Neprilysin Null Mice Develop Exaggerated Pulmonary Vascular Remodeling in Response to Chronic Hypoxia

Abstract

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. Chronic hypoxic pulmonary hypertension (PHTN) is a major clinical problem, complicating most lung and heart disorders. 1,2 In large animal models of chronic hypoxic PHTN that closely resemble human disease, the earliest pulmonary artery (PA) smooth muscle cell (SMC) proliferative changes occur at the medial/adventitial border. 3 Growth and migration of SMC and myofibroblasts in distal vessels is also a prominent feature. 4,5 These structural changes, together with derangements in vascular tone, are major contributors to the severity of chronic hypoxic PHTN. [1][2][3][4][5][6] However, mechanisms that regulate susceptibility to, and severity of, chronic hypoxic PHTN and vascular remodeling remain poorly understood. Currently available treatments for chronic hypoxic PHTN are also inadequate.

Decreased Neprilysin and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease

“…We investigated whether a reduction in NEP occurs (versus DPPIV) in human lungs with smoke-and hypoxia-induced vascular remodeling, similar to that observed in hypoxic mice (17). Advanced COPD lungs were obtained from patients with FEV 1 less than 30%, as worsening PHTN has been associated with decreased FEV 1 (8,27).…”

“…Staining (17) was with anti-human NEP, nitrotyrosine, CD31 (PECAM-1), or a-smooth muscle (SM) actin; counterstain was hematoxylin.…”

“…Density of distal PAs was measured in CD31-stained slides. For NEP-stained slides, intensity in the alveolar walls and distal vessels (nine areas per lung) was scored blindly on a scale of 0 to 41 (17).…”

“…NEP activity/ expression is decreased by CS (16), hypoxia (17,18), or reactive oxygen species (ROS) (19). Hypoxic NEP null mice develop greater PHTN, and PA SMCs from these mice grow faster than those from wild-type mice, suggesting that NEP protects against chronic hypoxic PHTN, in part by suppressing PA SMC growth and migration (17).…”

Mitogen-Activated Protein Kinase Phosphatase-1 Is a Key Regulator of Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Vessel Density in Lung

“…The following antibodies were used for staining of the lung sections: ␣SMA (1:100) (Sigma-Aldrich, St. Louis, MO) 19,20 ; phosphoP38 (1:50), phosphoJNK (1:100), and phosphoERK (1:100) (Cell Signaling Technology, Danvers, MA) 21,22 ; Ki-67 (1:100) (Epitomics, Burlingame, CA) 23,24 ; and VEGF (1:100) (Novus Biologicals, Littleton, CO). 25 Immunohistochemical staining was performed according to our previously described method.…”