Nitric oxide is required for lung alveolarization revealed by deficiency of argininosuccinate lyase

Abstract

Inhaled nitric oxide (NO) therapy has been reported to improve lung growth in premature newborns. However, the underlying mechanisms by which NO regulates lung development remain largely unclear. NO is enzymatically produced by three isoforms of nitric oxide synthase (NOS) enzymes. NOS knockout mice are useful tools to investigate NO function in the lung. Each single NOS knockout mouse does not show obvious lung alveolar phenotype, likely due to compensatory mechanisms. While mice lacking all three NOS isoforms display impaired lung alveolarization, implicating NO plays a pivotal role in lung alveolarization. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing L-arginine, the sole precursor for NOS-dependent NO synthesis. ASL is also required for channeling extracellular L-arginine into a NO-synthetic complex. Thus, ASL deficiency (ASLD) is a non-redundant model for cell-autonomous, NOS-dependent NO deficiency. Here, we assessed lung alveolarization in ASL-deficient mice. Hypomorphic deletion of Asl (Asl^Neo/Neo) results in decreased lung alveolarization, accompanied with reduced level of S-nitrosylation in the lung. Genetic ablation of one copy of Caveolin-1, which is a negative regulator of NO production, restores total S-nitrosylation as well as lung alveolarization in Asl^Neo/Neo mice. Importantly, NO supplementation could partially rescue lung alveolarization in Asl^Neo/Neo mice. Furthermore, endothelial-specific knockout mice (VE-Cadherin Cre; Asl^flox/flox) exhibit impaired lung alveolarization at 12 weeks old, supporting an essential role of endothelial-derived NO in the enhancement of lung alveolarization. Thus, we propose that ASLD is a model to study NO-mediated lung alveolarization.

urea cycle, argininosuccinate lyase, nitric oxide, alveolarization

Introduction

Lung alveolarization is the process to generate the alveolar gas exchange units in the lung. Defective lung alveolarization is a key histopathological hallmark in many lung diseases, such as bronchopulmonary dysplasia (BPD) [1, 2]. Thus, understanding the molecular mechanisms and the factors that influence lung alveolarization may facilitate the development of strategies to improve lung alveolarization and treat related diseases.

Nitric oxide (NO) is a signaling molecule that plays important roles in lung alveolarization. Both endogenous and exogenous NO has been shown to modulate branching morphogenesis in the rat lung [3]. Importantly, inhaled NO has been reported to improve lung alveolarization in neonatal rats exposed to hyperoxia or with BPD, preterm lambs with chronic ventilation-induced lung injury, and the baboon model with neonatal chronic lung disease [4–7]. Furthermore, there are potential benefits of inhaled NO in human preterm newborns [8–10]. However, the treatment effects have been variable and debatable, possibly due to the heterogeneous cohort of patients as well as various treatment strategies [11, 12]. Moreover, the mechanisms by which NO regulates lung alveolarization are largely unknown.

NO is produced by three isoforms of NO synthases (NOSs), neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS). Studies with global deletion of each single (nNOS, iNOS, or eNOS) or double (n/iNOSs, n/eNOSs, or i/eNOSs) NOS knockout mice did not have impaired lung alveolarization, while the triple n/i/eNOSs knockout mice displayed impaired lung alveolarization, implicating compensatory effects among different NOS isoforms [13, 14]. Nonetheless, eNOS KO mice are susceptible to both hypoxia and hyperoxia-induced inhibition of lung alveolarization [14–16]. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing L-arginine, the sole precursor for NOS-dependent NO synthesis. Moreover, ASL is also required for channeling extracellular L-arginine to NOS for NO production [17]. Therefore, our group and others utilized argininosuccinate lyase deficiency (ASLD) as a model to study cell-autonomous, NOS-dependent NO deficiency [17–21]. In the present study, we assessed lung alveolarization in a hypomorphic mouse model of ASLD. Furthermore, we deleted ASL in endothelial cells and tested whether endothelial-derived NO plays a role in lung alveolarization given that vascular endothelial cells are a major contributor to NO in the lung.

