Exogenous hydrogen sulfide attenuates hyperoxia effects on neonatal mouse airways

Abstract

Supplemental O₂ remains a necessary intervention for many premature infants (<34 wk gestation). Even moderate hyperoxia (<60% O₂) poses a risk for subsequent airway disease, thereby predisposing premature infants to pediatric asthma involving chronic inflammation, airway hyperresponsiveness (AHR), airway remodeling, and airflow obstruction. Moderate hyperoxia promotes AHR via effects on airway smooth muscle (ASM), a cell type that also contributes to impaired bronchodilation and remodeling (proliferation, altered extracellular matrix). Understanding mechanisms by which O₂ initiates long-term airway changes in prematurity is critical for therapeutic advancements for wheezing disorders and asthma in babies and children. Immature or dysfunctional antioxidant systems in the underdeveloped lungs of premature infants thereby heightens susceptibility to oxidative stress from O₂. The novel gasotransmitter hydrogen sulfide (H₂S) is involved in antioxidant defense and has vasodilatory effects with oxidative stress. We previously showed that exogenous H₂S exhibits bronchodilatory effects in human developing airway in the context of hyperoxia exposure. Here, we proposed that exogenous H₂S would attenuate effects of O₂ on airway contractility, thickness, and remodeling in mice exposed to hyperoxia during the neonatal period. Using functional [flexiVent; precision-cut lung slices (PCLS)] and structural (histology; immunofluorescence) analyses, we show that H₂S donors mitigate the effects of O₂ on developing airway structure and function, with moderate O₂ and H₂S effects on developing mouse airways showing a sex difference. Our study demonstrates the potential applicability of low-dose H₂S toward alleviating the detrimental effects of hyperoxia on the premature lung.

NEW & NOTEWORTHY Chronic airway disease is a short- and long-term consequence of premature birth. Understanding effects of O₂ exposure during the perinatal period is key to identify targetable mechanisms that initiate and sustain adverse airway changes. Our findings show a beneficial effect of exogenous H₂S on developing mouse airway structure and function with notable sex differences. H₂S donors alleviate effects of O₂ on airway hyperreactivity, contractility, airway smooth muscle thickness, and extracellular matrix deposition.

INTRODUCTION

Supplemental O₂ (hyperoxia) is almost always a necessary intervention following premature birth (<34 wk gestation): although the goal is to maintain physiological oxygenation, the airway tissues are exposed to a hyperoxic environment (1, 2). High O₂ concentrations (80%–90%) promote bronchopulmonary dysplasia (BPD), leading to the now-established clinical practice of moderate oxygen supplementation (30%–60% O₂) (3–5). However, even moderate O₂ (<60%) increases the risk for subsequent airway disease with airway hyperreactivity (AHR) and remodeling (proliferation, altered extracellular matrix) (6–10). Thus, early insults with premature birth predispose infants to pediatric asthma (11–14) involving chronic inflammation, AHR, and airway remodeling (12, 15), which narrow preterm infant airways and increase susceptibility to AHR/obstruction resulting from structural changes (16–18). With chronic airway disease being a short- and long-term consequence of prematurity, significance lies in understanding the effects of O₂ exposure during the perinatal period to identify targetable mechanisms that initiate and sustain detrimental airway changes.

Thickened airways are present in young children (<3 yr) with recurrent wheeze before the emergence of pediatric asthma (12, 19), suggesting that altered airway structure during the neonatal period contributes to asthma (15). Although multiple cell types [epithelium, airway smooth muscle (ASM), fibroblasts] contribute, the fundamental role of ASM in airway tone, contractility, and remodeling is indisputable. ASM in adult asthma is better explored, but developing ASM is less understood, particularly in the context of hyperoxia. Using fetal ASM (fASM) from 18- to 22-wk (canalicular stage) lungs, we previously showed that moderate hyperoxia (<60% O₂) increases ASM intracellular calcium and proliferation, and extracellular matrix deposition (20–23). In vivo studies using a neonatal mouse model of hyperoxia exposure have demonstrated that moderate O₂ increases ASM thickness, collagen deposition, and AHR in response to methacholine challenge (24, 25). However, mechanisms underlying detrimental neonatal O₂ effects on the airway are still being explored.

We propose that the novel gasotransmitter hydrogen sulfide (H₂S) attenuates O₂ effects on the developing airway. Although H₂S is toxic to the lung at high concentrations (26–28), it has recently been recognized as an important endogenous signaling molecular gas akin to NO or CO (28–36). At physiologically relevant levels, H₂S has been shown to be anti-inflammatory and cytoprotective, promoting vasodilation and resistance to oxidative stress. In the vasculature, H₂S blunts muscle tone, inflammation, metabolism, proliferation, and fibrosis (37–41): aspects that are also relevant to airway disease and involving pathways important in ASM. In adult asthmatics, serum and breath H₂S levels are decreased (42, 43). Lung H₂S levels are lower in a rat model of asthma (44) and negatively correlate with inflammatory cell portfolios and ASM hyperplasia. H₂S is also highly relevant to antioxidant pathways by regulating ROS, increasing GSH production, protecting against oxidative stress, and activating Nrf2 while regulating NF-κB signaling (45–47).

