Effects Of Recombinant Adenovirus-mediated Hypoxia Inducible Factor-1α Of Triple Mutant On Cell Proliferation And VEGF Expression In HMVECs

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) and percutaneous coronary intervention(PCI) develop dramatically, however,amount of patients having symptoms such as 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 find an alternative strategy for the treatment of ischemic heart disease.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 of ischemic heart disease.Now the angiogenic molecules mainly including VEGF,fibroblast growth factor(FGF),angiopoietin(Ang),platelet-derived growth factor(PDGF),insulin-like growth factor(IGF),and monocyte chemotactic protein-1 (MCP-1) are known as participating and regulating in the process of angiogenesis. VEGF and FGF have been studied deeply,and have shown promising results in amount of preclinical studies and safety in phaseâ… clinical trials,but they have shown either marginal or no efficacy in phaseâ…¡trials.The process of vascular growth is regulated by a series of growth factors,and single growth factor is not enough for inducing mature angiogenesis.Hypoxia inducible factor 1(HIF-1),an up-stream transcription factor,plays a key role in the cellular adaptive response to hypoxia.HIF-1 participates various physiological and pathological processes such as angiogenesis and erythropoiesis by modulating the transcription of various target genes including VEGF,Ang,erythropoietin(EPO),PDGF,heine oxygenase-1(HO-1), etc.It has been demonstrated in the preclinical studies that gene therapy with constitutively active form of HIF-1αmay result in physiologically functional neovascularization.The angiogenesis being induced by HIF-1αis not associated with inflammation reaction,vascular permeability and tissue edema,etc.So HIF-1αis considered to be one of the most prospective factors of angiogenesis.The advantage of HIF-1αin gene therapy of ischemic heart disease is that HIF-1αcan promote the process of angiogenesis by inducing the transcription of VEGF,Ang-1,Ang-2,Ang-4,PDGF and PlGF,etc.These factors have synergistic and contradictive effects,and lead to physiologically functional neovascularization avoiding excess proliferation of vessels.VEGF is the specific down-stream target gene which is directly modulated by HIF-1.It can promote the growth and proliferation of endothelium cell and increase the vascular permeability,providing the initial environment for angiogenesis and formation of primitive vessels,therefore, it plays an important role in survival of new vasculatureAng-1 and PDGF recruits pericytes and in this way stabilizes the maturing vascular system,so as Ang-4.PlGF and VEGF has synergistic effect,and reduces the vascular permeability.HIF-1 is consists of a constitutively HIF-1βsubunit and a HIF-1αsubunit,the expression of which is highly regulated by the cellular oxygen concentration.HIF-1 is active only when HIF-1αdimerizes with HIF-1β.The activated HIF-1 then associates with hypoxia response elements(HREs)in the regulatory regions of target genes and binds the transcriptional coactivators to induce gene expression.Tight regulation of the stability and subsequent transactivational function of HIF-1αis the chief way of HIF-1 modulation,which is both performed by the mechanism of oxygen-dependent degradation process.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(N-TAD) of HIF-1α.In order to make high expression of HIF-1αin normoxia,Mircea Ivan found that mutation of Pro564 to Ala564 block the ODDD/pVHL interaction,as a result,mutant HIF-1αwas more stable than that of wild type.Panu Jaakkola gained the similar result after mutation of Pro564 toGly564. Norma Masson analysed the expression of HIF-1αafter mutation of both Pro564 and Pro402 or respectively either proline point alone,and found that the expression of HIF-1αof mutation of either proline alone was approximately half of which under hypoxia condition,while mutation of both proline residues was approximately constitutively expressed in the presence of normal oxygen levels.