44 ASK-1 inhibition prevents hypoxia-induced pulmonary artery fibroblast proliferation and migration in an in vitro cellular model of pulmonary hypertension

Rationale Pulmonary arterial hypertension (PAH) involves progressive remodelling of the pulmonary arteries, increasing pulmonary vascular resistance leading to right ventricular dysfunction. The remodelling is due to increased pulmonary adventitial fibroblast proliferation, migration, and release of cytokines. These fibroblast responses involve members of the Mitogen Activated Protein Kinases (MAPK) signalling pathway. Apoptosis Signal-regulating Kinase-1 (ASK1) is an apical MAPK, which undergoes auto-phosphorylation and activation in response to oxidative stress, activating downstream p38 MAPK and c-Jun N-terminal kinase (JNK). The work presented here investigates the possible cellular mechanisms and downstream targets of ASK1 in the pulmonary artery fibroblast (PAF) in a model of PAH. Methodology PAF were obtained from adult Sprague Dawley rats. Hypoxia (5% O2) was used as an in vitro model of PAH. Proliferation was determined using [3H] thymidine incorporation. Migration was determined by scratch assay. Targets of ASK1 were determined by Western Blotting. ASK1 inhibition was achieved by the addition of a selective, small molecule inhibitor (GS-444217). Results Hypoxia increased PAF proliferation and migration; both were significantly reduced by the addition of 1 µM GS-444217. Hypoxia increased phosphorylation of both ASK1 and p38 MAPK, and increased Hypoxia-Inducible Factor 1 alpha (HIF1a) protein abundance; 1 µM GS-444217 attenuated these effects. ASK1 inhibition reduced hypoxia-induced expression of key mediators of vascular remodelling, tissue Inhibitor of Metaloproteinase-1 (TIMP-1), soluble intercellular adhesion molecule-1 (sICAM-1) and endothelin-1 (ET-1). Conclusions ASK1 inhibitor (GS-444217) reduced hypoxia-induced proliferation and migration of PAF, and reduced disease-associated signalling pathways. ASK1 inhibition may be a novel target for preventing maladaptive vascular remodelling.