Sodium nitrite augments lung S-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats RESEARCH ARTICLE Sodium nitrite augments lung S-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats Robert P. Jankov,1,2,3,4 Kathrine L. Daniel,1 Shira Iny,1 Crystal Kantores,4 Julijana Ivanovska,4 Nadya Ben Fadel,2 and Amish Jain5,6 1Molecular Biomedicine Program, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada; 2Faculty of Medicine, Department of Pediatrics, University of Ottawa, Ottawa, Ontario, Canada; 3Faculty of Medicine, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada; 4Translational Medicine Program, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada; 5Department of Physiology, University of Toronto, Toronto, Ontario, Canada; and 6Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Submitted 19 April 2018; accepted in final form 6 August 2018 Jankov RP, Daniel KL, Iny S, Kantores C, Ivanovska J, Ben Fadel N, Jain A. Sodium nitrite augments lung S-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats. Am J Physiol Lung Cell Mol Physiol 315: L742–L751, 2018. First pub- lished August 9, 2018; doi:10.1152/ajplung.00184.2018.—Deficient nitric oxide (NO) signaling plays a critical role in the pathogenesis of chronic neonatal pulmonary hypertension (PHT). Physiological NO signaling is regulated by S-nitrosothiols (SNOs), which act both as a reservoir for NO and as a reversible modulator of protein function. We have previously reported that therapy with inhaled NO (iNO) in- creased peroxynitrite-mediated nitration in the juvenile rat lung, although having minimal reversing effects on vascular remodeling. We hypothesized that sodium nitrite (NaNO2) would be superior to iNO in enhancing lung SNOs, thereby contributing to reversal of chronic hypoxic PHT. Rat pups were exposed to air or hypoxia (13% O2) from postnatal days 1 to 21. Dose-response prevention studies were conducted from days 1–21 to determine the optimal dose of NaNO2. Animals then received rescue therapy with daily subcutane- ous NaNO2 (20 mg/kg), vehicle, or were continuously exposed to iNO (20 ppm) from days 14 –21. Chronic PHT secondary to hypoxia was both prevented and reversed by treatment with NaNO2. Rescue NaNO2 increased lung NO and SNO contents to a greater extent than iNO, without causing nitration. Seven lung SNO proteins upregulated by treatment with NaNO2 were identified by multiplex tandem mass tag spectrometry, one of which was leukotriene A4 hydrolase (LTA4H). Rescue therapy with a LTA4H inhibitor, SC57461A (10 mg·kg�1·day�1 sc), partially reversed chronic hypoxic PHT. We conclude that NaNO2 was superior to iNO in increasing tissue NO and SNO generation and reversing chronic PHT, in part via upregulated SNO-LTA4H. cofilin; leukotriene; newborn; nitration; nitric oxide INTRODUCTION Chronic pulmonary hypertension (PHT), characterized by increased pulmonary vascular resistance (PVR), increased pul- monary arterial pressure, and arterial wall remodeling due to smooth muscle hyperplasia (46) is commonly observed in newborns and infants with bronchopulmonary dysplasia and other developmental lung disorders. Chronic PHT heralds a greatly increased risk of death and severe morbidity, and no therapies yet exist that are proven to modify the disease course (32). Abundant evidence implicates deficient nitric oxide (NO) signaling as central to the pathogenesis of chronic neonatal PHT (12, 13, 26, 50). In addition to endothelial NO synthase (NOS)-derived NO stimulating signaling pathways leading to vascular smooth muscle relaxation, physiological NO signaling is regulated by reversible S-nitrosylation of cysteine thiols (47), producing S-nitrosothiols (SNOs) (20). S-nitrosylation modifies protein structure, alters interactions with binding partners, or leads to changes in subcellular localization and degradation, causing a reversible alteration in protein function (2, 19, 24, 36, 49). Exogenous (inhaled) NO (iNO) is a commonly utilized NO-based therapy in the neonate. Unfortunately, iNO has not proven effective at reversing or slowing progression of chronic PHT in human infants (5, 11) and has been of variable efficacy in reversing established chronic PHT in neonatal rats (31, 50). A biochemical obstacle to effective NO-based therapy relates to the high potential for NO to be diverted to produce reactive nitrogen species such as peroxynitrite (31); particularly in the lung, which is directly exposed. Reactive nitrogen species cause irreversible oxidation and tyrosine nitration (51), which permanently alters protein function (6, 23, 59) and directly contributes to pulmonary vascular disease in neonatal rats (7, 15, 31, 35). Nitrite anion (NO2 �) is now recognized to contribute impor- tantly