There are currently little data regarding H₂S effects in developing airway, particularly with O₂. In experimental models of BPD, previous studies have demonstrated that exogenous H₂S donor GYY4137 restores arrested alveolarization (48), while knockout of two key enzymes involved in endogenous H₂S production, CSE (cystathionine γ-lyase), or CBS (cystathionine β-synthase), prevents alveolarization (49), overall suggesting a protective role of H₂S in the developing lung. Additionally, systemic CSE is lower in premature infants and newborns (50, 51), suggesting prematurity represents a state of deficient endogenous H₂S. We previously demonstrated that fASM has endogenous machinery involved in H₂S production and signaling and that H₂S production is blunted by O₂ exposure (52). These studies also showed that exogenous H₂S donors alleviate the effects of O₂ on [Ca²⁺]_i regulation (52).

In the present study, we hypothesized that exogenous H₂S donors would attenuate deleterious effects of O₂ on developing airway structure and function in neonatal mice exposed to hyperoxia. We used two exogenous H₂S donors, rapid-release NaHS and slow-release GYY4137, administered following neonatal hyperoxia exposure and recovery (Fig. 1). These studies show exogenous H₂S attenuates deleterious effects of O₂ on AHR (measured as airway resistance and respiratory system compliance), ASM thickness (but not epithelium thickness), and extracellular matrix composition (collagen and fibronectin deposition within airways). Furthermore, these studies reveal sex-specific effects of O₂ and attenuating effects of H₂S in the developing mouse airway. Ex vivo precision-cut lung slices (PCLS) support the beneficial effect of H₂S on contractility.

Figure 1.

Modeling neonatal hyperoxia exposure in mice. Newborn C57BL/6J pups were exposed to either 21% O₂ (room air) or 50% O₂ (hyperoxia chamber) on the day of birth for 7 days followed by 2-wk recovery in 21% O₂. From P8 to 21, neonates from both the 21% and 50% O₂ groups were intraperitoneally injected daily with either vehicle (PBS), 14 µM NaHS, 12.5 µM GYY4137, or 25 µM GYY4137. P21 pups were subject to pulmonary mechanics assessments and methacholine challenge via flexiVent or tissue harvested for precision-cut lung slices (PCLS) analysis. In addition, lung tissue was harvested for histological analysis (H&E and MT), and immunofluorescence (α-smooth muscle actin or fibronectin). (Image created with a licensed version of BioRender.com.) H&E, hematoxylin and eosin; MT, Masson trichrome; PBS, phosphate-buffered saline.

MATERIALS AND METHODS

Animal Protocols and Treatment Compounds

All animal studies were approved by the Institutional Animal Care and Use Committee at Mayo Clinic and performed in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. C57BL/6J mice were obtained from Jackson Labs. Both males and females were used in the studies. Mice were housed under constant temperature and light–dark cycles (12:12 light:dark) with food and water provided ad libitum. Newborn pups (P0) were exposed to either normocapnic, isobaric normoxia (room air; 21% O₂) or normocapnic, isobaric hyperoxia (40%–50% O₂) using a custom built, sealed plexiglass chamber with an inlet and a pressure relief valve (hyperoxic O₂ delivered via an oxygen/air blender and monitored using a digital oxygen monitor commonly used for clinical purposes). Dams were alternated on a daily basis between normoxia and hyperoxia conditions. Hyperoxia exposure was continued for the first 7 days of life, after which there was a 14-day room air normoxia recovery period with or without exogenous H₂S donors. At P21, neonates were subject to pulmonary function assessments via flexiVent, tissue was harvested, and structural studies conducted (histology and immunofluorescence).

From P8 to 21, after hyperoxia exposure, neonates (both the normoxia and hyperoxia groups) were intraperitoneally injected daily with vehicle (pharmaceutical grade PBS), 14 µM NaHS, 12.5 µM GYY4137 (for PCLS), or 25 µM GYY4137. NaHS was administered at 14 µM/kg/day. GYY4137 was administered at 12.5 or 25 mg/kg/day. The compounds were dissolved in pharmaceutical grade PBS and 100 µL was administered to each animal. Common dosing in rodents of GYY4137 is 25 mg/kg or 50 mg/kg administered concurrently with sepsis-induced acute lung injury (53), 50 mg/kg administered once in a 24-h period with LPS-induced acute lung injury (54), 25 mg/kg and 50 mg/kg administered once in a 24-h period with sepsis-induced acute lung injury model (55), 50 mg/kg administered before ischemia-reperfusion injury of the lung (56), 10, 25, or 50 mg/kg/day for 4 wk in a rat model of myocardial fibrosis (57), or 50 mg/kg/day for 2 wk in a mouse model of immobilization-induced muscle atrophy (58). Common dosing in rodents of NaHS is 10, 50, or 10 0 µmol/kg/day for 2 mo (59), 20 µmol/kg (1.12 mg/kg) twice a day for 2 wk in a mouse model of immobilization-induced muscle atrophy (58), and 1 dose of 10 mg/kg before a sepsis-induced lung injury model (60). Importantly, there were varying effects of exogenous H₂S donors based on the tissue evaluated (i.e., some tissues were more sensitive to H₂S effects at lower doses). Additionally, the length of time H₂S was administered varied considerably, suggesting the importance of balancing lower doses over longer periods of time versus higher doses given once. Lastly, these studies focused on adult mice. Since neonates are inherently more sensitive than adults (and the lung is not an exception), the goal is to obtain maximum effectiveness of treatments at the lowest possible dosing so that therapies are targeted appropriately for neonates. Thus, using the lower exogenous H₂S donor doses (14 µM/kg/day NaHS, 12.5 mg/kg/day GYY4137, or 25 mg/kg/day GYY4137), which demonstrated effectiveness in the neonatal lung and hyperoxia exposure, is justified in these studies.

