Abstract 18254: Pathogenic Role of STIM2 and Orai2 in Pulmonary Arterial Hypertension

Abstract

Pulmonary hypertension (PH) is a fatal disease attributed to increased pulmonary vascular resistance (PVR). Elevated PVR is partially due to sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling. Pulmonary arterial smooth muscle cells (PASMC) in their natural state are plastic and exist in two different phenotypes; a differentiated, contractile phenotype and a dedifferentiated, more proliferative phenotype. An increase in cytosolic free Ca 2+ concentration ([Ca 2+ ] cyt ) is a major stimulus for PASMC proliferation, contributing to pulmonary vascular remodeling. This study characterizes the phenotypic switch observed when freshly dissociated PASMC are cultured from normal male rats or in PASMC isolated from experimental PH rats. Western blot analysis showed freshly isolated pulmonary arteries (PA) express contractile markers that were decreased in cultured PASMC, demonstrating a switch to a proliferative phenotype. Cultured PASMC showed an increase in expression of store-operated and receptor-operated Ca 2+ channels Orai2, STIM2, and TRPC6 compared to freshly dissociated PASMC. Additionally, an increase in store-operated Ca 2+ entry (SOCE) and a decrease in voltage dependent Ca 2+ influx in cultured PASMC was observed when compared to freshly dissociated PASMC, suggesting SOCE plays a greater role in proliferating PASMC. Consistently, isometric force studies showed greater contraction induced by voltage-dependent Ca 2+ channel (VDCC) activation than SOCE induced contraction in freshly isolated pulmonary artery rings. Furthermore, Orai2, STIM2, and TRPC6 were upregulated in proliferative PASMC compared to quiescent PASMC. Knockdown of Orai2 was able to attenuate SOCE in proliferative PASMC demonstrating Orai2 is necessary for SOCE. These data suggest upregulation of STIM2 promotes the phenotypical transition of PASMC from a contractile to a proliferative phenotype by inhibiting VDCC and activating SOC, and that increased expression of Orai2 and TRPC6 channels mediate the increased [Ca 2+ ] cyt seen in proliferating cells. Understanding the molecular mechanisms which regulate [Ca 2+ ] cyt and PASMC proliferation is critical for the development of novel therapies for PH.