Neprilysin null mice develop exaggerated pulmonary vascular remodeling in response to chronic hypoxia.

Neprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder.

Neprilysin regulates pulmonary artery smooth muscle cell phenotype through a platelet-derived growth factor receptor-dependent mechanism.

Reduced neprilysin (NEP), a cell surface metallopeptidase, which cleaves and inactivates proinflammatory and vasoactive peptides, predisposes the lung vasculature to exaggerated remodeling in response to hypoxia. We hypothesize that loss of NEP in pulmonary artery smooth muscle cells results in increased migration and proliferation. Pulmonary artery smooth muscle cells isolated from NEP(-/-) mice exhibited enhanced migration and proliferation in response to serum and platelet-derived growth factor, which was attenuated by NEP replacement. Inhibition of NEP by overexpression of a peptidase dead mutant or knockdown by small interfering RNA in NEP(+/+) cells increased migration and proliferation. Loss of NEP led to an increase in Src kinase activity and phosphorylation of PTEN, resulting in activation of the platelet-derived growth factor receptor (PDGFR). Knockdown of Src kinase with small interfering RNA or inhibition with PP2, a src kinase inhibitor, decreased PDGFR(Y751) phosphorylation and attenuated migration and proliferation in NEP(-/-) smooth muscle cells. NEP substrates, endothelin 1 or fibroblast growth factor 2, increased activation of Src and PDGFR in NEP(+/+) cells, which was decreased by an endothelin A receptor antagonist, neutralizing antibody to fibroblast growth factor 2 and Src inhibitor. Similar to the observations in pulmonary artery smooth muscle cells, levels of phosphorylated PDGFR, Src, and PTEN were elevated in NEP(-/-) lungs. Endothelin A receptor antagonist also attenuated the enhanced responses in NEP(-/-) pulmonary artery smooth muscle cells and lungs. Taken together our results suggest a novel mechanism for the regulation of PDGFR signaling by NEP substrates involving Src and PTEN. Strategies that increase lung NEP activity/expression or target key downstream effectors, like Src, PTEN, or PDGFR, may be of therapeutic benefit in pulmonary vascular disease.