Flexivent

Lung mechanics were assessed on P21 neonates using flexiVent (Scireq; Montreal, QC, Canada). Airway resistance (Rrs) and compliance (Crs) were determined as described previously (24). Briefly, P21 mice were anesthetized with ketamine/xylazine, placed in the supine position, and tracheostomy performed for the insertion of a blunt tip metal cannula secured with a suture loop around exposed trachea. Appropriate anesthetic depth was verified before administering the paralytic (vecuronium). Once attached to the flexiVent system, lung mechanics measurements under static and dynamic conditions were determined using the manufacturer protocols. During measurements, animals were subjected to methacholine (MCh) challenge, starting with a baseline (0 mg/mL MCh), followed by the increasing doses of nebulized MCh (6.25, 12.5, 25.0, 50.0 mg/mL). Body temperature was consistently maintained at 37°C with a heating pad underneath the mouse. At the end of lung mechanics analysis, although still under anesthesia, lungs were harvested.

Histology

Following flexiVent analysis at day 21 for neonates exposed to either 21% or 50% O₂ with or without 14 µM NaHS or 25 µM GYY4137, mouse neonatal lungs were inflated at 25 cmH₂O with 4% paraformaldehyde. Formalin-fixed paraffin-embedded (FFPE) mouse neonatal lung sections were cut at 6 µm and stained with hematoxylin–eosin (H&E) or Masson trichrome (MT) using standard protocols. Stained sections were imaged using Motic EasyScan digital slide scanner. H&E images were analyzed using ImageJ (NIH) software to measure airway area and perimeter to calculate the airway thickness. Measurements were taken from a minimum of three airways per section with minimum two sections per animal (61, 62). MT images were analyzed using Orbit Image Analysis software (Idorsia Pharmaceuticals Ltd.; Allschwil, Switzerland) for collagen within airways (63, 64). A pixel-based classification model was used to identify and differentiate the amount of collagen (inclusion) versus background (exclusion). A training model in Orbit was used to categorize and quantify stained tissue within a specified region of interest (airway). The resulting ratio was multiplied by 100. All airways within a section were quantified regardless of shape or size to minimize bias.

Immunofluorescence

Immunofluorescence was performed on deparaffinized sections from female neonates exposed to 21% or 50% O₂ with or without 25 µM GYY4137 for α-smooth muscle actin (αSMA; 1:200; Sigma-Aldrich; St. Louis, MO: A2547; RRID: AB_476701), fibronectin (1:50; Abcam; Cambridge, UK; AB2413; RRID: AB_2262874), and DAPI mounted. The antibodies used and their concentrations and specificity have been characterized previously (24, 62). Control slides were processed concurrently but did not include primary antibody. Images were obtained on an inverted Keyence microscope (BZ-X800E; Osaka, Japan) after image acquisition parameters were set using background controls at each wavelength.

Precision-Cut Lung Slices

At P21, each PCLS was prepared according to a previously established protocol (65). In brief, following euthanization, the mouse with ketamine/xylazine and performing a tracheostomy, warm agarose was slowly injected to fill the lungs (P21 mouse volume ∼0.7–0.9 mL). Chilled HBSS was poured over the mouse to solidify the agarose. Mouse was placed in the refrigerator for 30 min to allow for adequate hardening of the agarose. Lungs were removed from the mouse and prepared in a mold using low melting point agarose. Once hardened, the mold was inserted into the vibratome with a cold HBSS bath prepared to cut lung sections. Slices were incubated at 37°C in culture media (DMEM/F-12 supplemented with 10% FBS and 1% A/A) overnight to melt any remaining agarose. The following day, slices were rinsed with HBSS and mounted using a slice holder, mesh, and coverslip secured with silicone. Slices were placed in a recording chamber (AmScope and MU300 camera) for live imaging of responses to tissue bath perfused with methacholine (MCh). Individual airways were identified and selected based on visibly intact epithelial layer and responsiveness to methacholine (to differentiate from vessels), without specific size exclusion. However, since the entire airway had to fit within the visible recording magnification (×10) to be usable for analysis, we inherently use 300–400 µm sized airways which represent 5th/6th level branches in 3–4 wk-old mice. All airways regardless of shape were quantified to minimize bias. PCLS contraction was measured using an ImageJ Macro in which the center of the airway and background were identified and designated. The baseline was determined before contraction, and the airway area was normalized. The lumen area (measured in pixels) was calculated as a fraction of the baseline, and the maximum contraction was calculated as a percentage. Greater decreases in lumen area were indicative of greater reactivity.

Statistical Analysis

Data were analyzed using either one- or two-way ANOVA, where appropriate, with Tukey’s multiple comparison test, using GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA). Outliers were determined by Grubb’s outlier test. The “n” values represent number of animals. Data are represented as means ± SE, and P < 0.05 was used for statistical significance.

RESULTS

Modeling Neonatal Hyperoxia Exposure in Mice

To model neonatal hyperoxia exposure experienced by premature infants in the NICU, we used our established regimen in mice (21, 24, 25, 62) (Fig. 1). Newborn, full gestation mice (P0) transition from the saccular to alveolar stage shortly after birth (∼P3–P4), the latter of which continues postnatally until around P21. On the other hand, human fetuses are already undergoing the alveolar stage before birth and complete alveolar maturation around 3 yr of age. Premature birth in humans thereby interrupts normal lung development in the saccular and alveolar stages (depending on extent of prematurity), resulting in much of the alveolar stage only occurring postnatally. Therefore, in terms of lung development, P0–7 mice approximate a premature infant receiving supplemental O₂. P7 neonatal mice approximate a newborn full-term human when therapeutic O₂ may be stopped. P21 mice represent an approximately 3-yr-old infant, a time period when children would manifest wheezing disorders/asthma.

