Effects Of Recombinant Adenovirus-mediated HIF-1α Mutants On The Chronic Ischemic Limb Of Rabbit

IntroductionBecause the changes of diet and the arrival of the aging society, the incidence of chronic peripheral ischemic diseases is increasing these years. Severe disabling symptoms of ischemic limbs related to ischemia are paid more attention, such as claudication, resting pain, and loss of tissue integrity in the distal limbs. Despite great advances in classic drugs, surgical and percutaneous revascularization techniques, treatment of patients with serious peripheral ischemic disease remains a significant clinical challenge. Currently available approaches for treating critical ischemic peripheral vascular disease are not satisfied. The occurrence of restenosis after revascularization discourages the application of this treatment. Furthermore, there is not an effective treatment for the distal and diffuse vascular stenosis. Other effective treatment is needed.Therapeutic angiogenesis maybe bring brightness for the patients. When the ischemia is induced by the stenosis or occlusion of blood vascular, the expressions of angiogenesis-related cytokines and the related acceptors are increased to induce the revascularization of blood perfusion, which are often less sufficient to improve the angiogenesis, blood perfusion and ischemic symptoms. Therapeutic angiogenesis is a procedure that prompts the formation of blood vessels, improve blood perfusion and alleviate the ischemic symptoms by giving exogenous angiogenic factors.Vascular endothelial growth factor(VEGF) and fibroblast growth factor (FGF) were the genes first be used for ischemic disease in preclinical and Phase Ⅰ clinical studies and achieved some satisfactory results. However, the results of Phase Ⅱ clinical trials were somewhat disappointing and hampered the further study. VEGF induced the formation of immature blood vessels, but leading to tissue edema and other adverse reactions. Proteinuria occurred in some patients with ischemic disease treated with FGF. It is necessary to find other genes more suitable for clinical application to promote angiogenesis closing to the physiological processes. Hypoxia inducible factor-1(hypoxia inducible factor-1, HIF-1) attracted the researchers’ attention as a’master switch’gene.HIF-1is a heterodimeric transcription factor composed of a and β subunits. The a subunit is the focal point of the research. The a subunit is unique to the hypoxic response and determines the activity of HIF-1. The β subunit is the constitutive composition of HIF-1and not subject to oxygen dependent regulation. Its main function is to form two mer with a variety of b HLH-PAS proteins, which binding DNA to initiate transcription. HIF-1α binding with HIF-1β and coactivators shifting from the cytoplasm to the nucleus, lead to the enhanced expression of angiogenic genes and initiate transcription. HIF-1α can directly control a variety of downstream target genes, which related to blood vessel growth, such as VEGF, inducible Nitric Oxide Synthase (iNOS), VEGF receptor1(Flt-1), Angiopoietin-1(Ang-1), Ang-2, Ang-4, Platelet-derived growth factor (PDGF) and placenta growth factor (PIGF). In the induction of HIF-1α, it is hopeful to achieve a more satisfactory angiogenesis result because of the complement of the domnstream target genes. In normoxia, the half life of HIF-1α is very short with reduced transcription activity. HIF-1α contains one oxygen-dependent degradation domain (ODDD) and two transactivation activation domains (TAD), naming N-TAD and C-TAD. ODDD mediates the oxygen-regulated stability, and two transactivation activation domains (TAD), N-TAD and C-TAD. The rapidly degradation of HIF-1α is achieved by the prolyl hydroxylases at residues P402and P564within ODDD in several minutes. The inactivation of transcription happens by an asparaginyl hydroxylase at residue N803in the C-TAD.Theoretically, mutation of Pro402, Pro564and Asn803in HIF-la will enhance its stability and transcriptional activity, furthermore, lead to improved angiogenic effects. Our study team had successfully constructed the recombinants of adenovirus-mediated hypoxia inducible factor-la by one, two or three point-mutations of Pro402, Pro564and Asn803. Our previous studies showed that,Our previous studies showed that, the recombinant adenovirus containing the three-point(402/564/803) mutation of HIF-la gene(HTF-1α-trip) expressed better stability and transcriptional activity than single and two points mutations in vitro. In acute ischemic peripheral vascular study in rabbit, Ad-HIF-1α-trip showed significant effects to improve blood perfusion, angiogenesis and ameliorate the prognosis.PurposeThe present study was undertaken to test the hypothesis whether the recombinant adenovirus-mediated HIF-1α mutants could effect the expression of the downstream angiogenic genes in the chronic ischemic limb of the rabbit by