Study Of The Effects And Related Mechanism Of ACEI On Ischemic Angiogenesis In Diabetic Rats

Objective: Currently, there are still divergences about the explanations of the impaired neovascularization in diabetic subjects. Because of angiotensin- converting enzyme inhibititors(ACEI)' remarkable therapeutic effect on the reduction of blood pressure and the protection of target organs, the clinical application of this kind of drugs is very widespread. But it is still elusive about the role and related molecular pathway of ACEI in limbs' postischemic revascularization. The aim of our study is to investigate the reasons of the impaired angiogenesis in diabetic ischemic hind limbs of rats,the role and related molecular mechanisms of ACEI in limbs' postischemic revascularization. It will provide the experimental foundation and rationale for the clarification of the mechamisms involved in the role of ACEI in vascular diseases.Methods: Diabetic mellitus was induced in male Wister rats by injection of streptozotocin. Hind limbs ischemia was then induced by right femoral artery and all side branches excised. The day after surgery, DM rats were randomly allocated to one of the following treatment: ACEI by perindopril; Perindopril in combination with NOS inhibitor; Perindopril in combination with BK-B1 receptor antagonist or saline. The normal rats were treated with perindopril or saline. The period of treatment is 1 or 4 weeks. Microvascular density was evaluated by HE and immunohistochemical stain. The mRNA and protein expression of eNOS,VEGF and bFGF were determined by RT-PCR and Western blot. NO content and cNOS activity were determined by NO and NOS testing boxes. All results are expressed as mean±SEM. One-way analysis of variance ANOVA and Post hoc Bonferroni t test comparisons were used to compare each parameter. P<0.05 was considered significant.Results:1. Comparison of general datas: Compared to control rats, diabetic rats showed decrease in body weight and increased plasma glucose levels (P<0.01) . The blood pressure showed no difference among each group (P>0.05) . 2. Analysis of postischemic neovascularization: At day 7 and 28 of treatment, no significant changes in MVD in non-ischemic hind limbs were observed in control and diabetic rats. But in ischemic hind limbs, the MVD in diabetic rats decreased 23% and 27% respectively at day 7 and 28 of treatment when compared with the control rats. Administration of perindopril didn't influence the MVD in non- ischemic hind limbs in control and diabetic rats, but increased the MVD in ischemic hind limbs in control and diabetic rats. The ACEI pro-angiogenic effect was blunted in the combination of NOS inhibitor or BK-B1 receptor antagonist.3. Molecular mechanisms: Compared with the control group, the mRNA and protein expression of eNOS,VEGF and bFGF,the content of NO and the activity of cNOS in ischemic hind limbs of diabetic rats decreased. Administration of perindopril enhanced above indexes in normal and diabetic rats. The combination of BK-B1 receptor antagonist made the expression of eNOS,VEGF and bFGF,the NO content and the cNOS activity decreased. In contrast, the combination of NOS inhibitor made the expression of eNOS and bFGF,the NO content and the cNOS activity decreased, but the expression of VEGF didn't changed. These finfings indicated that ACE inhibitor pro-angiogenic effect in diabetic rats was mediated, at least in part, by the BK-B1 receptor. Its downstream signal molecules included eNOS,VEGF and bFGF. And VEGF lies in the upstream of eNOS, bFGF lies in the downstream of eNOS.4. Correlation analysis: We found that the MVD in ischemic hind limbs was positively correlated with the mRNA and protein expression of eNOS,VEGF and bFGF,the content of NO and the activity of cNOS in ischemic muscles.Conclusions:Diabetes mellitus reduced neovascularization and related growth factors expression in rat ischemic legs. Treatment of perindopril improved angiogenesis in normal and diabetic ischemic leg. And this effect is more obvious in diabetic rats. This pro-angiogenic effect was related to the increase of expression of eNOS,VEGF and bFGF and the enhancement of cNOS activity. These results evidenced that ACE inhibitor pro-angiogenic effect in the ischemic legs of diabetic rats was mediated, at least in part, by the BK-B1R-related pathway, the activation of VEGF/eNOS/bFGF signals may involve in the ACEI-induced postischemic revascularization.