saline group,Ad-Null group,the Ad-HIF-1α-402/564 group,the Ad-HIF-1α-Trip group,VEGF group,intramuscular injection in ischemic myocardium by gene transfection.Gene transfection after 7 days,14 days,28 days on rat heart colour to exceed examination evaluation of cardiac function,take the rat heart pathological and immunohistochemical CD31 staining,observing angiogenesis;28 days after gene transfection executed in rats,take heart,TTC examination,assessment of infarction area.3.Gene transfection 1 week after extraction of venous blood determination ofmyocardial enzymes,troponin I,liver function,renal function,blood routine.4.Application of SPSS16.0 statistical software for data analysis.Results1.Ultrasonic results show: 7 days after gene transfection,LVEF and LVFS performance for Ad-HIF-1α-Trip group > VEGF/Ad-HIF-1α-402/564 group >Saline/Ad-Null group,with the extension of time,each index decreases with time trends,but Ad-HIF-1α-Trip group is relatively stable,prompt Ad-HIF-1α-Trip group of cardiac function is relatively stable,other groups have different degrees of heart function.2.7 days after gene transfection myocardial tissue immunohistochemical,CD31 positive cells were dyed tan or brown,scattered in myocardial infarction area and infarction peripheral area,Ad-HIF-1α-Trip group performance is most obvious.Microessel-density counts(Microvessel density,MVD)count,the order from high to low is: Ad-HIF-1α-Trip group >Ad-HIF-1α-402/564、VEGF group >Ad-Null group /Saline group,One way ANOVA analysis results show: contrast differences between groups was statistically significant(P < 0.05).Pairwise comparison results show there were no statistically significant difference between the Ad-Null group and Saline group,Ad-HIF-1α-402/564 and VEGF group(P > 0.05),more than two contrast differences were statistically significant(P < 0.05).14 days after gene transfection,MVD of Ad-HIF-1α-Trip group,VEGF group,the Ad-HIF-1α-402/564 group was obviously reduced,but still more than Ad-Null group and Saline group,to 28 days after gene transfection,5 groups of MVD in roughly the same level.3.Rat model of acute myocardial infarction after gene transfection,7 days in the rat blood routine and biochemical indexes comparison results show that: leukocyte groups of rats were characterized by increased slightly,but there was no statistically significant difference between the groups(P > 0.05),RBC,RBC,PLT,Hb,MCV,MCH counting were in normal range,there was no statistically significant difference between the groups(P > 0.05),c Tn I,CK,CK-MB concentration were higher than normal,from low to high performance as the Ad-HIF-1α-Trip group < VEGF/Ad-HIF-1α-402/564 group < Ad-Null/Saline group,the difference was statistically significant(P < 0.05).Indicators ofliver and kidney function is no obvious abnormal.4.HE staining showed: heart,liver,kidney,lung,spleen,testis,HE staining were observed under light did not see obvious pathological histology change.5.TTC staining results: 28 days postoperatively,TTC staining method is used to inspect each rat myocardial infarction area.Ad-Null group/Saline group > VEGF group >Ad-HIF-1α-402/564 group > Ad-HIF-1α-Trip group,the difference was statistically significant.Conclusions.1.Ad-HIF-1α-Trip group can promote rat ischemic myocardial angiogenesis,improve heart function.2.Ad-HIF-1α-Trip group transfection myocardial infarction rats did not cause obvious adverse effects to the important viscera function,have certain security.