HIF‐1α Expression is Decreased and Contractility is Enhanced in PASMC from PAH Patients

Abstract

OBJECTIVETo determine if hypoxia inducible factor‐1α (HIF‐1α) expression is decreased and myosin light chain (MLC) phosphorylation and contractility are enhanced in pulmonary artery smooth muscle cells (PASMC) from patients with pulmonary arterial hypertension (PAH).METHODSPASMC were isolated from patients diagnosed with PAH and controls. PASMC were examined by Western, RT‐PCR, and immunofluorescence to assess HIF‐1α and phosphorylated MLC levels. Prolyl‐hydroxylase domain (PHD) activity was examined by oxygen, iron (II) and ascorbate cellular content. Metabolism was assessed via formazan and lactate production. The relative contractile response was determined by collagen gel assay. Rho kinase and myosin light chain kinase (MLCK) activities were quantified by ELISA. Ca2+ imaging was employed to assess intracellular Ca2+ levels in HIF‐1α‐/‐ PASMC.RESULTSIn PAH SMC, HIF‐1α protein expression is decreased and MLC phosphorylation is increased. PHD inhibition increases HIF‐1α expression in PAH SMC. Furthermore, cellular oxygen and ascorbate levels are higher in PAH SMC. Formazan production is increased and lactate production is decreased in PAH SMC. The relative SMC contractile response is greater in PAH. MLCK activity is increased in PAH SMC. Loss of HIF‐1α in PASMC potentiates the hypoxia‐induced increase in cytosolic Ca2+.CONCLUSIONSIn PASMC from patients with PAH, HIF‐1α protein expression is decreased and MLC phosphorylation and functional contraction are increased via MLCK activation. Overall, these results demonstrate that PASMC contractility is increased in PAH, perhaps as a direct result of decreased HIF‐1α expression. Altered HIF‐1α expression may play an etiologic role in the development of PAH.