Effects Of Recombinant Adenovirus-mediated Hypoxia Inducible Factor-1α Of Triple Mutant On Modulating Angiogenesis

BackgroundNowdays coronary heart disease has become a serious threat to the health of human being, the incidence of which has increased every year. The coronary artery grafting(CABG),percutaneous transluminal coronary angioplasty(PTCA) and percutaneous coronary intervention(PCI) develope dramatically, however, amount of patients having symptoms such as multi-branches lesion, complete vascular occlusion, obstruction of the arteriole after PCI or CABG, refractory to medical treatment, and are unsuitable for conventional revascularization therapies.It is necessary to rebuild collateral circulation for the treatment of ischemic heart disease.The recent research found that angiogenic growth factors, which plays an important role in physiologic and pathologic angiogenesis, could contribute to neovascularization and promote collateral arterydevelopment in animal models of myocardial and hindlimb ischemia. Stimulation of angiogenesis represents an important new therapeutic approach for CAD.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 trial of therapeutic angiogenesis 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. By now there were many angiogenesis factors involved in modulating angiogenesis have be found,such as:vascular endothelial growth factor(VEGF), Fibroblast Growth Factor (FGF), angiopoietin, platelet derived growth factor(PDGF), Insulin-like growth factor (IGF), monocyte chemoattractant protein 1(MCP-1) et.al. The recent research finds that vascular endothelial growth factor (VEGF) can promote the progress of angiogenesis being transferred by a safe and effective vector or by directly injecting into ischemic myocardium, which may be a new alternative strategy for the treatment ofischemic heart disease. VEGF and FGF have been studied widely, there were evidences indicated that VEGF gene therapy could result in vascular permeability, tissue edemaetc and FGF therapy was associated with proteinuria. Hypoxia inducible factor 1(HIF-1), an upstream transcription factor, plays a key role in the angiogenesis response to hypoxia. HIF-1 participate various physiological and pathological processes such as angiogenesis and erythropoiesis by modulating the transcription of more than 60 target genes including VEGF, Ang, EPO, PDGF, HO-1, etc. It has been demonstrated in the preclinical studies that gene therapy with constitutively active formof HIF-1αmay result in physiologically functional neovascularization. Compared with VEGF, HIF-1 gene therapy was not associated with vascular permeability, tissue edema. So HIF-1αis considered to be one of the most prospective factors of angiogenesisHIF-1 is consists of a constitutively HIF-1βsubunit and a HIF-1αsubunit, the expression of HIF-1βis not regulated by the cellular oxygen concentration,the HIF-1αis the functional subunit,the stabilization and transcriptional activity of HIF-1αare highly regulated by the cellular oxygen concentration. Under Hypoxic conditions, HIF-1αwill move to cellular nucleus and form heterodimer with HIF-1βsubunits, then the transcription of HIF-1αis actived. Under normoxic conditions, oxygen mediates posttranslational hydroxylation of two proline residues Pro564 and Pro402 in the oxygen dependent degradation domain (ODDD) of HIF-1α. Hydroxylation occurs via the activities of three prolyl hydroxylase domain proteins (PHD1-3) and mediates binding of the tumor suppressor protein von Hippel-Lindau (VHL) to the N-terminal transactivation domain (TAD-N) of HIF-1α. Hydroxylation of the asparagines residue 803 (Asn803) in the C-TAD of HIF-1αby factor inhibiting HIF-1(FIH-1) prevented the interaction of HIF-1αwith CBP/P300, whereas hypoxia blocks degradation resulting in HIF-la stabilization and accumulation in the cell. Therefore, we finished the mutation of Pro402, Pro564 and Asn803 in HIF-la gene and successfully constructed the adenovirus vector of human hypoxia-inducible factor-1αof triple mutant (Ad-HIF-la564/402/803) in order to get high levels of gene expression under normoxic condition. In this study,we plated hMVEC on Matrigel after infected with