to local NO bioavailability and to SNO formation by acting as a stable, nonreactive endocrine pool of NO, formed from circulating and tissue-bound NO2 � reductases (1, 52). Unlike adults, in whom the majority of circulating NO2 � is derived from dietary nitrate, sources of nutrition in neonates are poor in NO2 � (33), resulting in lower plasma NO2 � levels (30) and a likely greater dependence upon NOS to generate NO. Inhaled ethyl (alkyl) nitrite, a volatile gas that acts as a NO2 � donor, was more effective than iNO in preventing hyper- oxic lung injury in neonatal rats (3) and was highly efficacious as an acute pulmonary vasodilator in human neonates (38). These observations support NO2 �-based therapy as a promising strategy. Systemic or inhaled sodium nitrite (NaNO2) is an Address for reprint requests and other correspondence: R. P. Jankov, Children’s Hospital of Eastern Ontario, 401 Smyth Rd., Ottawa, ON, K1H 8L1, Canada (e-mail: rjankov@cheo.on.ca). Am J Physiol Lung Cell Mol Physiol 315: L742–L751, 2018. First published August 9, 2018; doi:10.1152/ajplung.00184.2018. 1040-0605/18 Copyright © 2018 the American Physiological Society http://www.ajplung.orgL742 Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. http://doi.org/10.1152/ajplung.00184.2018 mailto:rjankov@cheo.on.ca alternative NO2 �-based therapy shown to be strongly protective in adult experimental models of chronic PHT (4, 17, 28, 41, 61). However, no previous studies, to our knowledge, have examined effects of NaNO2 on chronic PHT in the neonatal or juvenile animal. We hypothesized that therapy with NaNO2 will be superior to iNO in enhancing NO bioavailability and S-nitrosylation, thus allowing for rescue of chronic hypoxic PHT. Herein, we report that systemically administered NaNO2 both prevented and reversed chronic hypoxic PHT and in- creased lung NO and SNO contents to a far greater extent than iNO. Furthermore, unlike iNO, NaNO2 did not increase lung nitration. We identified several lung SNO proteins upregulated by NaNO2 therapy, including SNO leukotriene (LT) A4 hy- drolase (LTA4H). Rescue effects of NaNO2 were partially replicated by an LTA4H inhibitor, SC57461A, suggesting increased SNO-LTA4H as one mechanism by which NaNO2 reversed chronic PHT. MATERIALS AND METHODS Materials. Sodium nitrite (NaNO2; cat. no. S-2252) and SC57461A (cat. no. 3107) were purchased from Sigma-Aldrich (Oakville, Can- ada) and Tocris Biosciences (Bristol, UK), respectively. S-nitrosylated biotin switch (cat. no. 10006518) and nitrate/nitrite detection (cat. no. 780001) kits, peroxynitrite solution (cat. no. 81565), and C18 solid phase extraction cartridges (cat. no. 400020) were from Cayman Chemical (Ann Arbor, MI). Oxygen and NO exposure chambers and automated controllers, OxyCycler models A84XOV and AT2N, were from Biospherix (Parish, NY). NO 400-ppm cylinders (balance N2) were from Linde Canada (Mississauga, Canada). Iodo-tandem mass tag (TMT) labeling kits (cat. nos. 90100 and 90101), immobilized anti-TMT antibody resin (cat. no. 90076), and TMT elution buffer (cat. no. 90104) were from Thermo Fisher (Waltham, MA). Mass spectrometry grade Trypsin/Lys-C mix (cat. no. V-5072) was from Promega (Madison, WI). Precast Tris-glycine 4 –20% gradient gels (mini-PROTEAN TGX Stain-Free), chemiluminescent reagent (Clar- ity or Clarity Max Western ECL substrate), imaging system (Chemi- Doc Touch), and analysis software (Image Laboratory 6.0) were from Bio-Rad (Mississauga, Canada). Protease inhibitors were from Cal- biochem (cat. no. 539131-10-VL, San Diego, CA). Acids, alcohols, organic solvents, paraformaldehyde, Permount, and Superfrost/Plus microscope slides were from Thermo Fisher. Weigert’s resorcin- fuchsin stain was from Rowley Biochemical (cat. no. F-370-1, Dan- vers, MA). Avidin-biotin-peroxidase complex (cat. no. PK-6100) and 3,3-diaminobenzidine staining kits (cat. no. PK-4100) were from Vector Laboratories (Burlingame, CA). An antigen retriever device was from Aptum Biologics Ltd. (Antigen Retriever 2100, Southam- pton, United Kingdom). IHC-Tek antibody diluent (cat. no. IW-1000) and epitope retrieval solution (cat. no. IW-1100) were from IHC World (Woodstock, MD). Anti-LTA4H (cat. no. NBP-1-95835) was from Novus Biologicals (Littleton, CO). Anti-nitrotyrosine (cat. no. 06-284) was from EMD Millipore (Billerica, MA). Anti-cofilin-1 (cat. no. 5175) and goat anti-rabbit IgG-peroxidase (cat. no. 7074) anti- bodies were from Cell Signaling Technologies (Beverly, MA). Anti- phospho-serine 695 myosin phosphatase target (MYPT)-1 (cat. no. sc-33360) and goat anti-rabbit IgG-biotin (cat. no. sc-2040) secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pan-MYPT-1 was from BD Biosciences (cat. no. 