| IntroductionBecause the changes of diet, the arrival of the aging society, the intense social competition and more metabolic diseases, eg, hyperlipoidemia and diabetes, the incidence of ischemic diseases, such as coronary atherosclerosis heart disease, ischemic cerebrovascular disease and peripheral vascular disease, is increasing these years. Lower limb ischemic disease has been paid more attention recently, for the severe disabling symptoms related to ischemia, eg, claudication, resting pain, and loss of tissue integrity in the distal limbs. Despite great advances in classic drug, surgical and percutaneous revascularization techniques, treatment of patients with peripheral ischemic disease remains a significant clinical challenge. Currently available approaches for treating critical ischemic peripheral vascular disease are not satisfied. No medicine has been reported to favorably affect the natural history of critical limb ischemia. At the same time, the number of patients being not eligible for operative and percutaneous revascularization because of anatomic extent and sever arterial occlusive disease is growing. Furthermore, the occurrence of restenosis after revascularization restricts the application of this treatment at some degree. Amputation is a common solution to the serious cases, in particular excruciating ischemic rest pain of severe limb ischemia and severe ulcer. Consequently, the need for alternative treatment strategies in patients with critical ischemia is compelling. Therapeutic angiogenesis maybe bring encouraging results for us.When the ischemia was induced by the stenosis or occlusion of blood vessels, the expression of angiogenesis-related cytokines was increased to induce the revascularization of blood perfusion, which was often partially compensated. Recently, application of endothelial cytokines as proteins or genes has been of scientific and clinical interest to treat limb or myocardial ischemia. Therapeutic angiogenesis is a procedure that prompts the formation of blood vessels to improve blood supply to ischemic tissue. But protein is seldom chosen due to higher dose needed, shorter action time and repeated administration. In contrast, gene therapy is more popular in the research field of therapeutic angiogenesis, by taking the advantage of relatively long time expression in the local and often single administration needed.The process of angiogenesis is very complex, which need a variety of angiogenesis-related cytokines, acting by differing mechanisms to induce a more efficacious biological response. Vascular endothelial growth factor(VEGF) and fibroblast growth factor (FGF) were the genes first be used for ischemic disease in preclinical and PhaseⅠclinical studies and yielded some encouraging results. However,the results of PhaseⅡclinical trials were somewhat disappointing. VEGF induced the formation of immature blood vessels, leading to vascular leakage, tissue edema and other adverse reactions. Proteinuria occurred in some patients treated with FGF to promote angiogenesis. These unsatisfied outcomes limited the study of them in therapeutic angiogenesis. It is necessary to find other genes, which is more suitable for clinical application to promote angiagenesis. Hypoxia inducible factor-1 (hypoxia inducible factor-1, HIF-1) as a’master switch’gene caused the researchers’ attention.In recent years, studies have shown that:HIF-1 as an important nuclear transcription factor can directly or indirectly control a variety of downstream target genes, which related to blood vessel growth, vascular tension, sugar metabolism, and so on, furthermore, especially including those encoding angiogenic factors such as VEGF, placental growth factor (PLGF), angiopoietin 1 (ANGPT)1, ANGPT2, and stromal-derived factor (SDF)-1. HIF-1 is a heterodimeric transcription factors ofaandβsubunits. Theβsubunit is constitutive and not subject to oxygen dependent regulation. Theasubunit is unique to the hypoxic response, which determines the activity of HIF-1. HIF-la contains one oxygen-dependent degradation domain (ODDD) mediating oxygen-regulated stability, and two transactivation activation domains (TAD), N-TAD and C-TAD. C-TAD controls the major transactivation activity by interacting with co-activator. The rapidly degradation (several minutes) of HIF-1a is achieved by a prolyl hydroxylase at residues P402 and P564 within ODDD. However, the inactivation of transcription happens by an asparaginyl hydroxylase at residue N803 in its C-TAD, which blocks the combination of HIF-la and some coactivators. Under hypoxia condition, the activity of prolyl hydroxylase and asparaginyl hydroxylase are markedly reduced, and the lack of hydroxylation results in stable HIF-la binding with HIF-1βand coactivators, furthermore, leading to the enhanced expression of target angiogenic genes. Theoretically, mutation of Pro402, Pro564 and Asn803 in HIF-1a will enhance its stability and transcriptional activity, which lead to the improved expression of HIF-1a related angiogenic genes.Our study team had successfully constructed the recombinant adenovirus-mediated hypoxia inducible factor-la of triple mutant by point mutations of Pro402, Pro564 and Asn803. The enhanced stability of HIF-la and improved expression of HIF-1a related angiogenic genes had be documented by the study in vitro.PurposeThe present study was undertaken to test the hypothesis