Abstract 12150: Molecular Network Analysis Identifies NEDD9 as a Critical Node Regulating Vascular Fibrosis in Pulmonary Arterial Hypertension
Vascular fibrosis in pulmonary arterial hypertension (PAH) is associated with increased morbidity. In PAH, elevated levels of aldosterone (ALDO) increase fibrillar collagen in pulmonary artery endothelial cells (PAECs). However, ALDO also promotes adaptive fibrosis in skin wound healing, suggesting that cell type-specific pathways regulated by ALDO distinguish fibrosis subtypes. To explore this concept further, published mRNA array data were used to model an interactome of fibrosis-related genes. Betweeness centrality analysis identified the Crk-associated protein NEDD9 as a critical ALDO-regulated node distinguishing vascular from dermal fibrosis (P=7.5x10-7 vs. random network). To validate this observation, PAECs and dermal microvascular ECs were treated with ALDO (10-7 mol/L) or vehicle control (V) for 24 hr and NEDD9 levels were analyzed by immunoblot. Compared to V-treated cells, ALDO increased NEDD9 expression in PAECs (46 ± 12 vs. 90 ± 7 a.u., P<0.05) but not dermal microvascular ECs (P=NS). To determine the relevance of NEDD9 to fibrosis in PAH, anti-NEDD9 immunohistochemistry was performed on explanted lung tissue from patients. Compared to controls, PAH patients had increased pulmonary arteriole NEDD9 (32 ± 2.8 vs. 69 ± 12 a.u., P<0.05), which correlated with collagen deposition levels assessed by Gomori trichrome stain (r=0.73, P<0.02) and was confirmed by confocal microscopy imaging demonstrating increased overlap between collagen III and NEDD9 expression in PAH pulmonary arterioles. We next analyzed the functional effect of NEDD9 on ALDO-induced collagen III in PAECs in vitro. Compared to V-treated cells, ALDO increased collagen III expression (117 ± 16 vs. 225 ± 34 a.u., P<0.01) and the number of Gomori stain positive PAECs (2.1 ± 0.8 vs. 8.1 ± 0.6 cells/h.p.f., P<0.01). By contrast, si-NEDD9 decreased collagen III and fibrosis in ALDO-treated cells by 81% (P<0.03) and 70.5% (P<0.01), respectively. Overall, we used a systems approach to demonstrate NEDD9 targeting by ALDO as a key mechanism defining pathogenic fibrosis. Identifying fibrillar collagen regulation by NEDD9 in PAECs may have novel therapeutic implications for patients with PAH and other diseases characterized by pulmonary vascular fibrosis.
Author Disclosures: T.E. Stephens: None. R. Wang: None. A. Samokhin: None. M. Cao: None. E. Arons: None. S.O. Vargas: None. J. Loscalzo: None. J.A. Leopold: None. B.A. Maron: None.
- © 2016 by American Heart Association, Inc.