Molecular mechanism of limbs’ postischemic revascularization improved by perindopril in diabetic rats

Keywords: angiotensin-converting enzyme inhibitor·limb ischemia·diabetes mellitus·neovascularization
Abstract:
Background Currently, there are still divergent opinions about the mechanisms of the impaired neovascularization in diabetic subjects. Due to the remarkable therapeutic effect of angiotensin-converting enzyme inhibititors (ACEIs) on the reduction of blood pressure and the protection of target organs, the clinical application of this kind of drugs is very widespread. However, it is still not clear about the role and related molecular pathway of this kind of drugs in the limbs’ postischemic revascularization. It is of major therapeutic importance to resolve these questions. This study aimed to investigate the reasons of the impaired angiogenesis in the hind limbs of rats with diabetic ischemia, the role and related molecular mechanisms of ACEI in postischemic revascularization.
Methods Hind limbs ischemia was induced in diabetic rats by right femoral artery excision. Diabetic rats were randomly allocated to one of the following treatments for 4 weeks: ACEI by perindopril; perindopril in combination with a nitric oxide synthase (NOS) inhibitor; perindopril in combination with bradykinin (BK)-B1 receptor (B1R) antagonist or saline. The differences of angiogenesis, the mRNA and protein expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and basic fibroblast (bFGF), constitutive nitric oxide synthase (cNOS) activity and nitric oxide (NO) content were observed after treatment.
Results In non-ischemic hind limbs, no significant changes in capillary density, or the mRNA and protein expression of eNOS, VEGF and bFGF, or the NO content and the cNOS activity were observed among all groups. On the contrary, in ischemic hind limbs, the capillary density in diabetic rats decreased by 27% when compared with the control rats, so did the mRNA and protein expression of eNOS, VEGF and bFGF, or the NO content and the cNOS activity (P <0.05). The capillary density was increased by 1.65-fold in the perindopril treatment group in reference to untreared diabetic rats. Moreover, administration of perindopril enhanced the mRNA expression of eNOS, VEGF, and bFGF by 1.45-, 1.44-, and 1.33-fold, increased the protein content of the above indices by 1.55-, 1.30- and 1.50-fold compared with the untreated diabetic rats respectively. Perindopril also increased NO content and cNOS activity to 1.33- and 1.38-fold of that in untreated diabetic rats. The combination of BK-B1R antagonist significantly decreased the above indices (P <0.05). In contrast, the combination of NOS inhibitor decreased the expression of eNOS and bFGF, the NO content and the cNOS activity, while the expression of VEGF did not change.
Conclusions Diabetes mellitus reduces the neovascularization, related growth factors expression and activity in the diabetic rat ischemic legs model. Treatment of perindopril improves postischemic revascularization. This effect is mediated, at least in part, by the BK-B1R-related pathway, and the activation of VEGF/eNOS/bFGF signals may be involved in the pro-angiogenic effect.



2008;121(21):2129-2133
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It is generally accepted that angiotensin converting enzyme inhibitors (ACEIs) exert blood pressure reduction and extensive target organ protection effects. Numerous clinical and experimental trials have confirmed that ACE inhibitors have beneficial effects by inhibiting neovascularization in tumors and retina.1,2 However, the effect and related molecular mechanisms of this kind of drugs in ischemic angiogenesis is still elusive.3-6 In our study, we treated hind limb ischemic diabetic rats with perindopril alone or in combination with a nitric oxide synthase (NOS) inhibitor or bradykinin (BK)-B1 receptor (B1R) antagonist to investigate the effects of ACEIs and related molecular mechanisms in diabetic postischemic revascularization.


METHODS


Animal model
To induce diabetes, eight-week-old mail Wistar rats (n=32) were injected intraperitoneally with 45 mg/kg streptozotocin (STZ, Sigma) in 0.05 mol/L Na citrate. Three days after injection, blood glucose levels were measured. If serum glucose was >16.7 mmol/L, rats were selected for this study. On the first confirmation of hyperglycemia, 32 diabetic rats and 8 normal Wistar rats were anesthetized by pentobarbital, and unilateral hind limb ischemia was induced by excision of the right femoral artery and all side branches.


Grouping and treatments
The day after surgery, the diabetic rats were randomly allocated to one of the following treatments: ACEI by perindopril (2 mg∙kg-1∙d-1, Servier, France); perindopril in combination with NOS inhibitor (L-NAME, 10 mg∙kg-1∙d-1, Sigma, USA); perindopril in combination with BK-B1R antagonist (DALBK, 10 nmol∙kg-1∙d-1, Sigma) or saline. The normal rats were treated with saline. The period of treatment was 4 weeks.