612614; Mis- sissauga, Canada). Colorimetric competitive ELISA kits for biotin (cat. no. K811) and 3-nitrotyrosine (cat. no. K-4158) were from BioVision (Milpitas, CA). ELISA kits for LTB4 (cat. no. ADI-900- 068) were from Enzo Life Sciences (Farmingdale, NY). Unless specified, all other chemicals and reagents were from Sigma-Aldrich. In vivo experiments. All procedures involving animals were ap- proved by the Animal Care Committee at the University of Ottawa and conformed to the guidelines of the Canadian Council on Animal Care. A total of 36 timed-pregnant Sprague-Dawley rats were pur- chased from Taconic Farms (Germantown, NY). Experiments were conducted in multiples of four litters (2 litters in hypoxia and 2 litters in normoxia, one vehicle-treated, and one drug-treated). Except for samples used for mass spectrometry (n � 2 per group, performed twice), sample size estimates were based on previous work and set a priori [n � 8 for cardiac end points (pulmonary hemodynamics and Fulton index) and n � 6 for all other end points] incorporating animals from two to three litters per experimental group with equal- ized sex distribution. Analysis of all end points was conducted in a blinded fashion. Commencing on the day after birth [considered postnatal day (PND) 1], litters of Sprague-Dawley rat pups were chronically exposed to normobaric hypoxia (13% O2) or normoxia (21% O2) until PND 21. Automated controllers (Biospherix) main- tained O2 concentrations to within 0.1% of set point. The room in which chambers were housed had 12-h light/dark cycles. Chamber temperature was maintained at 25 � 1°C, humidity at 50 � 10%, and food and water were available ad libitum. Rat pups received daily subcutaneous NaNO2 (5, 10, 20, or 50 mg/kg in 0.9% saline vehicle; 5 �l per g body wt) or vehicle alone from PNDs 1–21 to determine the dose of NaNO2 that maximally prevented chronic PHT without significantly (�5%) increasing blood methemoglobin (MetHb). For rescue treatment studies, rats received daily subcutaneous NaNO2 (20 mg/kg), which was determined to be the dose that best met the above criteria, or SC57461A (LTA4H inhibitor) at a dose (10 mg· kg�1·day�1 sc) previously reported by our group to be effective in neonatal rats (16) or vehicle (0.9% saline or 20% DMSO in PBS, respectively) from PND 14 to PND 21. Separate animals were exposed to chronic hypoxia with or without iNO 20 ppm. Effects of rescue treatment reflect “reversal” of chronic PHT (rather than limit- ing further progression) as PHT markers have previously been deter- mined in this model to remain stable between PNDs 14 and 21 (54). Each litter was maintained at n � 12 pups throughout the exposure period to control for nutritional effects. At the end of the exposure/ treatment period, pups were either killed by pentobarbital overdose or were exsanguinated after inhalational or ketamine-induced anesthesia. Two-dimensional echocardiography and pulsed wave Doppler ultrasound. Two-dimensional echocardiography and pulsed wave Doppler ultrasound was performed as a noninvasive method to assess pulmonary hemodynamics as previously described (15), using a Philips Affiniti 50 cardiac ultrasound system with a small high frequency linear probe (L15-7io, Philips Healthcare, Richmond Hill, Ontario, Canada). The right ventricular ejection time (RVET) and pulmonary arterial acceleration time (PAAT) were measured using the pulmonary Doppler profile obtained from the parasternal short axis; RVET is the time from onset of systolic flow to completion of systolic flow and PAAT is from onset to peak pulmonary outflow velocity. A previously validated (15) index of PVR was calculated using the average ratio of RVET/PAAT from three systolic traces. Blood MetHb. At the end of each treatment period, pups were anesthetized as described above and the jugular vein isolated and severed. Blood was collected in a capillary tube and immediately analyzed using an ABL80 FLEX CO-OX blood gas analyzer (Radi- ometer America, Brea, CA). Right ventricular hypertrophy. Measurement of right ventricular hypertrophy (RVH) using the Fulton index (right ventricle/left ven- tricle � septum) is a well-established marker of PHT. The heart and lungs were separated, and the atria were removed inferior to the atrio-ventricular valves. The right ventricle was separated from the left ventricle and septum, freeze-dried, and weighed separately. Histological studies. Three males and three females (one from each of three litters per group) were euthanized by sodium pentobarbital overdose. Following the opening of the thoracic cavity and tracheal cannulation, the pulmonary veins were divided. The pulmonary cir- L743SODIUM NITRITE PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00184.2018 