Chronic blockade of endothelin receptors improves ischemia-induced angiogenesis in rat hindlimbs through activation of vascular endothelial growth factor-NO pathway

This study investigated in vivo the putative angiogenic role of endothelin (ET)-1 in a model of ischemia-induced angiogenesis. Ischemia was produced b y unilateral femoral artery occlusion in Wistar rats submitted to either ch ronic ET-1 infusion(2 nmol.k(-1).min(-1)) or to a dual ETA/ETB receptor ant agonist (bosentan, 100 mg.kg(-1).d(-1)) for 3 and 28 days. Arterial density was evaluated by microangiography and measurement of capillary and arterio lar density in hindlimb muscles. ET-1 infusion had no effect on ischemia-in duced angiogenesis and was associated with a slight decrease in vascular en dothelial growth factor (VEGF) content measured by Western blot analysis. C onversely, bosentan induced a marked increase in vessel density at 3 and 28 days (1.4-fold and 1.7-fold, respectively, compared with no treatment; P<0 .05), which was associated with an increase in VEGF and endothelial NO synt hase levels in ischemic legs (by 31<plus/minus>8% and 45 +/- 23%, respectiv ely, at 3 days and by 65 +/- 13% and 55 +/- 15%, respectively, at 28 days; P<0.05 versus nontreated rats). At day 28, the proangiogenic effect of bose ntan was abolished when NO synthesis inhibitor N-G-nitro-L-arginine methyl ester (10 mg.kg(-1).d(-1)) or VEGF-neutralizing antibody (2.5 <mu>g/kg, twi ce a week) were coadministered with bosentan. Those results provide the fir st evidence of an early and sustained proangiogenic effect of endothelin an tagonism associated with an upregulation of VEGF and endothelial NO synthas e in vivo.