In the present studies, newborn P0 C57BL/6J pups were exposed to either 21% O₂ (room air) or 50% O₂ (hyperoxia chamber) for 7 consecutive days. From P8 to 21, hyperoxia pups were transferred to room air, and all pups are kept in 21% O₂ for the “recovery” period. During P8–21, neonates from both oxygen exposure groups were intraperitoneally injected daily with either vehicle (PBS), 14 µM NaHS, 12.5 µM GYY4137, or 25 µM GYY4137. P21 mice were then subjected to lung mechanics testing using methacholine challenge and flexiVent (14 µM NaHS, 25 µM GYY4137, or vehicle) or tissue is harvested for precision-cut lung slice (PCLS) analysis (12.5 µM GYY4137, 25 µM GYY4137, or vehicle). Following functional measurements (flexiVent), lungs were harvested for histological analysis and immunofluorescence (Fig. 1).

Effects of O₂ and Exogenous H₂S Show Sex Differences in Airway Resistance and Compliance

Airway resistance (Rrs) is a quantitative measurement of dynamic resistance of airways (i.e., level of constriction in the lungs). Airway compliance (Crs) is a measurement inversely proportional to Rrs, indicating the overall elastic property of the respiratory system and therefore the ease with which it can extend during tidal breathing. We previously showed that neonatal mice exposed to moderate hyperoxia increases airway resistance and decreases airway compliance (24, 25). To determine the effects of H₂S on airway mechanics in neonatal mice exposed to hyperoxia, two exogenous H₂S donors were injected daily during the “recovery” period (P8–21): rapid-release NaHS (14 µm) or “chronic” slow-release GYY4137 (25 µM). Analysis of lung mechanics was performed on P21 pups using methacholine (MCh) challenge and flexiVent. Because recent published studies identified sex-specific outcomes in neonatal hyperoxic lung injury [using a high-O₂ (95%) BPD model] (66, 67) neonates were analyzed based on sex.

The lung mechanics results showed sex differences following moderate hyperoxia exposure and sex differences in the effect of exogenous H₂S donors. In considering sex differences between neonates exposed to 21% O₂ (vehicle), male neonates had increased airway resistance in response to MCh challenge compared with females (††††P < 0.0001) (Fig. 2A). Differences in airway resistance measurements between female and male neonates exposed to 50% O₂ were not evident (P = n.s.). Female neonates exposed to 50% O₂ from P0 to 7 showed increased airway resistance at P21 compared to female neonates exposed to 21% O₂ from P0 to 21 (***P < 0.001), whereas males did not demonstrate this effect (P = n.s.) (Fig. 2A). Administering 25 µM GYY4137 to female neonates did not significantly alter airway resistance compared to vehicle control, regardless of O₂ exposure condition (Fig. 2B). On the other hand, administration of 25 µM GYY4137 to male neonates showed reduced airway resistance in response to MCh challenge at P21 following either 21% O₂ (‡P < 0.05) or 50% O₂ (###P < 0.001) exposure (Fig. 2B). Administering 14 µM NaHS to female neonates did not significantly alter airway resistance compared to vehicle control, regardless of O₂ exposure condition (Fig. 2C). However, administering 14 µM NaHS to male neonates elicited reduced airway resistance in response to MCh challenge at P21 following 21% O₂ exposure (%P < 0.05), but not following 50% O₂ exposure (P = n.s.) (Fig. 2C).

Figure 2.

Effects of O₂ and exogenous H₂S show sex differences in airway resistance (Rrs) and compliance (Crs). Day 21 mice underwent analysis of lung mechanics (flexiVent), during which animals were subjected to methacholine (MCh) challenge (nebulized 0 mg/mL 6.25, 12.5, 25.0, 50.0 mg/mL MCh). Ordinary two-way ANOVA was used to compare O₂ and H₂S donor effects during methacholine challenge. Data are represented as means ± SE; n = 3–7 pups in each group. Airway resistance (Rrs): A: effect of sex in 21% O₂ (††††P < 0.0001), effect of sex in 50% O₂ (P = n.s.), effect of moderate hyperoxia (21% vs. 50% O₂ vehicle) in females (***P < 0.001) and males (P = n.s.); B: effect of 25 µM GYY4137 in 21% O₂ females (P = n.s.) and males (‡P < 0.05); effect of 25 µM GYY4137 in 50% O₂ females (P = n.s.) and males (###P < 0.001); C: effect of 14 µM NaHS in 21% O₂ females (P = n.s.) and males (%P < 0.05); effect of 14 µM NaHS in 50% O₂ females (P = n.s.) and males (P = n.s.). Airway compliance (Crs): D: effect of sex in 21% O₂ (††††P < 0.0001), effect of sex in 50% O₂ (!!P = 0.01), effect of moderate hyperoxia (21% vs. 50% O₂ vehicle) in females (****P < 0.0001) and males (P = n.s.); E: effect of 25 µM GYY4137 in 21% O₂ females (‡‡P < 0.01) and males (‡‡‡‡P < 0.0001); effect of 25 µM GYY4137 in 50% O₂ females (P=ns.) and males (####P < 0.0001); F: effect of 14 µM NaHS in 21% O₂ females (%%P < 0.01) and males (%%%P < 0.001); effect of 14 µM NaHS in 50% O₂ females (P = n.s.) and males ($P < 0.05). ANOVA, analysis of variance; H₂S, hydrogen sulfide.