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. culation was flushed with PBS and heparin (1 U/ml) to clear the lungs of blood, following which the lungs were perfusion-fixed with para- formaldehyde while air inflated at constant pressure (20 cm of H2O). Following dehydration and clearance in xylene, whole left lungs were embedded in paraffin, cut into 5-�m sections, mounted, air-dried, and baked overnight at 43°C. For elastin staining, sections were dewaxed by immersion in xylene, rehydrated in ethanol, rinsed in several washes of distilled water, and then left overnight in Weigert’s resor- cin-fuchsin (Hart’s elastin) stain. Slides were then washed with distilled water and counterstained with 0.25% (vol/vol) tartrazine in acetic acid followed by dehydration and mounting. Percentage medial wall area (%MWA) was measured on elastin-stained sections as a marker of pulmonary vascular remodeling. Pulmonary arteries (20 – 100-�m external diameter) were identified by the presence of an inner and outer elastic lamina and proximity to respiratory bronchioles. A minimum of 40 arteries per animal from 4 unique left lung sections were digitally captured and analyzed. Obliquely sectioned vessels where there was a greater than three times difference in perpendicular dimensions were excluded. As previously described (34), using the “Quick Selection” tool (Adobe Photoshop CS5, Adobe Systems Inc., San Jose, CA) the area of the inner lumen and the whole vessel was outlined and pixel count determined. The following formula was used to determine the percent medial wall area: [(whole vessel area�inner luminal area)/whole vessel area] � 100. For 3-nitrotyrosine immuno- staining, slides were incubated with anti-nitrotyrosine (1:100) in antibody diluent overnight at 4°C, followed by biotin-conjugated secondary antibody (1:1,000) for 2 h at room temperature, then washed, dehydrated, counterstained with hematoxylin, and mounted. Measurement of NO oxidation products. Nitrate/nitrite oxidation (NOx) was measured in lung tissue homogenized in PBS and depro- teinated by ultrafiltration (Amicon Ultra, EMD Millipore, Burlington, MA). Nitrate in deproteinated samples was enzymatically converted to nitrite (Cayman nitrite/nitrate detection kit) and quantified, along with nitrite standards, using a Sievers 280i NO analyzer (Zysense, Weddington, NC), according to the supplier’s instructions. Values were normalized to wet tissue weight. Quantification and identification of SNO proteins. SNO protein modifications, because of their labile nature, can be difficult to study employing traditional methods used for detecting phosphorylated proteins. For this reason, several “switch” assays have been developed to label SNO-modified cysteines with stable markers (18, 39). Total SNOs were quantified in lung tissue homogenates using a biotin switch assay and competitive biotin ELISA, both according to the manufacturer’s instructions. Specific SNO proteins were identified from lung tissue homogenates (two normoxia-exposed vehicle- treated, two hypoxia-exposed vehicle-treated, and two hypoxia-ex- posed NaNO2-treated) labeled with sixplex-TMT and affinity column purified according to the manufacturer’s instructions. Samples were quantified by multiplex liquid chromatography/TMT spectrometry (TMT-LC/MS/MS; Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, ThermoFisher Scientific) by the SPARC BioCentre (Hospital for Sick Children, Toronto), following previously reported methods (39). Reporter ions observed in the M2 mass spectrum (126 – 131), generated from higher-energy collisional dissociation fragmenta- tion, were used for relative quantification between samples. Mass spec- trometry data were searched using PEAKS software (58) version 8.0 (Bioinformatics Solutions, Waterloo, Canada) and the reporter ions quan- tified using Proteome Discoverer software (version 2.2; ThermoFisher Scientific). Seven SNO proteins were identified (Table 1) where content was altered (significance score � 2) by NaNO2 treatment with results that were consistent between two separate sets of samples and TMT-LC/ MS/MS runs. Two of the seven candidates (LTA4H and cofilin-1) were confirmed by Western blot, despite LC/MS/MS identifying one unique peptide with relatively low sequence coverage (9 and 7%, respectively; Table 1). Western blot analyses. Lung lysates from three males and three females per group (representing 3 litters) were TMT-labeled accord- ing to the manufacturer’s instructions, purified by agarose bead immunoprecipitation, and matched with unlabelled, unpurified lysates both of which were separated under reducing conditions by SDS- PAGE. Following electrophoresis, proteins were transferred to PVDF membranes. All