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-1αstabilization and accumulation in the cell.If mutation of Asn803,the interaction of C-TAD with CPB/p300 can not be affected in normoxia,thus can keep the complete transcriptinal activity.Therefore,we finished the mutation of Pro402,Pro564 and Asn803 in HIF-1αgene and successfully constructed the adenovirus vector of human hypoxia-inducible factor-1αof triple mutant(Ad-HIF-1α564/402/803) in order to get high levels of gene expression under normoxic condition.To identify the role of HIF-1αgene in modulating angiogenesis and the biological effect of Ad-HIF-1α564/402/803,we examined the cell proliferation and the expression of HIF-1αand VEGF after hMVECs infected by Ad-HIF-1α564/402/803.ObjectiveTo further study the role of HIF-1αgene in modulating angiogenesis and the biological effect of Ad-HIF-1α564/402/803,we examined the cell proliferation and the expression of HIF-1αand VEGF after hMVECs infected with Ad-HIF-1α564/402/803 in vitro.MethodThefirst part of this study:Recombinant adenovirus Ad-lacZ,Ad-Null,Ad-HIF-1αnature and Ad-HIF-1α564/402/803 were amplified in HEK293A cells and purified by ultracentrifugation in CsCl step gradient solutions,adenoviral titer was determined by End-Point Dilution Assay.The recombinant adenovirus was confirmed by polymerase chain reaction(PCR) and DNA sequence analysis.The second part of this study:The antigens of Fâ…§and CD31 were detected by immunofluorescence.The infection efficiency was observed with X-gal staining. The effect of recombinant adenovirus on cell proliferation was assessed by MTS assay.The expression of protein level of HIF-1αand VEGF in hMVECs after infected with recombinant adenovirus under normoxia condition at desirable time points was performed by Western blot.ResultThe first part of this study:High-titer adenovirus were produced after amplified. The titer of Ad-lacZ,Ad-Null,Ad-HIF-1αnature,and Ad-HIF-1α564/402/803 was 2.0×10¹³,2.5×10¹⁴,1.6×10¹²,2.0×10²¹pfu/ml respectively.HEK293A cells could be effectively infected by recombinant adenovirus in vitro,and cytopathic effect appear -ed just after 24 hours.The DNA of HIF-1α564/402/803 gene was extracted to confir -m 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 expectation.The second part of this study:The results of immunofluorescence detection of antigens of Fâ…§and CD31 were positive respectively.The hMVECs were infected by Ad-lacZ,and the optimal multiplicity of infection was 75pfu/cell by X-gal staining. In terms of MTS,there was statistical significance in factors of group and time (P=0.000 respectively),and there was intercept effect between factors of group and time(F=13.956,P=0.000),the values of absorptance of the four treated groups at the time points of the second,third,fourth,fifth day were significantly different(P= 0.000 respectively);Compared with control group,there was no significantly differe -nces in Ad-Null group(P=0.610,0.175,0.514 respectively);and the value of absorptance of Ad-HIF-1αnature group was significantly higher than that of control group(P=0.001,0.000,0.001,0.000 respectively;and at different desirable time points,the value of absorptance of Ad-HIF-1α564/402/803 group was significantly higher than that of Ad-HIF-1αnature group(P=0.000,0.000,0.001,0.000 respec -tively).There was statistical significance in factors of group and time(P=0.000 respectively) in terms of the expression of HIF-1αprotein,and there was intercept effect between factors of group and time(F=3.990,P=0.000).The expression of HIF-1αprotein of Ad-HIF-1α564/402/803 group was higher than that of blank group and Ad-Null group at the three desirable time points(P＜0.05 respectively);there was no statistical significance of the expression of HIF-1αprotein between groups of Ad-HIF-1α564/402/803 and Ad-HIF-1αnature at the time points of 48h and 96h (P=0.092,0.197 respectively).But at the time point of 72h,the expression of HIF-1αprotein of Ad-HIF-1α564/402/803 group was significantly higher than that of Ad-HIF-1αnature group(P=0.017),so as the expression of VEGF.The expression of HIF-1αand VEGF protein was related with the MOI of Ad-HIF-1α564/402/803, and they increased with the enhancement of MOI of Ad-HIF-1α564/402/803,but both of the protein expression decreased when the MOI was 300 pfu/cell.Conclusion(1) Recombinant adenovirus Ad-lacZ,Ad-Null,Ad-HIF-1αnature,and Ad-HI F-1α564/402/803 were amplified with high titer,high transfection efficiency.(2) The gene expression of Ad-HIF-1α564/402/803 was more effective and higher than that of Ad-HIF-1αnature.It can promote the proliferation of hMVECs and modulate the expression of VEGF protein effectively.