Angiogenesis Is Induced In A Rat Model Of Hindlimb Ischemia By Mutants HIF-1α Gene

IntroductionCoronary artery disease(CAD) is the most common type of heart disease.It is the leading cause of death in urban and rural residents in China and will become the most disease burden and primary death causes in the world in 2020.Now,the main treatment of CAD are medicine and revascularization,including percutaneous coronary intervention(PCI) and coronary artery bypass graft(CABG) surgery.A significant proportion of patients with coronary artery disease,were called end stage patients,have symptoms refractory to medical treatment,yet are unsuitable for conventional revascularization techniques,like PCI and CABG.Such patients are potential candidates for alternative forms of coronary revascularization,like therapeutic angiogenesis.Two major avenues for achieving therapeutic angiogenesis are currently under intense investigation:gene therapy(the introduction of new genetic material into somatic cells to synthesize proteins that are missing,defective, or desired for specific therapeutic purposes) and protein-based therapy (administration of the growth factors,instead of the genes encoding for the growth factors responsible for angiogenesis.) From 90's,it was found that angiogenic growth factors,which plays an important role in physiologic and pathologic angiogenesis,could contribute to neovascularization and promote collateral artery development in animal models of myocardial and hindlimb ischemia.Stimulation of angiogenesis represents an important new therapeutic approach for CAD. Vascular development involves vasculogenesis,in which endothelial cells form a primary tubular network,as well as angiogenesis,in which vessel size and structure are modified based upon flow and branching occurs to insure that all cells receive adequate O₂ delivery.In adults,angiogenesis occurs in response to tissue hypoxia/ ischemia and plays an important role in determining the progression of ischemic heart disease and cancer.A critical molecular pathway induced by hypoxia/ischemia is the activation of hypoxia-inducible factor 1,a transcriptional activator of genes encoding vascular endothelial growth factor and other important mediators of angiogenesis. Angiogenesis is a highly complex,orchestrated process that plays a critical role in normal development and in the pathophysiology of common diseases.It requires a balance of multiple angiogenic factors.Results of several randomized,double-blind. placebo-controlled trial of therapeutic angiogenesis by gene transfer have been reported that therapeutic angiogenesis strategies that involve administration of a single angiogenic factor was insufficient.Different angiogenic factors appear to play complementary roles in the process of vascular development.So combined gene therapy become the research hotspots of therapeutic angiogenesis.Hypoxia-inducible factor 1(HIF-1) is a heterodimeric transcription factor that functions as a master regulator of mammalian oxygen homeostasis.At a molecular level.HIF-1 is composed of an Q₂-regulated HIF-1αsubunit and a constitutively expressed HIF-1βsubunit.The HIF-1αsubunit is 826-amino-acid protein that is regulatory and is unique to the hypoxic response.In response to low level of oxygen, subunit HIF-1αexpression is up-regulated and transactivates its target genes essential (＞60) for glucose uptake,energy metabolism,erythropoiesis and vascular development.There are two key domains in HIF-1α,which named oxygen dependent degradation domain(ODDD) and transactivation domains(TAD).When cells maintained at low oxygen tension are returned to a nonhypoxic environment,HIF-1αprotein levels decay rapidly,as the protein is degraded via the ubiquitin-proteosome pathway.It was related to the prolyl hydroxylation of the two proline residus Pro564 and Pro402 in ODD.Under normoxic conditions,HIF-1αis hydroxylated on proline residues 402 and 564,and this modification functions as a molecular signal for ubiquitination and degradation of the protein.As a result,HIF-1αhas an extremely short half-life under normoxic conditions.Since O₂ is rate-limiting for the prolyl hydroxylases that perform this reaction,prolyl hydroxylation of HIF-1αis inhibited in hypoxic/ischemic tissues.This causes accumulation of the protein,which dimerizes with HIF-1β,generating a functional heterodimer that can bind to specific nucleotide sequences in target genes such as VEGF and activate their transcription.The