membranes were blocked with 5% skim milk for 1 h at room temperature, followed by incubation with primary antibody overnight at 4°C. Blots were then washed with Tris-buffered saline- Tween 20 and placed in secondary antibody for 1 h at room temper- ature. Dilutions of primary antisera were 1:10,000 for LTA4H (70 kDa) and secondary antiserum and 1:1,000 for Cofilin-1 (19 kDa), phospho-serine 695 and pan-MYPT-1 (80 kDa). Bands were quanti- fied by digital densitometry of nonsaturated images with background density removed. Bands were normalized to corresponding pan- protein content, each corrected for total protein/lane quantified by stain-free protein imaging (Bio-Rad). In preliminary experiments, we confirmed that stain-free protein imaging was equivalent to GAPDH as a marker of protein loading (data not shown). Raw values are expressed as a multiple or fraction of the normoxia-exposed, vehicle- treated group, which was assigned a value of 1. ELISA. Lung lysates from three males and three females per group (representing 3 litters) were purified and analyzed according to the manufacturer’s instructions. Values were normalized to protein con- tent (3-nitrotyrosine) or wet tissue weight (LTB4). Data presentation and statistical analysis. Data are expressed as means � SE after any outliers (�2 SD from mean value) were removed. Analyses were performed using Sigma Plot 13 (Systat software, San Jose, CA). Statistical significance (P 0.05) was determined by t-test or one-way ANOVA followed by Tukey post hoc test where significant intergroup differences were found or by Kruskal-Wallis one-way ANOVA for nonparametric data. Table 1. Candidate SNO proteins identified by TMT-LC/MS/MS Accession No. │ Uniprot ID (Protein name) Unique Peptides, n Sequence Coverage, % Known Functions P30349│LKHA4 (Leukotriene A4 hydrolase) 1 9 Epoxy hydrolase catalyzing biosynthesis of proinflammatory leukotriene B4. Also possesses amino-peptidase activity. P45592│COF1 (Cofilin-1) 1 7 Depolymerizes F-actin. Regulates cell morphology and cytoskeletal organization. B0BMY8 (Histone H3) 2 27 Nucleosomal protein. Q03348│PTPA (Serine/threonine-protein phosphatase 2A activator) 1 9 Regulation of apoptosis. Q63357│MYO1D (Unconventional Myosin-Id) 2 2 Cytoskeletal protein. Regulation of cell motility. Q9JLU4│SHAN3 (SH3 and multiple ankyrin repeat domains protein 3) 1 2 Cytoskeletal protein. Regulation of cell motility. O35244│PRDX6 (Peroxiredoxin 6) 1 5 Thiol-specific peroxidase enzyme/antioxidant. SNO, S-nitrosothiols; TMT-LC/MS/MS, multiplex liquid chromatography/TMT spectrometry. L744 SODIUM NITRITE PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00184.2018 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. RESULTS Systemic NaNO2 prevented and reversed chronic hypoxic PHT. Dose response prevention studies are shown in Fig. 1. NaNO2 at doses of 20 and 50 mg·kg�1·day�1 prevented chronic PHT as evidenced by significantly (P 0.001) de- creased PVR (Fig. 1A) and RVH (Fig. 1B). Blood MetHb was significantly greater (P 0.001; mean value 7.2%) at 50 mg/kg when compared with 20 mg/kg (mean value 1.75%). Rescue effects of NaNO2 20 mg·kg�1·day�1 are shown in Fig. 2. Rescue NaNO2 normalized PVR (Fig. 2A) and almost completely normalized RVH (Fig. 2B) and %MWA (Fig. 2C). In contrast, and as previously reported (31), iNO had no significant reversing effect on increased RVH (Fulton index 0.481 � 0.03; P 0.001 vs. NaNO2-treated, n � 8 animals per group) or %MWA (31.1 � 2.3%; P 0.001 vs. NaNO2- treated, n � 6 animals per group) in chronic hypoxia-exposed animals. MetHb values in animals receiving rescue NaNO2 were all 2% (data not shown). Rescue treatment with NaNO2 increased lung NOx, SNO protein content, and cGMP activity, without causing increased nitration. Lung nitrate/nitrite (NOx) was measured as a marker of total NO content. NaNO2 significantly increased (P 0.001) lung NOx (Fig. 3A) and SNO protein (Fig. 3B) contents in both normoxia- and hypoxia-exposed animals when com- pared with animals treated with vehicle- or hypoxia-exposed animals that were treated with iNO. Lung phospho-serine 695/pan-MYPT-1 ratio was used as a marker of cGMP-medi- ated protein kinase G signaling (PKG), as previously described (42). As shown in Fig. 3C, rescue treatment with both NaNO2 and iNO significantly increased lung cGMP-PKG activity in chronic hypoxia-exposed animals. Chronic exposure to hyp- oxia or treatment with NaNO2 had no effect on total lung 3-nitrotyrosine content (Fig. 3D). In contrast, and as previously reported (31), lung 3-nitrotyrosine was greatly increased by rescue treatment with iNO (P 0.001 vs. all other groups; Fig. 3D). Representative images of 3-nitrotyrosine immunohisto- chemistry are shown in Fig. 