J012 Cyclooxygenase-2-dependent isoprostane production in hypoxia-induced pulmonary hypertension

This study investigates the contribution of contractile prostanoids in hyper-reactivity of pulmonary arteries to vasoconstrictors, in a mice model of hypoxic pulmonary arterial hypertension.Male C57BL/6 mice were exposed or not to hypobaric hypoxia (0.5atm) for 21 days. Extrapulmonary arteries were removed and used for evaluation of vasomotor responses (using wire myograph), for expression and localisation of cyclooxygenases and thromboxane A2 (TXA2)-synthase (by western blotting and immunofluoresnce) and for release of vasoactive prostanoids (by ELISA).In pulmonary arteries from hypoxic mice (but not in those from normoxic mice), arachidonic acid (30μM) induced a contractile effect, that was converted into relaxation in the presence of SQ29548 (0.5μM), a thromboxane receptor (TP) antagonist. In these arteries, contraction to phenylephrine (3μM) was enhanced about 1.8 fold increase compared to controls, in both endothelium-intact and denuded preparations. This hyper-reactivity to phenylephrine was diminished by SQ29548, by the selective COX-2 inhibitor NS398 (1μM), but not by the phospholipase A2 inhibitor AACOCF3 (30μM), the COX-1 inhibitor SC560 (0.1μM) or the TXA2-synthase inhibitor furegrelate (100μM). None of these agents affected contraction to phenylephrine in pulmonary arteries from normoxic mice. Expression of COX-1, which was found in all layers of pulmonary arteries, was decreased by chronic hypoxia as well as expression of TXA2-synthase. COX-2 expression was restricted to the medial layer of pulmonary arteries. Hypoxia decreased the release of TXA2 and the release of PGI2 from pulmonary arteries, while it increased the release of 8-iso-PGF2α (an isoprostane derivative that is a marker of oxidative stress). NS398 abolished hypoxia-induced elevation of 8-iso-PGF2α release from pulmonary arteries. Finally, 8-iso-PGF2α induced a contractile effect in pulmonary arteries, which was blunted by SQ29548. Moreover, 8-iso-PGF2α markedly potentiated contraction to phenylephrine.These data show that following chronic hypoxia, pulmonary arteries exhibited alterations in arachidonic acid pathway, and hyper-responsiveness to phenylephrine. The latter is likely mediated by COX-2-dependent production of 8-iso-PGF2α, which in turn activates TP receptor. Such mechanisms probably contribute to elevation in pulmonary arterial resistance in hypoxia-induced pulmonary arterial hypertension