Endothelin-1 impairs angiogenesis in vitro through Rho-kinase activation after chronic intrauterine pulmonary hypertension in fetal sheep

BACKGROUND: Endothelin-1 (ET-1) and Rho-kinase (ROCK) increase vascular tone in experimental persistent pulmonary hypertension of the newborn (PPHN). Whether ET-1 activates ROCK to decrease angiogenesis in the developing lung remains unknown. METHODS: Proximal pulmonary artery endothelial cells (PAECs) were harvested from fetal sheep after partial ligation of the ductus arteriosus in utero (PPHN) and controls. Growth and tube formation were assessed after ET-1 treatment. The effect of ET-1 antagonism on tube formation was studied using ET-1 small interfering RNA (siRNA), ET-1 monoclonal antibodies (ET-1mAbs), BQ-123 (an ETA blocker), and bosentan (an ETA/ETB blocker). ET-1 gene and protein and ETA/ETB receptor protein expression were measured in normal and PPHN PAECs. ET-1 ROCK interactions were assessed by measuring ROCK activity after ET-1, ET-1 siRNA, and bosentan treatments, and tube formation with ET-1 and Y-27632 (ROCK inhibitor). RESULTS: ET-1 did not affect growth but decreased tube formation in normal and PPHN PAECs. ET-1 protein and gene expression were increased and ETB receptor protein decreased in PPHN PAECs. ET-1 siRNA, ET-1mAbs, and bosentan, but not BQ-123, increased tube formation. ROCK activity was increased in PPHN PAECs and decreased with ET-1 siRNA and bosentan treatments. Y-27632 prevented the decrease in tube formation with ET-1. CONCLUSION: ET-1 activation of ROCK impairs angiogenesis of fetal PAECs. Disruption of ET-1 ROCK interactions may increase vascular growth in PPHN.