Materials and methods

Mice

Asl^Neo/Neo mice and Asl^flox/flox mice were previously generated and described [17, 18]. Heterozygous Asl^Neo/+ mice were bred to generate WT mice and Asl^Neo/Neo mice. In addition, regular water was replaced with sodium nitrite water (60 mg/l) to supplement NO in the breeding cages. To generate Asl ^Neo/Neo;Cav-1^+/− mice, Asl^Neo/+ mice were crossed with Caveolin-1 KO mice (B6.Cg-Cav1^tm1Mls/J from the Jackson Laboratory). To generate endothelial-specific Asl KO mice, VE-Cadherin-Cre mice (B6.FVB-Tg(Cdh5-cre)7Mlia/J from the Jackson Laboratory) were crossed with Asl^flox/flox mice. All experiments were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine.

Figure 1

Heterozygous deletion of Cav-1 rescues lung alveolarization in Asl^Neo/Neo mice. Lungs were inflated through the trachea and fixed with 4% PFA for H&E staining at postnatal day 21 to day 23 male mice. At least 10 images from three sections were assessed for alveolarization for each mouse and the mean linear intercept (MLI) was calculated using image J (NIH). (A) Representative H&E staining images. (B) MLI quantification. WT mice (n = 8); Cav-1^+/− mice (n = 5); Asl^Neo/Neo mice (n = 9); Asl^Neo/Neo;Cav-1^+/− mice (n = 7). ^**P < 0.01; ^***P < 0.005; ns, not significant (P > 0.05). Results are shown as means ± s.d.

Lung inflation and mean linear intercept (MLI) quantification

After mice euthanization, lungs were equally inflated under 25 cm pressure with 4% PFA, through a butterfly needle inserted into the trachea. After inflation, the suture was closed at the trachea. Lungs were paraffin-embedded, sectioned, and stained with H&E. A total of 10 images of histological fields per mouse were taken from three sections and used for quantification. We quantified the lung MLI using a modified ImageJ software (National Institutes of Health) as previously described [22, 23]. We manually removed the vessels, large airways, and other nonalveolar structures in each image.

Biotin switch assay to detect S-nitrosylated proteins

We performed the Biotin Switch assay using lung tissues with minor modifications [24]. All steps before the biotin switch were performed under indirect light. Briefly, proteins were extracted from lung tissue by homogenizing in HENS buffer. 20 mM N-ethylmaleimide (NEM; Thermo Scientific PI23030) was used to block free thiols. Next, NEM was removed by acetone. After ascorbic acid (20 mM) reduction of S-nitrosothiols, 0.5 mM EZ-Link HPDP-Biotin (Thermo Scientific PI21341) was used to simultaneously label the biotinylation of the previous S-nitrosylated (SNO) proteins in the lung lysate. For negative controls, ascorbic acid was omitted. Biotinylated proteins were detected by immunoblotting with anti-biotin (Cell Signaling; #7075) antibody.

Western blot

Lungs were harvested, snap frozen in liquid nitrogen, and stored at −80°C until protein extraction. Protein was extracted via homogenizing (IKA T25 Basic Ultra-Turrax Homogenizer) in RIPA buffer (Thermo Fisher Scientific; Cat #89900). A standard western blot was performed. Samples were resolved in an SDS polyacrylamide gel, transferred to PVDF membrane, and incubated with the primary antibodies Caveolin-1 antibody (Cell Signaling; #3267s), and α-tubulin antibody (Invitrogen, #T5168) as internal control. Then incubated with HRP-conjugated secondary antibodies. Western blot images were quantified with ImageJ (NIH).

Statistical analyses

Statistical significance between the two groups was determined by 2-tailed Student’s t test. Comparisons between multiple groups were determined by one-way or two-way ANOVA, followed by Tukey’s multiple comparisons test. ^*P < 0.05; ^**P < 0.01; ^***P < 0.005; ns: not significant P > 0.05. All results are represented as means ± s.d.