In considering sex differences in airway compliance between neonates exposed to 21% O₂ (vehicle), male neonates had decreased Crs compared to females (††††P < 0.0001) (Fig. 2D). Furthermore, males exposed to 50% O₂ from P0 to 7 had decreased airway compliance in response to MCh challenge compared to female littermates (!!P = 0.01) (Fig. 2D). Female neonates exposed to 50% O₂ from P0 to 7 showed decreased airway compliance at P21 compared to female neonates exposed to 21% O₂ from P0 to 21 (****P < 0.0001), whereas males did not demonstrate this effect (P = n.s.) (Fig. 2D). Administering 25 µM GYY4137 to female neonates decreased airway compliance compared to vehicle control, and only in those exposed to 21% O₂ from P0 to 21 (‡‡P < 0.01) (GYY4137 did not show an effect in female neonates exposed to 50% O₂; P = n.s.). On the other hand, administration of 25 µM GYY4137 to male neonates showed increased airway compliance in response to MCh challenge at P21 following either 21% O₂ (‡‡‡‡P < 0.0001) or 50% O₂ (####P < 0.0001) exposure (Fig. 2E). Administering 14 µM NaHS to female neonates decreased airway compliance compared to vehicle control, and only in those exposed to 21% O₂ from P0 to 21 (%%P < 0.01) (NaHS did not show an effect in female neonates exposed to 50% O₂; P = n.s.). Administering 14 µM NaHS to male neonates elicited increased airway compliance in response to MCh challenge at P21 following 21% O₂ exposure (%%%P < 0.001) and 50% O₂ exposure ($P < 0.05) (Fig. 2F).

Exogenous H₂S Blunts Effects of O₂ on ASM Thickness

Because lung mechanics data showed differential effects of O₂ and exogenous H₂S based on sex, histological analysis was therefore assessed accordingly. H&E staining showed increased ASM thickness at P21 in both male and female neonates exposed to moderate hyperoxia from P0 to 7 compared to neonates exposed to normoxia from P0 to 7 (P < 0.05) (Fig. 3A). Neither male nor female neonates showed significant effects of hyperoxia on the thickness of the airway epithelium (Fig. 3B). In both male and female mice exposed to 21% O₂ from P0 to 21, exogenous H₂S donors had negligible effects on ASM thickness, regardless of donor used (Fig. 3A). However, both male and female neonates exposed to 50% O₂ from P0 to 7 revealed a reduction in ASM thickness at P21 in the presence of exogenous H₂S donor GYY4137 (25 µM; P < 0.05). Exogenous NaHS (14 µM) also showed a reduction in ASM thickness at P21, but results were not significant. Representative H&E images are shown (Fig. 3C).

Figure 3.

Exogenous H₂S blunts effects of O₂ on ASM thickness. Lungs were harvested from day 21 mice following the neonatal hyperoxia exposure model and treatments with or without exogenous H₂S donors (Fig. 1). Formalin-fixed paraffin-embedded sections were used for hematoxylin and eosin (H&E) staining. Quantitative analysis (ImageJ) measured the lumen, epithelial layer, and smooth muscle layer to determine the airway thickness and values were normalized to the airway diameter. Female and male neonates were measured for ASM (A) and epithelial (B) thickness. C: representative images are shown. ImageJ was used to adjust white balance for consistency. Two-way ANOVA with Tukey’s multiple comparison test was used to compare ASM or epithelial thickness in the 21% and 50% O₂ groups, either male or female neonates, exposed to 25 µM GYY4137 or 14 µM NaHS. Data are represented as means ± SE; n = 5–7 (female) or 3–5 (male) pups in each group; *P < 0.05. ANOVA, analysis of variance; ASM, airway smooth muscle; H₂S, hydrogen sulfide.

Altered Extracellular Matrix in Hyperoxia is Attenuated by Exogenous H₂S

We previously showed that 50% O₂ alters airway extracellular matrix, both in our neonatal hyperoxia exposure model and using human ASM cells in vitro (20, 23–25, 62). Here, MT staining showed that female neonates exposed to 50% O₂ from P0 to 7 have increased collagen deposition in airways at P21 compared to female neonates exposed to 21% O₂ from P0 to 21 (P < 0.01) (Fig. 4A). Although male neonates exposed to 50% O₂ from P0 to 7 showed increased collagen deposition in airways in P21 compared to male neonates exposed to 21% O₂ from P0 to 21, this increase was not significant. Administration of 25 µM GYY4137 or 14 µM NaHS had negligible effects on collagen deposition in both male and female neonates exposed to 21% O₂ from P0 to 21. However, the administration of 14 µM NaHS to P8–21 female pups that were exposed to 50% O₂ from P0 to 7 showed a reduction in collagen deposition within airways, similar to the collagen levels of pups in normoxia from P0 to 21 (P < 0.01) (Fig. 4A). The administration of 25 µM GYY4137 also decreased collagen deposition within airways of female pups exposed to 50% O₂ from P0 to 7, but results were not significant. Interestingly, neither exogenous H₂S donor altered airway collagen deposition in male pups exposed to 50% O₂ from P0 to 7. Representative images are shown (Fig. 4B).

Figure 4.