3E. Rescue treatment with NaNO2 increased lung contents of SNO-LTA4H and SNO-cofilin-1. As shown in Fig. 4, A and B, SNO-LTA4H and SNO-Cofilin-1 were significantly increased by rescue treatment with NaNO2 in hypoxia-exposed animals. There were no significant differences in pan-LTA4H (P � 0.125, by ANOVA; data not shown) or pan-Cofilin-1 (P � 0.15, by ANOVA; data not shown) between groups. Increased SNO-LTA4H in NaNO2-treated hypoxia-exposed animals was associated with a nonsignificant (P � 0.093) trend toward decreased LTA4H activity, as indicated by lung LTB4 content, when compared with hypoxia-exposed vehicle-treated animals (Fig. 4D). Rescue treatment with SC57461A, a LTA4H inhibitor, par- tially reversed chronic hypoxic PHT. Treatment with SC57461A partially but significantly (P 0.05), decreased PVR (Fig. 5A) RVH (Fig. 5B) and %MWA (Fig. 5C) in chronic hypoxia-exposed animals. These effects were accom- panied by significantly decreased lung LTB4 content (7.2 � 0.4 pg per lung wet wt in hypoxia-exposed vehicle-treated animals versus 5.4 � 0.2 in hypoxia-exposed, SC57461A- treated animals, n � 6 per group; P 0.01, by t-test). Fig. 1. Dose-response studies. From postnatal days 1–21, rat pups were exposed to normobaric hypoxia (13% O2) or normoxia (21% O2). Beginning on day 1, pups received daily subcutaneous injections of sodium nitrite (5, 10, 20, or 50 mg/kg) or 0.9% saline vehicle. A: pulmonary vascular resistance (PVR) index. Ratio of right ventricular ejection time (RVET) to pulmonary arterial acceleration time (PAAT), n � 8 animals per group. B: right ventric- ular hypertrophy (RVH) assessed by right ventricle (RV)/left ventricle (LV) � septum (S) weight ratio (Fulton index), n � 8 animals per group. C: blood percentage methemoglobin levels, n � 6 animals per group. Bars represent means � SE; *P 0.001, by ANOVA, vs. vehicle-treated group. #P 0.001, by ANOVA, vs. all other groups. L745SODIUM NITRITE PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00184.2018 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. DISCUSSION We present the first data, to our knowledge, to demonstrate therapeutic efficacy of NaNO2 and superiority to iNO in reversal of chronic PHT in the juvenile animal. NaNO2 has many properties that are favorable for clinical translation, including stability, low cost, and ability to be administered systemically or by inhalation. As confirmed in this study, NaNO2 leads to upregulation of physiological NO signaling [increased SNOs (9, 10, 62)] without an apparent concurrent propensity to cause nitration (14). In humans, an injectable form of NaNO2 is marketed as therapy for cyanide poisoning (25), and nebulized NaNO2 was safe and well tolerated in healthy adults (44) and acutely reduced PVR in pulmonary hypertensive patients with -thalassemia (56). In rats chronically exposed to hypoxia from birth, we report that increased PVR, RVH, and pulmonary arterial wall remod- eling were almost completely reversed by rescue treatment with systemic NaNO2, in contrast to iNO, which had no significant reversing effect (31). Furthermore, rescue NaNO2 increased lung NOx and total SNO protein contents to a much greater extent than iNO, without causing nitration. In addition to avoidance of nitration, increased bioavailability of NO and enhanced cGMP-PKG signaling, a likely mechanism behind the efficacy of NaNO2 was reversible and specific modification in protein function that arises from S-nitrosylation (27). Inter- estingly, we observed that rescue iNO greatly increased cGMP-PKG activity, which was surprising in light of the lack of reversing effect on markers of chronic PHT. Possible ex- planations are that enhanced nitration may have negated any benefits of enhanced cGMP signaling and/or that increased S-nitrosylation is of greater importance to reversal of pulmo- nary arterial remodeling in this model. As there is currently no knowledge on the functional role/s of specific SNO proteins in neonatal cardiopulmonary disease, Fig. 2. Rescue sodium nitrite reversed chronic pulmonary hypertension. From postnatal days 1–21, rat pups were exposed to normobaric hypoxia (13% O2; hypoxia) or normoxia (21% O2). From days 14 –21, pups received daily subcutaneous injections of sodium nitrite (NaNO2) 20 mg/kg or 0.9% saline vehicle or were continuously exposed to 20 ppm inhaled nitric oxide (iNO). A: pulmonary vascular resistance (PVR) index. Ratio of right ventricular ejection time (RVET) to pulmonary arterial acceleration time (PAAT), n � 8 animals per group. B: right ventricular hypertrophy (RVH) assessed by right ventricle (RV)/left ventricle (LV) � septum (S) weight ratio (Fulton index), n � 8 animals per group. C: pulmonary arterial medial wall area as a marker