Indices determination
At the end of the experiment, systolic arterial blood pressure was assessed through carotid intubation by the biology function experiment system (Changchun Tianyue Technology Company, China). Ischemic and nonischemic muscles were dissected and progressively frozen in isopentane solution cooled in liquid nitrogen. Capillary density in the hind limb was evaluated by HE and immunohistochemical staining. Sections (5 μm) were incubated with rabbit polyclonal antibody directed against factor VIII-related antigen (von Willebrand factor, dilution 1:50, ABCAM, USA) to identify capillaries. Capillary density (ncap/mm2) was then calculated in randomly chosen fields of a definite area using OptiLab Pro software (Graftek, Mirmande, France).


The mRNA and protein expression of eNOS, vascular endothelial growth factor (VEGF), and basic fibroblast (bFGF) in ischemic and nonischemic legs were determined by RT-PCR and Western blotting. NO content and constitutive nitric oxide synthase (cNOS) activity were determined by NO and NOS testing boxes (Nanjing Jiancheng Bioengineering Institute, China).


Statistical analysis
All statistical analyses were performed using SPSS 13.0. Data were expressed as mean ± standard deviation (SD). Differences between the five groups were determined by one-way analysis of variance (ANOVA). The 2-side t test for paired samples was used to determine the significance of differences between several measurements in ischemic and nonischemic legs. Bivariate correlations were calculated using Spearman's correlation coefficients. A P value less than 0.05 was considered statistically significant.


RESULTS


Comparison of serum glucose and blood pressure
There was a comparable hyperglycemia in all diabetic groups when compared with controls. The levels of blood pressure showed no difference among the groups. The levels of systolic arterial blood pressure were similar in all groups. The blood pressure of control, diabetic untreated, ACEI treatment, ACEI in combination with NOS inhibitor, and ACEI in combination with BK-B1R antagonist treatment group were (101±6)/(70±3) mmHg, (105±5)/(73±4) mmHg, (100±6)/(69±3) mmHg, (104±5)/(72±4) mmHg, and (102±6)/(73±5) mmHg, respectively.


Analysis of revascularization
After 4 weeks of treatment, the capillary density in nonischemic hind limbs was similar among all groups. On the contrary, in ischemic hind limbs, the capillary density in diabetic rats decreased by 27% when compared with that in the control rats. Perindopril improved capillary density by 1.65-fold in ACEI-treated diabetic rats compared with that in untreated diabetic rats. In contrast, The ACEI pro-angiogenic effect was blunted to some degree by the combination with a NOS inhibitor or BK-B1R antagonist. The combination of NOS inhibitor or BK-B1R antagonist caused a capillary density decrease by a similar percentage (46% and 45% respectively) (Figure).


view in a new window Figure. Angiogenesis in hind limbs among the groups (immunohistochemical stain, original magnification ×200). A: the immunohistochemical graph. B: the strip graph. *P <0.05 vs the control group, △P <0.05 vs the diabetic group, #P<0.005 vs the DM+ACEI group, ▲P <0.05 vs the ACEI+NOS inhibitor group, ☆P <0.05 vs the ACEI+BK-BIR antagonist group.





Molecular mechanisms
eNOS
After 28-day treatment, in nonischemic hind limbs, eNOS mRNA and protein levels were unaffected in all groups. In contrast, in the ischemic hind limbs, the endothelial nitric oxide synthase (eNOS) mRNA and protein contents decreased by 24% and 28% respectively in untreated diabetic rats compared with controls. Administration of perindopril raised eNOS mRNA and protein levels by 1.45- and 1.55-fold compared with those of untreated diabetic rats. Conversely, the combination of NW-nitro-L-arginine methyl ester (L-NAME) decreased eNOS mRNA and protein levels by 85% and 83% compared with those of ACEI-treated diabetic rats. The combination of DALBK made the above indices decreased by 72% and 67% in reference to the ACEI-treated group (Table).


view in a new window Table. Comparisons of eNOS, VEGF and bFGF expression





VEGF
After treatment, in the nonischemic leg, the VEGF mRNA and protein levels were similar among all groups. In the ischemic leg, VEGF mRNA and protein levels decreased by 23% and 25% respectively in untreated diabetic rats compared with the control group. In ACEI treatment group, VEGF mRNA and protein expression increased by 1.44- and1.30-fold compared with untreated diabetic rats. The combination of L-NAME had no influence on the above indices, while the combination of DALBK downregulated the VEGF mRNA and protein levels to 75% and 79% of those in ACEI-treated group respectively (Table).

bFGF
In the nonischemic leg, there were no significant differences in bFGF mRNA and protein levels in any groups after treatment. In the ischemic leg, the bFGF mRNA and protein contents decreased by 30% and 23% respectively in untreated diabetic rats compared with the control group. After treatment with perindopril, the above indices were improved by 1.33- and 1.5-fold respectively compared with untreated diabetic animals. Both L-NAME and DALBK reduced the bFGF mRNA and protein contents obviously. The addition of L-NAME made the above indices decreased to 64% and 63% of those in ACEI-treated animals respectively. Similarly, DALBK made the above indices decrease to 67% and 69% of those in ACEI-treated group respectively (Table).


cNOS activity
In the nonischemic leg, cNOS activity was similar among all groups. In the ischemic leg, cNOS activity was reduced by 24% in diabetic rats when compared with controls. ACEI administration improved this index by 1.38-fold in reference to untreated diabetic group. The combination of L-NAME or DALBK made cNOS activity decreased by 25% and 19% compared with ACEI-treated group respectively (Table).