activaty of transcription of HIF-1 is related to the hydroxylation of Asn803 in TAD.These dates identified the function of two primary domains in HIF-1α."Discovery of these could open up new therapeutic possibilities for the many diseases such as ischemic heart disease and strock"(Science).We have constructed the adenovirus mutant HIF-1α-Ala564(Ad-H₅₆₄), adenovirus mutant HIF-1α-Ala564-Ala402(Ad-H_564/402) and adenovirus mutant HIF-1α-Ala564-Ala803(Ad-H_564/803) in last 3-4 years.In subsequent investigations, we establish the model of rat hindlimb ischemia,intramuscular transfect mutant HIF-1 a genes respectively,and evaluate the function of these mutant HIF-1αgenes.The transfection efficiency of genes could be promoted by vector.Adenoviruses is the vectors most often studied likely because of the ease of production,reasonable transfection efficiency,and expression in nonproliferating cells.The life cycle does not normally involve integration into the host genome,rather they replicate as episomal elements in the nucleus of the host cell and consequently there is no risk of insertional mutagenesis.It is more suitable for cardiomyocyte because of its specific features.ObjectTo investigate the gene expression and compare the function of mutant HIF-1αadenoviral-mediated gene in various sites(Ad-H₅₆₄,Ad-H_564/402 and Ad-H_564/803) in the rat model of hindlimb ischemia.Methods (1) In the first experiment,mutant HIF-1αgene adenovirus vector was amplified in HEK293A cells and purified by ultracentrifugation in CsC1 step gradient solutions and transfect into the hela cells.Then the adenovirus was determined by electron microscope,PCR and sequencing,and the amount of viral particles was determined by spectrophotometry.(2) In the second experiment,the acute rat ischemic hindlimb models were produced and the Ad-LacZ,Ad-H₀,Ad-H₅₆₄,Ad-H_564/402,Ad-H_564/803 and normal saline (NS) 500μl were administered respectively intramuscularly into the ischemic limb in 5 sites.The gene expression was evaluated by reverse transcriptase polymerase chain reaction(RT-PCR) after 1,3,5,7 days of intramuscular gene transfer in skeletal muscles of rat in vivo.(3) The protein expression of HIF-1αand CD31 were observed by immunohistochemistry stain after 28 days of intramuscular gene transfer in skeletal muscles of rat in vivo.(4) Selective internal iliac angiograghy was performed by the same angiogragher to illustrate the microvessel density at the 28^th day after intramuscular gene transfer.Results(1) The correct mutant adenovirus HIF-1αwas obtained after being amplified and purified.The last titre of adenovirus was about 10¹¹ OPU/ml.Not found the wild type adenovirus.(2) The results of RT-PCR showed that mutant adenovirus HIF-1αimprove the gene expression in ischemic muscle in vivo,and the relative expression was the highest at the 7^th day after intramuscular gene transfer(F=24.942,P=0.000).It is higher in all groups of mutant adenovirus HIF-1αthan that of control groups (F=99.380,P=0.000).The relative expression in group Ad-HIF-1α_564/402 was the highest(0.7952±0.1563,P=0.000) in three groups of mutant adenovirus HIF-1α, then in group Ad-HIF-1α_564/803(0.5815±0.0994,P=0.000),then the Ad-HIF-1α₅₆₄ (0.5447±0.1413)。 (3) Many positive cells of HIF-1αand CD31 by immunohistochemistry stain were found in skeletal muscles and interstitial tissues in groups of mutant HIF-1αgene at the 28^th day after intramuscular gene transfer,and the positive cells was more densely around the blood vessels.The positive cells in all groups of mutant HIF-1αgene was more densely than that of nature HIF-1αgene and was the most densely in group of Ad-H_564/803.The more minimal the vessel is,the darker the positive cells was been stained.(4) The results of angiograghy and vascular cast showed that mutant adenovirus HIF-1αcould induce angiogenesis and promote collateral artery development in vivo after 28 days of gene transfer,and the function of mutant adenovirus HIF-1αis more powerful than that of the nature HIF-1α.The microvessel density in group Ad-H_564/803 was the greatest in three groups of mutant adenovirus HIF-1α.ConclusionThe mutant adenovirus-mediated gene transfer of HIF-1αimproves the gene expression of HIF-1αmRNA and HIF-1αprotein in vivo and contributes to neovascularization and promotes collateral artery development in rat models of hindlimb ischemia.The function of the Ad-HIF-1α_564/803 is more powerful than that of Ad-HIF-1α₅₆₄ and Ad-HIF-1α_564/402.