Altered extracellular matrix in hyperoxia is attenuated by exogenous H₂S. FFPE sections from day 21 mice following the neonatal hyperoxia exposure model with or without exogenous H₂S donors (Fig. 1) were used to assess collagen deposition via Masson trichrome (MT) staining. A: tissue sections were quantified with Orbit Image Analysis software to determine the collagen deposition within airways using a pixel-based classification model to differentiate the amount of collagen (inclusion) vs. background (exclusion) within a specified region of interest. The resulting ratio was multiplied by 100. B: representative sections illustrate the collagen staining (blue) and noncollagen tissue (red/pink). ImageJ was used to adjust the white balance for consistency. Two-way ANOVA with Tukey’s multiple comparison test was used to compare the airway collagen deposition in the 21% and 50% O₂ groups, both males and females, exposed to 25 µM GYY4137 or 14 µM NaHS. Data are represented as means ± SE; n = 5–7 (female) or 3–5 (male) pups in each groups; **P < 0.01. FFPE, formalin-fixed paraffin-embedded; H₂S, hydrogen sulfide.

To further investigate the effects of O₂ and 25 µM GYY4137, we assessed α-smooth muscle actin (αSMA) and fibronectin, the latter of which is an extracellular matrix protein highly expressed in developing lungs and is indicative of airway remodeling. To determine changes in αSMA and fibronectin, immunofluorescence was performed on lung sections from female neonates from 21% or 50% O₂ with either vehicle or 25 µM GYY4137 treatment. Moderate O₂ exposure increased αSMA and fibronectin in female neonatal airways (Fig. 5). Importantly, female neonates exposed to hyperoxia from P0 to 7 showed reduced αSMA and fibronectin immunofluorescence at P21 when 25 µM GYY4137 was administered during the recovery period (Fig. 5).

Figure 5.

Increased αSMA in female neonates exposed to moderate hyperoxia is attenuated by exogenous H₂S. Immunofluorescence was used on FFPE lung tissue sections to determine the changes in α-smooth muscle actin (αSMA; green) and fibronectin (red) in airways from female neonates exposed to either 21% or 50% O₂ with or without 25 µM GYY4137. Images were captured using Keyence microscopy (n = 1–3 animals/group). Representative images show αSMA in airways (A) and vessels (V) with DAPI mount. DAPI, 4′,6-diamidino-2-phenylindole; FFPE, formalin-fixed paraffin-embedded; H₂S, hydrogen sulfide.

Precision-Cut Lung Slices Support Alleviating Effects of H₂S Donors on O₂-Enhanced Contractility

To support the previous findings of alleviating effects of H₂S on O₂-induced AHR, PCLS were separately prepared from mice exposed to the neonatal hyperoxia model. Here, mice were collectively assessed with both males and females within the group. In mice exposed to 21% O₂ from P0 to 21, 12.5 µM GYY4137 increased contraction of the airway. Neonates exposed to 50% O₂ from P0 to 7 showed increased airway contraction and decreased lumen area following methacholine exposure at P21 (P < 0.05). Importantly, 25 µM GYY4137 administered during the recovery period elicited a reduction in maximum airway contraction, trending toward significance, that was at similar levels as control mice in normoxia (Fig. 6). In contrast, 12.5 µM GYY4137 did not have such an effect, as the contraction was similar to hyperoxia exposure alone (Fig. 6). Representative videos of airways in PCLS suitable for airway responsive analysis are provided in Supplemental Fig. S1.

Figure 6.

Precision-cut lung slices support the alleviating effects of H₂S donors on O₂-enhanced contractility. Mice that did not undergo lung mechanics testing were used for the preparation of precision cut lung slices (PCLS) and ex vivo functional analyses. Brightfield microscopy and a recording chamber were used to capture the live imaging of responses to a tissue bath perfused with increasing concentrations of methacholine (MCh). Airway contraction was measured (ImageJ) by determining the baseline and airway lumen prior to contraction, normalizing airway area, and calculating lumen area as a fraction of baseline. Maximum contraction was calculated as a percentage. Airways were selected based on visibly intact epithelial layers and responsiveness to MCh (to differentiate from vessels). Airways sized 300–400 µm (representing 5th/6th level branches in 3- to 4-wk-old mice) fit within the visible recording magnification of ×10, and thus were used for analysis. Representative images show airways (A) in PCLS suitable for airway responsive analysis (V, vessel). Unpaired t test was used to compare 21% and 50% O₂ groups. Data are represented as means ± SE; n = 3–4 pups in each groups. *P < 0.05. Representative videos are provided in Supplemental Fig. S1. H₂S, hydrogen sulfide.

DISCUSSION

Understanding the mechanisms of early hyperoxia insults that elevate the risk of premature infants developing chronic airway disease is critically important for advancing long-term therapeutic outcomes. The recognition that gasotransmitter H₂S may offer therapeutic benefit provides a promising strategy worthy of investigation. However, the application of H₂S is in its infancy and therefore warrants a deep dive into its signaling pathways and downstream effects. The neonatal hyperoxia exposure model enables studies on the use of exogenous H₂S donors within a whole system, the data of which support numerous in vitro studies focusing on H₂S in developing ASM. We previously showed that human fetal ASM express the core machinery necessary for H₂S production and catabolism, and that exposure to moderate hyperoxia blunts these pathways. In addition, we demonstrated that the loss of H₂S is particularly detrimental in the context of hyperoxia, and exogenous H₂S donors alleviate the deleterious effects of O₂ on ASM function.

