PROJECT ABSTRACT Chronic hypoxic pulmonary hypertension (PHTN) is a major clinical problem, in the general and Veteran populations, and complicates most lung and heart disorders, including chronic obstructive pulmonary disease (COPD). Many factors, including cigarette smoke (CS), oxidant stress, inflammation and eventually hypoxia, contribute to the observed vascular dysfunction and remodeling in this form of chronic PHTN. Proliferation of pulmonary artery (PA) smooth muscle cells (SMCs) at the medial/adventitial border and migration to more distal sites are prominent features of the structural change. Neprilysin (NEP) is a transmembrane protein with a cell surface peptidase activity that degrades select pro- and anti-inflammatory neuropeptides and may take part in signaling cascades by directly coupling to intracellular proteins via peptidase-independent mechanisms. Novel substrates of NEP have also recently been described. There is growing evidence that lung NEP activity and expression decrease in response to chronic CS and hypoxia by as yet unknown mechanisms. We now have preliminary evidence that NEP is also selectively reduced in lungs of patients with advanced COPD. NEP null mice have exaggerated pulmonary vascular remodeling and PHTN in response to chronic hypoxia, and resident PA SMCs have increased growth compared to their wild type counterparts. The decrease in human and mouse NEP is most striking in distal vessels where early PA SMC proliferation and migration occurs. We have also observed that CS extract (CSE) and hypoxia decrease NEP activity and expression in isolated human and mouse PA SMCs. These observations support the idea that NEP may exert a protective effect on the lung circulation and could be a disease modifying gene for chronic forms of PHTN. To extend these observations, we will investigate the role of NEP in chronic CS- and hypoxia-induced PHTN, in complementary human and mouse tissue and cell preparations. We will test the Overall Hypothesis that: decreased NEP predisposes to the development of increased pulmonary vascular remodeling in chronic lung disorders, like COPD. Chronic CS (like hypoxia) reduces NEP activity and/or expression, ultimately leading to increased proliferation of resident PA SMCs, vascular dysfunction, pulmonary vascular remodeling, and PHTN. In contrast, increased NEP protects the lung vasculature from the development of pulmonary vascular remodeling and PHTN in response to chronic CS (and hypoxia) at least in part, by decreasing the growth of resident PA SMCs. Our Aims are: 1) determine the involvement of NEP activity and expression in human and mouse pulmonary vascular remodeling due to CS;2) determine key mechanisms by which CS exerts an attenuating effect on NEP activity and expression (focus on reactive oxygen and nitrogen species [ROS/RNS] leading to protein degradation by lysosomes and proteasomes and decreased gene expression mediated by the hypoxia-induced transcription factors HIF-1 and HIF-2). This project will draw on human lung tissue and PA SMCs from 'control'patients and those with severe COPD, genetically engineered mouse models, and unique lentiviral vectors to study CS-induced pulmonary vascular remodeling. We will sequentially examine how oxidant stress inactivates NEP peptidase activity, initiates degradation of NEP protein in the lysosome and proteasome, and later decreases NEP gene expression by induction/activation of HIF-1 and HIF-2. These studies may help increase our understanding of mechanisms that control susceptibility to chronic CS- and hypoxia-induced PHTN and could also identify new therapeutic strategies (like directly or indirectly increasing NEP in the lung) to limit or reverse this important clinical problem. PUBLIC HEALTH RELEVANCE: Project Narrative: Pulmonary hypertension, or high pressure in the lung blood vessels, is often caused by exposure to cigarette smoke and low oxygen levels (hypoxia);it is a major clinical problem in the Veteran population, complicating lung disorders like COPD and heart failure. Smooth muscle cells in the walls of lung arteries may grow abnormally in response to smoke and hypoxia, leading to a reduction in vessel diameter and increased work for the right side of the heart. A cell surface protein, Neprilysin, may play an important role in preventing this abnormal growth. This project will draw on lung tissue from patients with pulmonary hypertension, human vascular cells, genetically engineered mouse models, and unique reagents to determine the extent to which Neprilysin can protect against this disease process. These studies may help to increase our understanding of mechanisms that control susceptibility to smoke and hypoxia-induced pulmonary vascular disease and may ultimately lead to the identification of new therapeutic strategies for this important clinical problem.