of vascular remodeling, n � 6 animals per group. Bars represent means � SE; *P 0.001, by ANOVA, vs. all other groups. D: high-power photomicrographs demonstrating elastin-stained pulmonary arteries. Bar length � 50 �m. L746 SODIUM NITRITE PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00184.2018 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. Fig. 3. Rescue sodium nitrite increased lung nitric oxide oxidation (NOx), S-nitrosylated (SNO) proteins, and cGMP-PKG activity, without increasing nitration. From postnatal days 1–21, rat pups were exposed to normobaric hypoxia (13% O2; Hypoxia) or normoxia (21% O2). From days 14 –21, pups received daily subcutaneous injections of sodium nitrite (NaNO2) 20 mg/kg or 0.9% saline vehicle or were continuously exposed to 20 ppm inhaled nitric oxide (iNO). A: lung NOx products as a marker of tissue nitric oxide abundance, n � 6 animals/group. B: total lung SNO proteins, n � 6 animals per group. C: Western blot analyses of lung phospho-serine 695/pan-myosin phosphatase target (MYPT)-1 ratio as a marker of cyclic guanosine monophosphate-protein kinase G signaling, n � 6 animals per group. Representative immunoblots show two contiguous lanes for each group. D: lung 3-nitrotyrosine content, n � 6 animals per group. Bars represent means � SE; *P 0.001, by ANOVA, vs. all other groups. #P 0.05, by ANOVA, vs. normoxia groups. †P 0.001, by ANOVA, vs. vehicle-treated groups. E: representative medium-power photomicrographs of lung 3-nitrotyrosine immunoreactivity, as a marker of nitric oxide-derived reactive nitrogen species-mediated nitration. Bar length � 100 �m. Inset: positive control section pretreated with 100 mM peroxynitrite. L747SODIUM NITRITE PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00184.2018 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. we undertook TMT-LC/MS/MS and identified seven lung SNO proteins that were consistently upregulated by treatment with NaNO2. Despite there being relatively low sequence coverage for most peptides identified by LC/MS/MS, changes in two of these proteins, LTA4H and cofilin-1, were able to be confirmed by Western blot analyses. S-nitrosylation is the covalent mod- ification of cysteine thiols by addition of a nitrosyl group (by either NO or NO2 �). As a result, cysteine-rich proteins contrib- ute importantly to physiological NO signaling by acting as a circulating and tissue reservoir for NO (2, 19, 24, 36, 49). In addition, S-nitrosylation possesses the essential criteria for a signaling modification (akin to phosphorylation), including a rapid reaction, specificity for particular cysteine residues, and enzymatic removal [by S-nitrosoglutathione reductase and the thioredoxin reductase system (8)]. The functional implications for the majority of the �3,000 SNO proteins identified to date remain obscure. We have previously reported that LTA4H inhibition pre- vented vascular remodeling and chronic PHT in bleomycin- exposed neonatal rats (16). In the present study, we observed that rescue NaNO2 significantly decreased lung LTB4 content (product of LTA4H epoxy hydrolase activity) and that rescue therapy with SC57461A partially reversed chronic hypoxic PHT. A potential role for S-nitrosylation in modulation of LTA4H activity has not, to our knowledge, been previously described. In addition to production of LTB4, which is known to stimulate inflammation, production of matrix proteins, and to increase smooth muscle contractility and airway/vascular remodeling in the lung (43, 48), LTA4H is also known to act as an aminopeptidase, products of which may also contribute to Fig. 4. Rescue treatment with NaNO2 increased lung contents of SNO-leukotrieneA4 hydrolase (LTA4H) and SNO-cofilin-1. From postnatal days 1–21, rat pups were exposed to normobaric hypoxia (13% O2; Hypoxia) or normoxia (21% O2). From days 14 –21, pups received daily subcutaneous injections of sodium nitrite (NaNO2) 20 mg/kg or 0.9% saline vehicle. Western blot analyses of lung SNO-LTA4H (A) and SNO-cofilin-1 (B). Representative immunoblots for SNO and pan-proteins (C). Unlabeled control � purified normoxia-exposed, vehicle-treated sample not labeled with TMT. Lung Leukotriene B4, as a marker of LTA4H activity, quantified by ELISA (D). Bars represent means � SE for n � 5– 6 samples per group. *P 0.05, by Kruskal-Wallis one-way ANOVA, compared with all vehicle-treated groups. #P 0.05, by Kruskal-Wallis one-way ANOVA, compared with all other groups. H, heavy chain; L, light chain; SNO, S-nitrosothiols; TMT, tandem mass tag. L748 SODIUM NITRITE PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00184.2018 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. lung inflammation and injury separate from the