In the present study, we applied this concept to our in vivo model to test our hypothesis that H₂S alleviates O₂-induced effects in the developing airway. Here, our data show the applicability of both rapid- and slow-release exogenous H₂S donors on attenuating O₂-induced AHR and contractility, ASM thickening, and extracellular matrix deposition. Additionally, these findings indicate the importance of neonatal sex in understanding the effects of O₂ and H₂S in the developing lung. In brief, we show that moderate hyperoxia exposure deleteriously affects female neonatal airway resistance and airway compliance more robustly than their male neonatal counterparts. Although female neonates are more affected by moderate hyperoxia, male neonates even without hyperoxia exposure (i.e., normoxia group) have worse lung mechanics at baseline. Our data show that the exogenous H₂S donor GYY4137 (25 µM) has a more profound alleviating effect on male neonates exposed to either 21% or 50% O₂ by reducing airway resistance and increasing airway compliance. Similar effects of exogenous H₂S donor NaHS (14 µM) are shown in male neonates. Interestingly, exogenous H₂S donors may have a worsening effect on airway compliance in female neonates exposed to 21% O₂. The assessment of lung mechanics is supported by H&E and MT staining in which ASM thickness (but not epithelial thickness) and collagen deposition are increased by 50% O₂ and alleviated by exogenous H₂S donors. Exogenous GYY4137 attenuated the increase in αSMA and fibronectin shown in female neonates exposed to O₂. Lastly, PCLS data show increased contraction in O₂ that was alleviated by 25 µM GYY4137. Interestingly, a lower concentration of GYY4137 (12.5 µM) increased contraction in response to MCh in both normoxia and hyperoxia conditions.

Although these studies show a beneficial and sex-specific effect of exogenous H₂S on developing mouse airway structure and function, there are limitations to this study. Beneficial effects of exogenous H₂S are evident, as shown by our data, but underlying mechanisms remain elusive. Understanding how exogenous H₂S impacts lung function during the perinatal period will help target therapeutic approaches. In addition, the timing and dosage of exogenous H₂S donors become particularly important when considering physiological levels because too much H₂S can become cytotoxic (68). For example, one explanation for differential effects of GYY4137 versus NaHS in our PCLS data may be due to H₂S offering a unique role in hyperoxia. Our previous work using an in vitro model of human developing airway indicated that exogenous H₂S has a beneficial specific role only under moderate O₂, and that this O₂ exposure detrimentally impacts H₂S production (52). Therefore, manipulating endogenous H₂S in normoxia, when its production and pathways are not deleteriously impacted by O₂, may inadvertently shift H₂S out of its physiologically safe range. Future studies may elucidate O₂-specific effects of exogenous H₂S. Lastly, our present model was structured “therapeutically” rather than “preventatively” (i.e., exogenous H₂S donors were administered in the 2 wk following hyperoxia exposure). In the present studies, H₂S was administered during the 2-wk “recovery” period in normoxia following 1 wk of hyperoxia exposure. Therefore, these studies served as “therapeutic” mimics. When considering potential future clinical approaches, it is important to understand the effects of exogenous H₂S concurrently with moderate hyperoxia exposure during the first week of life, thus mimicking a neonatal infant receiving preventative therapies in the NICU. Determining the immediate and long-term effects of “preventative” exogenous H₂S therapies may be more applicable to future clinical approaches. In addition, it would be beneficial to investigate whether nebulized H₂S administration, either during (“preventative”) or after (“therapeutic”) hyperoxia exposure, would be as effective. Our studies set the stage for future investigation of H₂S administration of H₂S, which is logistically feasible and clinically relevant.

The importance of including sex-specific analyses in scientific investigation has become increasingly apparent and rightfully expected. With respect to the neonatal period, sex differences are more likely due to sex chromosomes rather than sex hormones (however, exposure to maternal sex hormones prenatally are also a relevant factor). Recent studies indeed highlight a key role of chromosomal sex in response to neonatal hyperoxia exposure using a BPD neonatal hyperoxia model (95% O₂—high O₂, rather than moderate O₂), and the Four Core Genotypes mice (female chromosomal sex with either male or female sex hormones and male chromosomal sex with either male or female sex hormones): researchers identified that chromosomal male sex, irrespective of sex hormones, deleteriously impacts hyperoxia effects on alveolarization and vascularization in the neonatal lung (66). The sex-specific effect of neonatal hyperoxia exposure, using the high-O₂ model, was supported in single-cell resolution such that all lung cell subpopulations showed sex-specific differences in transcriptional changes and endothelial to mesenchymal transition following hyperoxia exposure (67, 69). Although these studies use 95% O₂ in a mouse model, it demonstrated the importance of considering sex as a biological variable in our studies. Thus, we used our established clinically relevant moderate O₂ mouse model shown to elicit deleterious functional and structural changes in developing airways. Our work shows that moderate levels of O₂ impact the lung in a sex-specific manner, and that therapeutic interventions such as exogenous H₂S have sex-specific effects as well. Our findings that male neonates have worse lung function at baseline (i.e., 21% O₂ vehicle) support clinical studies that males are at a particular disadvantage of neonatal mortality and major morbidities when born prematurely (70).

In adults, nitric oxide (NO) reduces airway tone, proliferation, and remodeling (71–73), but NO effects in the premature airway are less conclusive; clinical evidence in preterm infants shows NO to be less effective in bronchodilation and vasodilation (74–76), and inhaled NO is not effective in preventing airway disease in prematurity (77–79). This may be due to dysfunctional soluble guanylate cyclase (sGC), an enzyme that produces cGMP, normally responds to the well-known bronchodilator NO, and dilates airways while limiting ASM proliferation. Dysfunctional sGC in the context of O₂ thereby blocks NO effects. Regardless, there is consensus that enhancing endogenous NO benefits the lung and airway development (80). H₂S may be a potential avenue for enhancing endogenous NO and thereby promoting beneficial NO effects in the premature lung. H₂S may regulate sGC redox state, therefore altering its responsiveness to NO donors. Several studies have supported this concept by showing that H₂S is indeed an enhancer of NO production and its downstream signaling effects, at least with respect to the vasculature (81, 82). It is thought that H₂S stimulates eNOS activity through increasing eNOS synthesis (83–85), calcium mobilization (81), AKT-mediated phosphorylation (86, 87), direct sulfhydration (88), and inhibition of cGMP phosphodiesterase (PDE) activity (87, 89). Much less is known about the relationship between H₂S and NO in ASM, but these previous studies provide insight into the potential therapeutic benefit of H₂S in the context of the premature airway. Future studies include investigation into H₂S-enhanced NO production and its downstream effects that may reduce remodeling and aid bronchodilation in the premature developing lung.

