Abstract 12577: Pulmonary Endothelial Sulfatase-1 is Regulated by NEDD9 and Increased in Pulmonary Arterial Hypertension

Abstract

Introduction: Oxidation of the scaffolding protein NEDD9 by aldosterone (ALDO) increases NEDD9 bioactivity in human pulmonary artery endothelial cells (HPAECs), leading to pulmonary arterial hypertension (PAH). In tumor cells, oxidizing conditions alter Sulfatase-1 (SULF-1) activity. In turn, SULF-1 catalyzes 6-O-sulfate removal from heparan sulfate proteoglycans that releases cytokines implicated in the pathogenesis of PAH. Hypothesis: NEDD9 regulates SULF-1 expression in HPAECs, which is associated with pulmonary arterial remodeling in PAH. Methods: HPAECs were treated with ALDO (10 -7 mol/L) for 24 hr and then untransfected (Un-Tx) or transfected with siRNA against NEDD9, and cell lysates were used to isolate mRNA for RNA-Seq. Separately, SULF1 expression was measured by immunoblot in HPAECs transfected with si-NEDD9 or scrambled control siRNA. Paraffin-embedded lung specimens from donor controls (N=5), patients with PAH (N=5), or the monocrotaline (MCT) (inflammatory) and SU-5416/hypoxia (angioproliferative) experimental rat models of PAH were analyzed by immunofluorescence to quantify SULF1, NEDD9, and endothelial PECAM-1. Results: In ALDO-treated HPAECs, si-NEDD9 decreased SULF1 mRNA quantity vs. Un-Tx (logFC -6.39, N=3, P<0.001, FDR<0.001). Transfection with si-NEDD9 also decreased SULF1 protein compared to si-Scr (14 ± 2.0 vs. 2.7 ± 1.0 arb. units, N=3, P<0.01) in vitro . In remodeled pulmonary arterioles from MCT-PAH and SU-5416/hypoxia-PAH, SULF1 was increased by 5.6-fold (P<0.01) and 6.6-fold (P<0.01), respectively, and correlated strongly with NEDD9 protein (r=0.91, N=9, P<0.001). The translational relevance of these data was assessed next: compared to donor controls, pulmonary endothelial SULF1 was increased in remodeled pulmonary arterioles from PAH patients in situ (0.03 ± 0.01 vs. 0.14 ± 0.03 arb. units, P<0.05). Conclusions: SULF1 is regulated by the scaffolding protein NEDD9 in HPAECs in vitro , and remodeled pulmonary arterioles from two experimental PAH models and PAH patients express increased NEDD9-SULF1. Identifying the molecular mechanism(s) regulating NEDD9-dependent regulation of SULF1 may have important implications for therapeutic target discovery to prevent vascular remodeling in PAH.