Results

Asl^Neo/Neo mice exhibit impaired lung alveolarization

Asl hypomorphic mice (Asl^Neo/Neo) have 25% residual RNA, 25% residual protein and 16% residual enzyme activity and die within 4 weeks of age [17]. We examined lung alveolar development of WT and Asl^Neo/Neo mice between postnatal day 21 to day 23. After euthanization, the lung was inflated and fixed with 4% paraformaldehyde (PFA). Hematoxylin-eosin (H&E) staining was performed on lung sections (Fig. 1A). The calculation of lung Mean Linear Intercept (MLI) showed that Asl^Neo/Neo mice exhibited impaired lung alveolarization, as evidenced by increased lung intercept lengths in Asl^Neo/Neo mice compared to WT controls (Fig. 1B).

Asl^Neo/Neo mice exhibit decreased total S-nitrosylation level

S-nitrosylation is a covalent post-translational modification of protein cysteine thiols by adding nitrogen monoxide group, which translates a large part of the ubiquitous influence of NO on cellular signal transduction and function [25–27]. Using Biotin Switch assay, we detected decreased total S-nitrosylation level in whole lung extracts of Asl^Neo/Neo mice compared to WT controls (Fig. 2), indicating NO signaling is attenuated in the lung of Asl^Neo/Neo mice.

Figure 2

Heterozygous deletion of Cav-1 reverses total S-nitrosylation of lungs in Asl^Neo/Neo mice. Lungs were collected at the age of postnatal day 21 to day 23. Biotin switch assay followed by immunoblotting of biotin was performed using lung tissues. The result was representative of three independent experiments. ASC: Ascorbic acid.

Heterozygous deletion of Cav-1 reverses S-nitrosylation as well as alveolarization in the lung of Asl^Neo/Neo mice

Caveolin-1 (Cav-1) has been reported to form a protein complex with NOS and inhibit NO production [28, 29]. In contrast, ASL is part of a positive NO synthetic complex [17, 19]. We examined Cav-1 protein level in the lung by western blot. We did not observe any alterations of Cav-1 at the protein level between WT and Asl^Neo/Neo mice (Supplementary Fig. 1). We hypothesized that genetic deletion of a copy of Cav-1 could enhance NO production and thereby rescue lung alveolar development in Asl^Neo/Neo mice. Indeed, we observed that total lung S-nitrosylation level was reversed in Asl^Neo/Neo;Cav-1^+/− mice compared to Asl^Neo/Neo mice (Fig. 2). Further MLI quantification of lung sections showed that lung alveolarization was also rescued in Asl^Neo/Neo;Cav-1^+/− mice (Fig. 1B). Heterozygous deletion of Cav-1 did not change lung MLI in Cav-1^+/− mice as compared to WT mice (Fig. 1A and B).

NO supplementation partially rescues lung alveolarization in Asl^Neo/Neo mice

To directly test the hypothesis that impaired lung alveolarization in Asl^Neo/Neo mice is due to impaired NO production, we replenished NO by the supplementation of sodium nitrite water (60 mg/l) in the breeding cages, during both gestation and lactation stages, and assessed lung alveolarization of offsprings at the age of postnatal day 21 to day 23 (Fig. 3A). Quantification of lung MLI showed that NO supplementation did not improve lung alveolarization in WT mice, however, it could partially restore lung alveolarization in Asl^Neo/Neo mice (Fig. 3B).

Figure 3

Partial rescue of lung alveolarization of Asl^Neo/Neo mice by sodium nitrite water supplementation. The breeding cages were supplemented with regular water or sodium nitrite (60 mg/l) water. Lungs were inflated and harvested at postnatal day 21 to day 23 male pups and alveolarization was quantified. (A) Representative H&E staining images. (B) MLI quantification. WT control (n = 8; data adapted from Fig. 1); WT sodium nitrite (n = 7); Asl^Neo/Neo control (n = 9; data adapted from Fig. 1); Asl^Neo/Neo sodium nitrite (n = 5). ^*P < 0.05; ^**P < 0.01; ^***P < 0.005. Results are shown as means ± s.d.