An important consideration related to the mechanisms of H₂S involves mitochondrial function and antioxidation. Although several mechanisms likely contribute to initial and subsequent airway disease during prematurity, immature antioxidant systems are an appealing option. Antioxidant systems (e.g., catalase, glutathione peroxidase, superoxide dismutase) mature later in gestation in the fetal lung, with antioxidant systems coupled to surfactant production in preparation of the postnatal transition (90–92). In utero development occurs in a relatively hypoxic environment [fetal $\text{P}{\text{a}}_{{\text{O}}_2}$ ∼25–30 mmHg vs. maternal $\text{P}{\text{a}}_{{\text{O}}_2}$ ∼80–90 mmHg (90)], so premature transition to ex utero life places immediate, greater demands on already underdeveloped lungs to adapt to higher ambient O₂ that is exacerbated by supplemental O₂. Accordingly, the preterm lung is highly susceptible to oxidative stress. Although antioxidant pathways have been a popular potential target in the past, antioxidant therapies have failed clinically (e.g., NAC). However, with the more recent understanding of the cytoprotective and antioxidant capabilities of H₂S, there lies immense potential to leverage readily available exogenous H₂S donors for therapeutic benefit. H₂S signaling is highly relevant to antioxidant pathways, reducing ROS, increasing glutathione production, protecting against oxidative stress, and activating Nrf2 while regulating NF-κB signaling (45–47).

H₂S production and catabolism are linked to the mitochondria, and an important link between H₂S and antioxidant systems is through glutathione (GSH). Synthesis involves homocysteine, cystathione, and cysteine, and the enzymes CBS, CSE, and mercaptopyruvate sulfurtransferase (3-MST). H₂S is catabolized via mitochondrial enzymes SQR and ETHE1. The regulation of CBS and the localization of enzymes may be important in H₂S regulation. There is an important connection between H₂S and the antioxidant glutathione (GSH) in the mitochondria. H₂S helps redistribute GSH to mitochondria toward cytoprotection (93). H₂S increases GSH production and protects against oxidative stress in neurons (94). H₂S increases glutamate transport, cysteine production, and γ-GC expression, all vital for GSH production (93). GSH can function in multiple cellular compartments and can either directly eliminate free radicals (reduce H₂O₂) or act as cofactor for GSH-dependent enzymes that detoxify products generated by ROS (95). GSH levels have been shown to be lower in patients with asthma (96, 97), and there is increasing interest in increasing GSH as a therapy for lung disease (97–99). It is plausible that exogenous H₂S would increase GSH and restore antioxidant systems in the developing lung.

The role of H₂S in oxygen sensing is another mechanism worthy of our investigation. Studies in vascular responses to hypoxia have shown that H₂S is an important mediator through its own metabolism in the mitochondria, particularly in smooth muscle (100). Others propose that there is a balance between how much H₂S is produced and O₂ available for H₂S oxidation, contributing to how much biologically active H₂S is available for cell processes. In pulmonary arteries, H₂S levels appear low under normoxic conditions and increase under hypoxic conditions; decreased O₂ availability reduces mitochondrial H₂S oxidation and thereby increases biologically active H₂S, which promotes cellular adaptation to hypoxia (100, 101). Both H₂S production and catabolism appear to be O₂-dependent. Importantly, these previous studies have primarily been performed in hypoxia conditions, leaving H₂S in the context of hyperoxia (and the developing lung) previously unexplored.

In summary, we initially proposed that exogenous H₂S would attenuate the effects of O₂ on airway contractility, thickness, and remodeling in mice exposed to hyperoxia during the neonatal period. Our use of functional (flexiVent; PCLS) and structural (histology; immunofluorescence) approaches revealed differential sex effects of O₂ and exogenous H₂S. This study found that moderate O₂ affects female neonatal lung function more so than males, but that male neonates have worse lung function even without hyperoxic insult. Furthermore, exogenous H₂S donors tend to benefit neonatal lung function in male neonates in particular. Considering that human males born preterm are at a particular disadvantage compared to their female preterm counterparts, these findings highlight the importance of considering neonatal sex in understanding and approaching future therapeutic strategies.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL DATA

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.c.6709092.

GRANTS

These studies are supported by American Heart Association Grant 20POST35210002 (to C. M. Bartman) and by NIH National Heart, Lung, and Blood Institute Grants R01 HL160570 (to C. M. Pabelick) and R01 HL056470 (to Y. S. Prakash).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.M.B., M.S., Y.S.P., and C.M.P. conceived and designed research; C.M.B., M.S., L.N., and K.K.L. performed experiments; C.M.B. and M.S. analyzed data; C.M.B., Y.S.P., and C.M.P. interpreted results of experiments; C.M.B. prepared figures; C.M.B. drafted manuscript; C.M.B., M.S., Y.S.P., and C.M.P. edited and revised manuscript; C.M.B., M.S., L.N., K.K.L., Y.S.P., and C.M.P. approved final version of manuscript.