Role of Nitric Oxide Synthases in Respiratory Health and Disease: Insights from Triple Nitric Oxide Synthases Knockout Mice

Abstract

The system of nitric oxide synthases (NOSs) is comprised of three isoforms: nNOS, iNOS, and eNOS. The roles of NOSs in respiratory diseases in vivo have been studied by using inhibitors of NOSs and NOS-knockout mice. Their exact roles remain uncertain, however, because of the non-specificity of inhibitors of NOSs and compensatory up-regulation of other NOSs in NOS-KO mice. We addressed this point in our triple-n/i/eNOSs-KO mice. Triple-n/i/eNOSs-KO mice spontaneously developed pulmonary emphysema and displayed exacerbation of bleomycin-induced pulmonary fibrosis as compared with wild-type (WT) mice. Triple-n/i/eNOSs-KO mice exhibited worsening of hypoxic pulmonary hypertension (PH), which was reversed by treatment with sodium nitrate, and WT mice that underwent triple-n/i/eNOSs-KO bone marrow transplantation (BMT) also showed aggravation of hypoxic PH compared with those that underwent WT BMT. Conversely, ovalbumin-evoked asthma was milder in triple-n/i/eNOSs-KO than WT mice. These results suggest that the roles of NOSs are different in different pathologic states, even in the same respiratory diseases, indicating the diversity of the roles of NOSs. In this review, we describe these previous studies and discuss the roles of NOSs in respiratory health and disease. We also explain the current state of development of inorganic nitrate as a new drug for respiratory diseases.

Keywords: asthma; bone marrow; mice; nitrate; nitric oxide; nitric oxide synthases; pulmonary emphysema; pulmonary fibrosis; pulmonary hypertension.

Figure 1

Schematic diagram showing the chemical reactions catalyzed by NOSs. NO is synthesized from L-arginine, oxygen, and NADPH by NOSs along with generation of L-citrulline, water, and NADP⁺. NO is oxidized to nitrite (NO₂⁻), and, in turn, to nitrate (NO₃⁻). Nitrate is rich in green leafy vegetables, such as beetroot, lettuce, and spinach. Nitrate is reduced to nitrite by bacteria in the oral cavity and xanthine oxidase, and nitrite is reduced to NO by xanthine oxidoreductase, deoxyhemoglobin, myoglobin, respiratory chain enzymes, vitamin C, Polyphenols, and protons.

Figure 2

Compensatory interactions among each NOS isoform (A) and plasma NOx levels in triple-n/i/eNOSs-KO mice (B). (A) Schematic diagram showing compensatory interactions among each NOS isoform. (B) Plasma NOx levels in wild-type C57BL/6 and 129SV, single-nNOS-KO, -iNOS-KO, -eNOS-KO, double-n/iNOSs-KO, -n/eNOSs-KO, -i/eNOSs-KO, and triple-n/i/eNOSs-KO mice. * p < 0.05, ^# p < 0.01, ^† p < 0.001 vs. C57BL/6. Data from [28]. Copyright 2005 National Academy of Sciences.

Figure 3

Spontaneous pulmonary emphysema in triple-n/i/eNOSs-KO mice. (A) Hematoxylin and eosin staining of lung tissues. Scale bars in upper and lower pictures indicate 2500 and 250 μm, respectively. (B) Hematoxylin and eosin staining of lung tissues in WT and triple-n/i/eNOSs-KO mice. Arrowheads indicate destroyed alveoli. Scale bar = 50 μm. (C) Microscopic computed tomography images of the lung. Eight-week-old male WT mice, three strains of single-NOS-KO mice, three strains of double-NOSs-KO mice, and triple-n/i/eNOSs-KO mice were studied. Data from [32].

Figure 4

Deterioration of pulmonary fibrosis and increased inflammatory cell number in the bronchoalveolar lavage fluid in triple-n/i/eNOSs-KO mice at 2 weeks after bleomycin treatment. (A) Hematoxylin–eosin (H.E) staining in normal saline-treated mice. Scale bar = 100 μm. (B) Hematoxylin–eosin staining, Masson’s trichrome staining, α-smooth muscle actin (SMA) staining, MAC-2 staining in bleomycin-treated mice. Scale bar = 100 μm. (C) Fibrotic tissue area (blue-stained). (D) Collagen content in lung tissues. White and black bars indicate normal saline-treated (n = 3) and bleomycin-treated mice (n = 5), respectively. * p < 0.05 vs. bleomycin-treated WT mice. (E–H) Total, macrophage, and lymphocyte cell counts and total protein concentrations in the bronchoalveolar lavage fluid. White and black bars indicate normal saline-treated (n = 3) and bleomycin-treated mice (n = 5), respectively. * p < 0.05 vs. bleomycin-treated WT mice. Data from [33].