Endothelial cell-specific deletion of Asl in VE-cad Asl cKO mice exhibits decreased lung alveolarization

Previous studies from our group showed that Asl^Neo/Neo mice have elevated blood pressure [17]. We further demonstrated that endothelial dysfunction is a primary driver of increased blood pressure in ASLD [18]. Since accumulating evidence suggests pulmonary endothelial cells are involved in lung alveolarization [30, 31], we next investigated whether loss of NO in endothelial cells affected lung alveolarization. We crossed Asl^flox/flox mice with vascular endothelial (VE)-Cadherin (Cad)-Cre mice to generate VE-Cad Asl conditional knockout mice (VE-Cad Asl cKO or VE-Cad Cre;Asl^flox/flox). We first examined lung alveolar development of Asl^flox/flox control mice and VE-Cad Asl cKO mice at postnatal day 21 to day 23 (Fig. 4A and C). The results showed no alterations of lung MLI at this age (Fig. 4A and C). We next assessed lung alveolarization at 12 weeks old and found that VE-Cad Asl cKO mice exhibited increased MLI, indicating impaired alveolarization compared to Asl^flox/flox control mice (Fig. 4B and D). These data suggest that endothelial-derived NO plays a role in lung alveolarization in early adulthood. However, the defect of lung alveolarization was mild in VE-Cad Asl cKO mice, since the endothelial-specific knockout mice may have compensatory effects of NO from the surrounding un-targeted cells.

Figure 4

Endothelial-specific deletion of Asl in VE-cad Asl cKO mice exhibits decreased lung alveolarization at 12 weeks old. Lungs were inflated and harvested at postnatal day 21 to day 23 (A and C) and 12-week-old mice (B and D) from Asl^flox/flox (control) mice and VE-cad Asl cKO male mice. Postnatal day 21 to day 23: Control (n = 5); VE-cad Asl cKO (n = 4). 12 weeks old: Control (n = 5); VE-cad Asl cKO (n = 6). ^**P < 0.01. Results are shown as means ± s.d.

Discussion

The present study demonstrates that Asl^Neo/Neo mice spontaneously develop impaired lung alveolarization and that NO supplementation could reverse the defective lung alveolarization. Furthermore, the results support that endothelial-derived NO plays a role in enhancing lung alveolarization as mice with ASL-specific deletion in endothelial cells result in mild but significantly decreased lung alveolarization under basal normoxia conditions in early adulthood. Therefore, we propose that ASLD could be used as a unique model to investigate NO regulation of lung alveolarization, given that the deletion of ASL is equal to the deletion of three NOS isoforms. Moreover, mice with ASL conditional alleles (Asl^flox/flox mice) are available, it will be of interest to study the cell-specific function of NO using ASL conditional knockout mice.

The importance of Cav-1, the primary coat protein of caveolae, in the suppression of eNOS activity and NO production has been studied in recent years [28, 32]. As complete deletion of Cav-1 in mice results in systemic manifestations including abnormal lipid metabolism, pulmonary hypertension, and cardiomyopathy [33–35], in this study we generated heterozygous deletion of Cav-1 in Asl^Neo/Neo mice and found the rescue of lung alveolarization. We have recently shown that heterozygous loss of Cav-1 restores NO synthesis in osteoblasts and the low bone mass in Asl^Neo/Neo mice [19]. Both findings support the hypothesis that manipulating the balance of positive and negative proteins that form a NO synthetic complex could be used as a therapeutic approach to modulate NO production.

One limitation of the VE-Cad Cre transgenic mice that we used is the Cre also targets bone marrow hematopoietic lineage cells [36]. Given that hematopoietic cells can induce lung alveolarization through paracrine signaling mechanisms, it is possible that the effect seen in the conditional knockout mice may be mediated by the loss of NO in non-endothelial cells [37, 38].

It will be of interest to explore the molecular mechanisms by which NO regulates lung alveolarization using ASLD mice. In addition, whether ASLD mice are more susceptible to stress-induced impaired alveolarization, such as under hypoxia or hyperoxia conditions, needs further investigation.

Author contributions

Z.J. designed the experiments, performed most of the experiments, analyzed data, and wrote the manuscript. M.M.J. performed the sectioning and staining of the lungs. B.L. supervised this study.

Conflict of interest statement

The authors have declared that no conflict of interest exists.

Funding

This work was supported by the National Institutes of Health NIH/NIAMS R01AR071741 and NIDDK R01DK102641. Research reported in this publication was also supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Number P50HD103555 Core facilities. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.