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 by unilateral femoral artery occlusion in Wistar rats submitted to either chronic ET-1 infusion (2 nmol. kg(-1). min(-1)) or to a dual ET(A)/ET(B) receptor antagonist (bosentan, 100 mg. kg(-1). d(-1)) for 3 and 28 days. Arterial density was evaluated by microangiography and measurement of capillary and arteriolar density in hindlimb muscles. ET-1 infusion had no effect on ischemia-induced angiogenesis and was associated with a slight decrease in vascular endothelial growth factor (VEGF) content measured by Western blot analysis. Conversely, 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 synthase levels in ischemic legs (by 31+/-8% and 45+/-23%, respectively, 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 bosentan 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 micro/kg twice a week) were coadministered with bosentan. Those results provide the first evidence of an early and sustained proangiogenic effect of endothelin antagonism associated with an upregulation of VEGF and endothelial NO synthase in vivo.