NO content
NO content was similar among all groups in the nonischemic leg. In the ischemic leg, NO content decreased to 70% of that in controls. ACEI enhanced by 1.33-fold of NO content in reference to untreated diabetic group. L-NAME and DALBK decreased to 71% and 76% of those in ACEI-treated group respectively (Table).


Correlation analysis
By correlation analysis, we found that the capillary density in ischemic hind limbs was positively correlated with the mRNA and protein expression of eNOS (r=0.87, 0.84), VEGF (r=0.85, 0.81) and bFGF (r=0.85, 0.83), and with the content of NO and the activity of cNOS (r=0.85, 0.82) in ischemic muscles.


DISCUSSION


Angiogenesis is the growth of new blood vessels from preexisting ones and is a complex process regulated by numerous factors, such as VEGF and FGF. After vascular occlusion, ischemic angiogenesis is an important reparative mechanism and can ameliorate the outcome of ischemic disease. Numerous studies have confirmed the impaired postischemic angiogenesis in diabetic statement.7-10 Our previous study also drew the same conclusion. This deficit might explain why vascular occlusion evolves more severely among diabetic patients.11


Currently it is still controversial about the mechanisms of impaired ischemic angiogenesis in diabetic statement. Previous studies have demonstrated that it might involved alterations in VEGF signaling.12 But recently, Tanii et al13 found that diabetic microangiopathy in ischemic limb was not caused by the impaired expression of angiogenic factors. Our findings showed that in ischemic hind limbs, the eNOS, VEGF and bFGF mRNA and protein expression, NO content and the cNOS activity decreased significantly in diabetic rats compared with those of the control animals. Correlation analysis demonstrated that the capillary density in ischemic hind limbs was positively correlated with above indices. These results indicated that ischemia-induced neovascularization was impaired in diabetic subjects. And this defect might be associated with the expression and activity decrease of angiogenic growth factors. These discrepant results are likely caused, at least partly, by the difference in species.


There is accumulating evidence that the RAS components are upregulated in diabetic vascular tissue, and the production of AngⅡis increased. It is well-known that AngⅡ is a pro-angiogenic factor, which has been shown to promote cells proliferation and blood vessel growth.14-16 Nevertheless, studies which focused on the effect of ACEI in ischemic angiogenesis had reached different conclusions.3-6,17-19 It is now well established that ACEI can improve ischemic myocardial angiogenesis.17-19 However, the role of ACEI on angiogenesis in the ischemic limbs is still controversial. Many studies showed that this kind of drugs can stimulate angiogenesis in ischemic limbs, and this pro-angiogenic effect might be mediated by the bradykinin-related pathway.5 But it is still elusive that which receptor subtype may mediate the pro-angiogenic effect and related molecular mechanisms.20,21 Li et al3 evidenced that B1R antagonist suppressed the imidapril-induced angiogenesis in AT1a receptor knockout (AT1aKO) mice to an extent even lower than that of nontreated AT1aKO mice. A NOS inhibitor moderately attenuated the imidapril-mediated angiogenesis. In the present study, we provide evidence that ACEI improves impaired postischemic neovascularization in diabetic rats. The combination of BK-B1R antagonist or a NOS inhibitor can partly abrogate the pro-angiogenic effect of ACEI. The combination of a BK-B1R antagonist made the expression of eNOS, VEGF and bFGF, the NO content and the cNOS activity decreased. In contrast, the addition of NOS inhibitor made the expression of eNOS and bFGF, the NO content and the cNOS activity decreased, but did not influence the expression of VEGF. These findings indicated that an ACEI pro-angiogenic effect in diabetic rats was mediated, at least in part, by the BK-B1R. Its downstream signal molecules included eNOS, VEGF and bFGF. And VEGF lies upstream of eNOS and bFGF lies downstream of eNOS.


Because ACEI belongs to hypotensive drugs, the changes of capillary density in ischemic legs that resulted from ACEI might relate to the reduction of systemic blood pressure. However, how high blood pressure per se may impair the angiogenic process remains unclear. Moreover, in the present study the blood pressure levels between control and treatment group are similar, therefore the pro-angiogenic effect of an ACEI might be independent of blood pressure.


In conclusion, our study shows that an ACEI pro-angiogenic effect in the ischemic limbs of diabetic rats was mediated, at least in part, by the BK-B1R -related pathway, and that the activation of VEGF/eNOS/bFGF signals may be involved in the ACEI-induced postischemic revascularization. These findings are of major therapeutic importance for the prevention and treatment of peripheral vascular complications of diabetes.


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