Abstract 16739: Impaired Nedd9-smad3 Binding Upregulates Collagen III Synthesis in Human Pulmonary Artery Endothelial Cells in vitro to Promote Pulmonary Arterial Hypertension in vivo

Genetic mutations that affect Smad-TGF-β signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is characterized by vascular fibrosis and hyperaldosteronism (ALDO). We developed a fibrosome network from published mRNA array data, which predicted that the Smad3 target NEDD9 is a critical ALDO-regulated node underpinning vascular fibrosis in silico (P=7.5x10-7 vs. random network). Our bioinformatics analysis suggested Cys18 in the Smad3 docking region of NEDD9 is a highly redox sensitive cysteinyl thiol based on its pKa and accessible surface area of 6.65 and 17.2 A2, respectively. To test this prediction, purified wild type (WT) human NEDD9 (1μM-1mM) or a mutant NEDD9 with an alanine for cysteine substitution at position 18 (NEDD9-C18A)(0.5 μM -1mM) was incubated with Smad3 (20 nM) for 20 min. Compared to vehicle (V) control, treatment with hydrogen peroxide (H2O2)(250 μM) diminished WT NEDD9-Smad3 binding (2.89 x 10-5 vs. 5.20 x10-5 Kd) assessed by microscale thermophore...