Early mortality in pulmonary arterial hypertension (PAH) is due, in part, to fibrotic remodeling of pulmonary arterioles that increases vascular resistance, although effective therapies for vascular fibrosis in PAH do not exist currently. Overlap among established signaling pathways that regulate collagen function in reparative (e.g., dermal wound healing) and pathogenic (i.e., causing end-organ damage) fibrosis suggests that reductionist methods are limited for identifying key mechanisms mediating vascular fibrosis in PAH. In this NIH Research Project Grant Program proposal, we use network theory to develop a protein-protein interactome (fibrosome) and analyzed genes in silico according to their association with dermal wound healing or vascular fibrosis. The pro-oxidant hormone aldosterone (ALDO) promotes collagen synthesis in dermal wound healing. However, ALDO also increases collagen in human pulmonary artery endothelial cells (HPAECs) in vitro to induce vascular fibrosis in PAH in vivo. Thus, we propose that segregating ALDO-regulated genes in the fibrosome according to their association with pathogenic or adaptive fibrosis will allow novel molecular targets responsible for arterial fibrillar collagen synthesis in PAH to emerge. From our network analysis, we predicted that regulation of the Cas-L protein NEDD9 by ALDO delineates pathogenic from reparative fibrosis. We show that oxidation of a functionally essential cysteinyl thiol at position 18 of NEDD9 by ALDO prevents binding of NEDD9 with Smad3, which is required for normal proteolytic degradation of NEDD9. Impaired NEDD9-Smad3 binding, in turn, was associated with increased NEDD9 as well as NEDD9- dependent fibrillar collagen III levels and collagen matrigel contraction in ALDO-treated HPAECs in vitro, but not in cell types of reparative fibrosis. However, exosomes from ALDO-treated HPAECs increased NEDD9 in co-cultured human pulmonary artery smooth muscle cells, which was consistent with our observation that NEDD9 was increased globally in fibrotic pulmonary arterioles from PAH patients. Therefore, the central hypothesis of the current proposal is that oxidative modification of NEDD9-Cys18 by ALDO is a key molecular mechanism underpinning NEDD9 accumulation in HPAECs in vitro to promote pathogenic pulmonary vascular fibrosis and PAH in vivo. We postulate further that NEDD9 upregulation and transcellular signaling involving endothelial exosomes are two novel molecular mechanisms by which HPAECs promote vascular fibrosis in PAH. The study Aims are: (1) use Raman spectroscopy to demonstrate definitively that NEDD9-Cys18 oxidation by ALDO is an essential molecular mechanism underpinning a fibrotic phenotype in HPAECs in vitro, (2) test the hypothesis that HPAECs contribute to vascular fibrosis via intercellular signaling through a mechanism involving exosomes, and (3) use transgenic mice and gain-of-function methods to demonstrate that NEDD9 is a critical mediator of pulmonary vascular fibrosis and pulmonary hypertension in vivo. These studies aim to identify redox regulation of NEDD9 as a novel treatment target for PAH and other diseases characterized by pathogenic vascular fibrosis.

Public Health Relevance

Collagen is a form of connective tissue that is implicated in the development of cardiovascular diseases. Elevated blood pressure in the lung arteries (pulmonary hypertension) due to abnormal lung artery collagen (pulmonary vascular fibrosis) is a major cause of illness and early death in patients with various disorders of the heart, lung, blood, and joints, although effective treatments targeting pulmonary vascular fibrosis in pulmonary hypertension patients do not exist. Based on our preliminary research, we propose that the pro- fibrotic protein NEDD9 represents a potential new treatment target to prevent or reverse pulmonary vascular fibrosis in the large patient population afflicted with pulmonary hypertension.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Respiratory Integrative Biology and Translational Research Study Section (RIBT)
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Xiao, Lei
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Brigham and Women's Hospital
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Abman, Steven H; Lovering, Andrew T; Maron, Bradley A (2018) Pulmonary Vascular Disease Across the Life Span: A Call for Bridging Pediatric and Adult Cardiopulmonary Research and Care. Am J Respir Crit Care Med :
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