intramuscular injection; whether Ad-HIF-1α-Trip is more effective than Ad-HIF-1α-564/803to promote angiogenesis. We also investigated the partial safety of the adenovirus-mediated HIF-la mutants transfection in the rabbit model of hind limb ischemiaMethods1. In the first stage of our study, Ad-HIF-1α-Nature and the mutants of Ad-HIF-1α (Ad-HIF-1α-564/803and Ad-HIF-1α-Trip) were amplified in HEK293A cells and purified by ultracentrifugation in CsCl concentration gradient solutions. The viral titers were evaluated by end-point dilution method. Furthermore, the adenovirus was determined by gene sequencing2.7days after ligation of the left femoral artery,40Zealand Whites Rabbits were randomly divided into4groups immediately:Saline group, Ad-HIF-1α-Nature group, Ad-HIF-1α-564/803group and Ad-HIF-1α-Trip group. Saline or recombinant adenovirus-mediated HIF-1α was given by intramuscular injection.7days after treated with corresponding intramuscular injection, Real-time PCR was chosen to evaluate the expression of HIF-1α and its downstream genes (VEGF and bFGF); On the day of gene transfection and7,14as well as28days after gene transfection, contrast enhanced ultrasound (CEU), vascular ultrasound and Doppler calf blood pressure measurement were used to estimate the blood perfusion of hindlimb and the blood flow of internal iliac artery. On the28th day, immunohistochemistry of CD31was used to evaluate the degree of micro vascular density (MVD).3. The chronic peripheral ischemic models of rabbits were divided into four groups. The day before gene transfection (Oday) and48hours,72hours,7days,14days,28days after gene transfection, venous blood was drew off to evaluate the blood routine count, the NT-proBNP,the liver function and renal function.4. SPSS13.0statistical software was used for data analysis. Measurement data were showed by x±s. Measurement data, such as blood routine count, the NT-proBNP, the liver function, and renal function, the normalized value of A×β, the normalized blood flow of internal iliac artery and the normalized calf blood pressure were analysised by reapeated measurement or one-way ANOVA. Multiple comparisons between groups were analysied by LSD method or Games-Howell test. The count of MVD, the angiography score and the relative expression of HIF-1α mRNA were analysised by one-way ANOVA. The difference was statistically significant when P<0.05.Results1. The titers of Ad-HIF-1α-Nature, Ad-HIF-la-564/803and Ad-HIF-1α-Trip were3.1×1012PFU/ml,1.9×1012PFU/ml and2.5×1012PFU/ml respectively by End-point dilution method. The results of gene sequencing showed that the virus carried the integrity of mutated genetic information The virus amplified reached the requirement of our study.2. On the7th day after gene transfection in the chronic ischemic limb, the results of Real-time PCR showed that the expression of HIF-1α, VEGF and bFGF at mRNA level from higher to lower was Ad-HIF-1α-Trip group、Ad-HIF-1α-564/803group、 Ad-HIF-1α-Nature group and Saline group (P<0.01), but there was no statistical difference between Ad-HIF-1α-Nature group and Saline group.3. The blood routine test and serum biochemical tests showed that:the gene transfection did not cause serious impact on the blood cell count, the heart function, the liver function and renal function from the beginning to28days after gene transfection.4. The results of Doppler calf blood pressure measurement, the contrast enhanced ultrasound (CEU) and the vascular ultrasound showed that the blood perfusion and angiogenesis were improved in each group with the time prolonged. The mutants of Ad-HIF-1α (Ad-HIF-la-564/803, Ad-HIF-1α-Trip), especially Ad-HIF-1α-Trip showed outstanding effects of angiogenesis and improving blood perfusion in chronic peripheral ischemic limb at every time point (P<0.01), but there was no statistical difference between Ad-HIF-1α-Nature group and saline group.5. On the28th day after gene transfection, in the rabbit model of chronic ischemic hind limb, the angiography score of the mutants of Ad-HIF-1α groups (Ad-HIF-1α-564/803, Ad-HIF-1α-Trip), especially Ad-HIF-1α-Trip group, were higher than other groups, but there was no statistical difference between Ad-HIF-la-Nature group and Saline group. The result of MVD evaluated by CD31showed the same trend.Conclusion1. The mutants of Ad-HIF-1α (Ad-HIF-1α-564/803, Ad-HIF-1α-Trip) expressed well in the ischemic limb of rabbit by local intramuscular injection. They promoted revascularization and blood perfusion in the ischemic hindlimb of rabbit. Furthermore, the results of Ad-HIF-1α-Trip was more outstanding than Ad-HIF-1α-564/803.2. The mutants of Ad-HIF-1α (Ad-HIF-1α-564/803, Ad-HIF-1α-Trip) administrated by intramuscular injection didn’t damage the important organs.3. Intramuscular injection of Ad-HIF-1α-Trip is a feasible and effective treatment of chronic peripheral ischemic disease, which maybe be used clinically in the future hopefully.