Ad-HIF-1α-564/402/803, Ad-HIF-1α-nature, Ad-Null, to compare the number of capillary-like tube structures in vitro, and we use the chick embryo chorioallantoic membrane (CAM) model to observe angiogenesis effect of Ad-HIF-1α-564/402/803.ObjectiveTo further study the role of HIF-1αgene in modulating angiogenesis and the biological effect of Ad-HIF-1α564/402/803, we plate hMVEC on Matrigel af ter infected with Ad-HIF-1α-564/402/803,Ad-HIF-1α-nature, Ad-Null, to comp are the number of capillary-like tube structures in vitro. Then we establish the model of CAM to evaluate the function of these mutant HIF-la genes.MethodThe first part:Recombinant adenovirus Ad-lacZ, Ad-Null, Ad-HIF-1αnatur e, Ad-HIF-la564/402/803 were'amplified in HEK293A cells and purified by ul tracentrifuggation in CsCl step gradient solutions, adenoviral titer wasdetermine d by End-Point Dilution Assay. The recombinant adenovirus was confirmed by polymerase chain reaction (PCR) and DNA sequence analysis.The second part:1. Gelled Matrix Growth Factor Reduced if placed at on ice for 24-48 ho urs before use, BD Matrigel must be re-liquified 4℃on ice before use, and u se pre-cooled pipettes, tips, and tubes. Keeping culture plates on ice, add 75μL per of 96 well plate. Place plates at 37℃for 30 minutes. Plates are ready to use when the matrigel become gel. Then hMVEC were seeded into 96 well plates and incubated with Ad-HIF-la-564/402/803, Ad-HIF-1a-nature, and Ad-Null. Compare the number of capillary-like tube structures for per 6 hours. Th e data obtained were analyzed for one way ANOVA using SPSS 13.0 software package.2.8-day fertilized eggs were placed in 37.8±0.5℃constant temperatur e incubator, biochemical, relative humidity 50%～60%. After 24 hours, make a small hole in the air chamber with the injection needle then, open a square window with the Ophthalmological tweezers. With a careful eye tweezers to unfold the inner shell membrane, the above procedure was performed under ste rile operating manner. Outer shell membrane of the CAM was detached from t he shell and the underlying CAM vessels are disclosed. Ad-HIF-1a-564/402/803,Ad-HIF-la-nature,Ad-Null and PBS was added in vector the respectively and the window is sealed with transparent adhesive paper and incubation goes on for 72 hours. then open the window again and record the image of blood vessels with camera; using Image Pro Plus6.0 software to collect the image an d calculate the ratio of vascular area and CAM area. At last, the data obtaine d were analyzed for one way AN OVA using SPSS 13.0 software package.ResultThe first part of this study:High-titer adenovirus were produced after amplified.The titer of Ad-HIF-1α564/402/803,Ad-HIF-1αnature,Ad-Null,Ad-LacZ, was 1.5×1012,1.2×1011,2.0×1012,6.3×1011pfu/ml respectively.HEK293A cells could be effectively infected by recombinant adenovirus in vitro, and cytopathic effect appeared just after 24 hours. The DNA of HIF-la564/402/803 gene was extracted to confirm the presence of three mutant points by PCR, and the size of PCR products were 380bp,460bp and 214bp respectively, the result of DNA sequence analysis was in excellent accord with expection. The hMVECs were infected by Ad-lacZ, and the optimal multiplicity of infection was 100 pfu/cell by X-gal staining.The second part of this study:1.The number of capillary-like tube strucures of Ad-HIF-la-564/402/803 group was significantly more than that of Ad-HIF-la-nature group,Ad-Null group and control group (P=0.000 respectively). The microvessel density in group Ad-HIF-la-564/402/803 was higher than that in group Ad-HIF-la-nature as well as Ad-Null and PBS.2. The microvessel on the chick embryo chorioallantoic membrane(CAM) in group Ad-HIF-la-564/402/803 was more than the other group,and form the leaf vein-like vasculature.The ratio between angiogenesis area and CAM area in group Ad-HIF-1α-564/402/803 was higher than the other group(P-0.01,0.000,0.000 respectively).ConclusionThe triple mutant Ad-HIF-1α-564/402/803 plays an important role in promoting the formation of capillary-like tube structures in vitro and modulating angiogenesis in the CAM model. It proved that the triple mutant Ad-HIF-la-564/402/803promoted the angiogenesis in vitro under normoxia condition.