whether intramuscular injection of a modified, constitutively active form of HIF-1a (Ad-HIF-la-trip) could improve the expression of itself and some other angiogenic genes, such as VEGF in the ischemic muscle of rabbit; whether Ad-HIF-la-trip could augment functional mature neovascular without much leakage in the model of rabbit limb ischemia.Methods1. In the first stage of our experiment, Ad-HIF-1a-Trip and Ad-Null were amplified in HEK293A cells and purified by ultracentrifugation in CsCl concentration gradient solutions. The amount of viral particles was determined by spectrophotometry and the number of plaque formation unit was determined by end-point dilution method. Then, the adenovirus was determined PCR and gene sequencing2. After underwent ligation of their left femoral artery,27 Zealand Whites Rabbits were randomly divided into 3 groups immediately:saline group, Ad-Null group (2×1010PFU) and Ad-HIF-1a group(2×1010PFU).10 days after treated with corresponding intramuscular injection, Real-time PCR and Western blot were applied to detect the expression of HIF-la and its target angiogenic genes;On 0,7, 14 and 28 days after gene transfection, contrast enhanced ultrasound (CEU) and vascular ultrasound were used to estimate the blood perfusion of hindlimb and the blood of internal iliac artery. On the 28th day, immunohistochemistry were carried out to detect the microvascular density(MVD); the anatomy revascularization was observed by CTA and arterial vascular casting.3. The acute hind-limb ischemic models of rabbit were performed and diveded into three groups. The specfic method of intramuscular injection and dosage of virus was consistent with method 2.10 days after gene transfection, HIF-la gene expression in skeletel muscle, heart, liver, kidney and lung was detected by Real-time PCR.28 days after genes transfection, the permeability of the neovasculature in the ischemic skeletal muscle was evaluated by Evans blue uptake assay.4. SPSS13.0 statistical software was used for data analysis. Measurement data were analysised by reapeated measurement or one-way ANOVA. Multiple comparisons between groups was analysied by LSD method or Games-Howell test. Numeration data were analysised by multi-level non-parametric test (Kruskal-Wallis Test). The difference was statistically significant when P<0.05.Results1. The titres of Ad-Null and Ad-HIF-la-Trip, after being amplified and purified, were 2.6×1013 VP/ml and 3.9×1013VP/ml respectively by OD260; 2.2×1012 PFU/ml and 3.7×1012PFU/ml respectively by End-point dilution method. The results of Real-time PCR and sequencing showed that the virus carries the integrity of the genetic information, without mutation or loss. The virus obtained reached the requirement of our study.2. On the 7th day after gene transfection, HIF-la mRNA expression was detected by Real-time PCR in the ischemic muscle, and the expression level in the rabbit treated with Ad-HIF-la-Trip is significant higher than treated with saline or Ad-Null (P <0.01); the results of western blot showed that, the protein level of HIF-la and VEGF in ischemic muscle administrated with Ad-HIF-la-Trip is obviously higher than with saline or Ad-Null.3. CEU revealed better blood perfusion in Ad-HIF-lagroup than the group treated with saline or Ad-Null, and there were significant difference on the 7th,14th and 28th day after gene transfection (P<0.01). The results of vascular ultrasound showed that, the internal iliac artery blood flow in Ad-HIF-la-Trip groups increased most obviously in the three groups (P<0.01). Furtherly, there were significant differences whether on day7, day14 or day28 (P<0.01).4. On the 28th day after gene transfection, in the rabbit model of acute ischemic hind limb, the Ad-HIF-la-Trip increased more recognizable collateral vessels measured by CT angiography and vascular casting (P<0.01), as well as more histological identifiable capillaries verified by immunohistology compared to Ad-Null and saline (P<0.01).5. There were no significant differences of the above indexes between Ad-Null group and saline group.6. On the 10th day after gene transfection, the ectogenic HIF-la mRNA expression was detected by Real-time PCR in ischemic muscle, but there was no expression in the non-target organs (heart, liver, kidney, lung, testes and contralateral skeletal muscle). Furthermore, there were no abnormal vascular network, tumor-like structures and significant morphological changes observed in the HE staining pathological sections of these organs. Quantification of the leakage levels of Evans blue dye demonstrated that there were no obvious differences among three groups.Conclusion1. Ad-HIF-1a-Trip expressed well in the ischemic limb of rabbit by local intramuscular injection; Ad-HIF-la-Trip promoted revascularization in rabbit hindlimb ischemia and enhanced ischemic tissue perfusion; the vascular permeability was not abnormal increased.2. Ad-HIF-1a-Trip administrated by intramuscular injection had no expression in the non-targets (heart, liver, kidney, lung,testis and contralateral skeletal muscles), and didn’t induce apparent formation of tumor-like structures and abnormal vascular networks3. Intramuscular injection of Ad-HIF-la-Trip is a feasible and effective treatment of peripheral ischemic disease, which provides a new way to treat other ischemic diseases. |
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