Abstract 13336: Rasa3 Affects Key Proliferation and Angiogenic Pathways in and Around Pulmonary Artery Endothelial Cells

Abstract

Introduction: Despite improvements in quality of life and functional status, pulmonary hypertension (PH) remains a highly morbid disease with poor life expectancy with no targeted treatments available. RASA3 is a ubiquitously expressed GTPase activating protein that is integral to angiogenesis and barrier function in endothelial cells (EC). In this study, we sought to explore the differential expression of RASA3 in response to PH-relevant stimuli and downstream markers of EC health when RASA3 expression is lost. Methods: Human pulmonary artery cells (PAECs) from Lonza were grown in culture and treated with 1% hypoxia, increasing concentrations of TNF-α for 24 hours or IL-1β for 48 hours and then harvested. Cells were transfected with siRNA directed against RASA3 or non-targeting control and harvested to evaluate downstream markers of EC health, or plated on Matrigel to evaluate angiogenic capabilities. Non-contact co-culture experiments were performed using human pulmonary artery smooth muscle cells (PASMC) with PAECs that were transfected with siRNA against RASA3 or non-targeting control. Results: We found that when untreated PAECs were exposed to 1% hypoxia for up to 96 hours, RASA3 expression was significantly decreased (p=0.046). When treated with increasing concentrations of TNF-α for 24 hours, RASA3 expression decreased (p=0.0002). When PAECs were treated with IL-1β 10ng/mL for 48 hours, RASA3 expression significantly increased (p=0.001). Silenced RASA3 PAECs had significantly less PCNA (p=0.0474), a trend towards increased HIF-2α expression (p=0.08) and increased VEGFR2 expression (p=0.0878). Cells that had silenced RASA3 expression when plated on Matrigel were significantly less able to form rings compared to non-targeting control (p=0.0152). PASMCs that had been co-cultured with silenced RASA3 had significantly increased PCNA compared to PASMCs co-cultured with non-targeting control PAECs (p=0.0467). Conclusions: RASA3 is essential to PAEC homeostasis, and loss of RASA3 affects endothelial cell health as well as affects its local environment. Further studies are ongoing to determine how loss of RASA3 contributes to the development of PH.