leukotriene pathway (40). Our present data provide indirect evidence for inhibitory effects of S-nitrosylation on LTA4H activity. The identity of specific cysteine residues on LTA4H that are prone to S-nitrosylation and whether inhibition of hydrolase or ami- nopeptidase pathways predominated in reversal of chronic PHT awaits further study. Cofilin is an intracellular actin-modulating protein that binds and depolymerizes filamentous F-actin. Cofilin activity is in- creased in hypertrophic cardiomyocytes (29) and in chronic hypoxia-exposed murine lung (53) and is activated down- stream of upregulated RhoA and Rho-kinase activity [via enhanced LIM kinase-mediated phosphorylation (45)]. Patho- logical Rho-kinase activity is a critical mediator of chronic neonatal PHT in the present model (37, 55, 60), in bleomycin- exposed neonatal rats (34, 37) and in pulmonary hypertensive fetal sheep (21, 22). S-nitrosylation is known to modulate the activity of cofilin-1 in endothelial cells (57). In the present study, we observed that lung SNO-cofilin-1 was decreased by chronic exposure to hypoxia and restored by rescue NaNO2. As most SNO protein modifications lead to inhibition of protein function, we speculate that smooth muscle cofilin-1 activity was reduced by enhanced S-nitrosylation, potentially also con- tributing to reversal of vascular remodeling. There are several limitations to this study. First, it is likely that multiple SNO protein modifications contributed to NaNO2-mediated reversal of chronic PHT; however, we chose to explore LTA4H as a candidate because of amenability to pharmacological inhibition and the known effects of the leu- kotriene pathway in mediating PHT (16). Of the other candi- date SNO proteins identified, all may contribute to the patho- genesis of chronic PHT (modulators of actin depolymerization, oxidative stress, cell survival) and warrant further exploration. Second, although we were able to measure total and specific lung SNOs in whole tissue, we were unable to localize them in situ. Commercially available SNO-nitrosocysteine antibodies are available; however, we were unable to obtain any mean- ingful data employing several of these antibodies. Third, we Fig. 5. Rescue treatment with SC57461A, a leukotrieneA4 hydrolase (LTA4H) inhibitor, partially reversed chronic hypoxic pulmonary hypertension (PHT). From postnatal days 1–21, rat pups were exposed to normobaric hypoxia (13% O2) or normoxia (21% O2). From days 14 –21, pups received daily subcutaneous injections of SC57461A 10 mg/kg or 20% DMSO in PBS (vehicle). A :pulmonary vascular resistance (PVR) index. Ratio of right ventricular ejection time (RVET) to pulmonary arterial acceleration time (PAAT), n � 8 animals per group. B: right ventricular hypertrophy (RVH) assessed by right ventricle (RV)/left ventricle (LV) � septum (S) weight ratio (Fulton index), n � 8 animals per group. C: arterial medial wall area as a marker of vascular remodeling, n � 6 animals per group. Bars represent means � SE; *P 0.05, by ANOVA, vs. all other groups. #P 0.05, by ANOVA, vs. normoxia groups. D: high-power photomicrographs demonstrating elastin-stained pulmonary arteries. Bar length � 50 �m. L749SODIUM NITRITE PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00184.2018 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. did not explore the relative contributions of NO-cGMP versus SNO signaling in the therapeutic effects of NaNO2. This could be explored in future studies employing a smooth-muscle- specific soluble guanylate cyclase knockout model, either in vitro or in vivo. Finally, although pulmonary-selective (inha- lation) therapy with NaNO2 has the greater translational po- tential, we chose to limit the present study to systemic therapy as proof of concept of efficacy and superiority to iNO. In conclusion, we provide the first preclinical evidence, to our knowledge, for efficacy of NaNO2 in the immature animal. Ongoing and future studies will examine the utility of the inhalational route of therapy and efficacy in alternative neona- tal models that replicate bronchopulmonary dysplasia and other developmental lung disorders. ACKNOWLEDGMENTS We thank Dr. Jonathan Krieger (SPARC BioCentre, Hospital for Sick Chil- dren, Toronto, Canada) for valuable assistance with multiplex liquid chromatog- raphy/TMT spectrometry S-nitrosothiol protein measurement and analysis. GRANTS This work was supported by operating funding from the Physicians’ Services Incorporated Foundation and the Heart and Stroke Foundation of Canada and by infrastructure funding from the Canada Foundation for Inno- vation (all to R. Jankov). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. 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