Figure 5

The protective role of NO derived from myelocytic NOSs in chronic hypoxia-induced pulmonary hypertension in mice. (A) An inverse correlation between pulmonary artery systolic pressure and NOx (nitrite plus nitrate) levels in bronchoalveolar lavage fluid in patients with idiopathic pulmonary fibrosis. Pulmonary artery systolic pressure estimated by Doppler echocardiography was significantly and negatively correlated with NOx concentrations in bronchoalveolar lavage fluid in patients with idiopathic pulmonary fibrosis. Statistical analysis was performed using the Pearson correlation coefficient. BALF, bronchoalveolar lavage fluid. (B) Hematoxylin and eosin staining, elastic van Gieson staining, and α-smooth muscle actin staining of small pulmonary arteries. Scale bar = 50 μm. Medial thickness of small pulmonary arteries (50–150 µm in diameter) (n = 5–10). Statistical analyses were performed by an ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. * p < 0.05 vs. normoxia; ^† p < 0.05 vs. wild type; ^‡ vs. eNOS^−/−. H&E, hematoxylin and eosin staining; EVG, elastic van Gieson staining; α-SMA, α-smooth muscle actin staining. (C) Immunofluorescent staining in the lungs of wild-type and triple-n/i/eNOSs^−/− mice transplanted with bone marrow cells isolated from green-fluorescent-protein-transgenic mice after chronic hypoxic exposure. Green-fluorescent-protein-positive green fluorescence and green fluorescent protein/α-smooth-muscle-cell-double-positive white fluorescence were observed (n = 5). Scale bars = 50 μm. DAPI, 4′,6-diamidino-2-phenylindole (nuclear staining); GFP, green fluorescent protein; α-SMA, α-smooth muscle actin. (D–G) Plasma NOx levels (D), weight ratio of the right ventricle to the left ventricle plus the interventricular septum (RV/[LV + S]) (E), right ventricular systolic pressure (RVSP) (F), and medial thickness of small pulmonary arteries (50–150 µm in diameter) (G) after chronic hypoxic exposure (n = 5–6). Statistical analyses were performed by an ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. * p < 0.05 vs. wild-type mice transplanted with wild-type bone marrow cells; ^† p < 0.05 vs. triple-n/i/eNOSs^−/− mice transplanted with triple-n/i/eNOSs^−/− bone marrow cells. (H) Heat maps of differentially expressed mRNAs categorized as immunity and inflammation in the Subio platform. RNA sequencing was performed to obtain the data. (I) A schematic diagram showing the protective role of NO derived from myelocytic NOSs in chronic hypoxia-induced pulmonary hypertension. Data from [6]. Reprinted with permission of the American Thoracic Society. Copyright© 2024 American Thoracic Society. All rights reserved. The American Journal of Respiratory and Critical Care Medicine is an official journal of the American Thoracic Society.

Figure 6

Pathological findings and cytokine and chemokine mRNA expression levels in the lungs of WT and triple-n/i/eNOSs-KO mice that were sensitized and challenged with ovalbumin (OVA). (A–D) Hematoxylin and eosin staining of lung sections from wild-type (WT) (A) and triple-n/i/eNOSs-KO mice (B) and periodic acid–Schiff staining of lung sections from WT (C) and triple-n/i/eNOSs-KO mice (D). (E–L) mRNA expressions of interferon (IFN)-γ (E), interleukin (IL)-4 (F), IL-5 (G), IL-10 (H), IL-13 (I), monocyte chemoattractant protein (MCP)-1 (J), eotaxin-1 (K), and thymus- and activation-regulated chemokine (TARC) (L) in the lungs of WT (n = 4) and triple-n/i/eNOSs-KO mice (n = 4). Data are represented as the ratio to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). * p < 0.05 by t test. Data from [34].

Figure 7

Diverse roles of NOSs in respiratory diseases (A) and impact of NOSs in different cell types and dietary nitrate in pulmonary health (B). (A) Schematic diagram showing the diverse roles of NOSs in respiratory diseases. NOSs play a protective role in the pathogenesis of spontaneous pulmonary emphysema, pulmonary fibrosis, and pulmonary hypertension, whereas they play a harmful role in the pathogenesis of asthma. (B) Schematic diagram indicating the impact of NOSs in different cell types and dietary nitrate on pulmonary health. NO derived from NOSs in non-bone-marrow-derived cells, such as lung cells and vascular wall cells, elicits vasodilation and reduces pulmonary vascular resistance, preventing pulmonary hypertension; NO derived from NOSs in bone marrow cells inhibits pulmonary vascular remodeling, preventing pulmonary hypertension; and NO converted from dietary nitrate (e.g., a beetroot, lettuce, and spinach) via the nitrate–nitrite–NO pathway provides an alternative source of NO, maintaining pulmonary health and protecting against the development of pulmonary hypertension.