Exogenous hydrogen sulfide (H2S) protects alveolar growth in experimental O2-induced neonatal lung injury

Abstract

Background: Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, remains a major health problem. BPD is characterized by impaired alveolar development and complicated by pulmonary hypertension (PHT). Currently there is no specific treatment for BPD. Hydrogen sulfide (H2S), carbon monoxide and nitric oxide (NO), belong to a class of endogenously synthesized gaseous molecules referred to as gasotransmitters. While inhaled NO is already used for the treatment of neonatal PHT and currently tested for the prevention of BPD, H2S has until recently been regarded exclusively as a toxic gas. Recent evidence suggests that endogenous H2S exerts beneficial biological effects, including cytoprotection and vasodilatation. We hypothesized that H2S preserves normal alveolar development and prevents PHT in experimental BPD.

Methods: We took advantage of a recently described slow-releasing H2S donor, GYY4137 (morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate) to study its lung protective potential in vitro and in vivo.

Results: In vitro, GYY4137 promoted capillary-like network formation, viability and reduced reactive oxygen species in hyperoxia-exposed human pulmonary artery endothelial cells. GYY4137 also protected mitochondrial function in alveolar epithelial cells. In vivo, GYY4137 preserved and restored normal alveolar growth in rat pups exposed from birth for 2 weeks to hyperoxia. GYY4137 also attenuated PHT as determined by improved pulmonary arterial acceleration time on echo-Doppler, pulmonary artery remodeling and right ventricular hypertrophy. GYY4137 also prevented pulmonary artery smooth muscle cell proliferation.

Conclusions: H2S protects from impaired alveolar growth and PHT in experimental O2-induced lung injury. H2S warrants further investigation as a new therapeutic target for alveolar damage and PHT.

Figure 1. H₂S protected human pulmonary artery endothelial cells (HPAECs) from O₂-induced toxicity.

(A) H₂S promotes endothelial network formation. Quantitative assessment of cordlike structure formation shows a significant decrease in the number of intersects and the total length of cord-like structures in hyperoxia. H₂S preserved the number of intersects (B) and total cord-structure length (C). (n = 3 per group, *P<0.0001 hyperoxia vs. other groups, scale bar 65 µm). (D) HPAECs were cultured for 48 hours in room air (Normoxia) or 95% hyperoxia. Mean data of cell viability as assessed by measuring the mitochondrial-dependent reduction of colorless 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) shows that hyperoxia significantly decreases HPAECs viability as compared with room air–exposed cells. H₂S treatment significantly improved HPAECs viability in hyperoxia (n = 7, *P<0.001). (E) After 48 hours culture in hyperoxia (95%), ROS activity evaluated by measuring the dichlorofluorescein (DCF) shows that hyperoxia increases the ROS production in HPAECs, treatment with GYY4137 significantly decreased the ROS (n = 6/group, *P<0.005 Hyperoxia vs O₂+H₂S).

Figure 2. In vivo H₂S treatment prevents arrested alveolar growth in experimental O₂-induced lung injury.

Representative (A) hematoxylin and eosin (H&E)-stained (scale bar 130 µm) lung sections at P21 showing larger and fewer alveoli in hyperoxia-exposed lungs as compared with lungs of room air housed rat pups. Treatment of hyperoxia-exposed animals with H₂S preserved alveolar structure. (B) The mean linear intercept confirms arrested alveolar growth in untreated O₂-exposed animals and preserved alveolar structure with H₂S treatment (n = 5 per group, *P<0.0001 hyperoxia vs. other groups).

Figure 3. In vivo H₂S treatment prevents O₂-induced arrested lung vascular growth.

A. Representative photomicrographs showing von Willebrand (vWF) factor staining (brown) in RA (room air), RA+H₂S, hyperoxia (O₂) and O₂+H₂S exposed lungs. Arrows highlight vWF-positive vessels; scale bars represent 100 µm. B. Mean data quantifying the number of vWF positive vessels between groups. The decrease in the number of vessels per high-power field (HPF) after hyperoxia exposure was prevented by H₂S treatment (n = 5–7/group, *P<0.005 Hyperoxia vs O₂+H₂S). C. Representative immunoblot and densitometric (D) analysis for endothelial marker CD31 in lung homogenates from control and H₂S treated animals. H₂S treatment preserved the expression of CD31 in hyperoxic rats compared with hyperoxic control (n = 3/group, *P<0.005 Hyperoxia vs O₂+H₂S).

Figure 4. H₂S prevents pulmonary hypertension associated with O₂-induced lung injury.

(A) Pulmonary arterial acceleration time/right ventricular ejection time (PAAT/RVET). Representative echo Doppler and mean PAAT/RVET showing a characteristic notch indicating PHT (arrow) in hyperoxic-exposed rat pups and a significantly decreased PAAT/RVET as compared with rat pups housed in room air. (B) H₂S significantly increased PAAT/RVET as compared with untreated hyperoxic rat pups (n = 6 animals per group, *P<0.005 hyperoxia vs. other groups). (C) Pulmonary arterial medial wall thickness (MWT). Representative H&E stained sections of pulmonary arteries from the four experimental groups and % mean MWT. Hyperoxic-exposed rats had a significant increase in %MWT as compared with room air–housed rat pups. (D) H₂S significantly reduced %MWT (n = 5 animals per group, *P<0.0001, hyperoxia vs. other groups, scale bar 65 µm). (E) Right ventricular hypertrophy (RVH). Hyperoxic-exposed rats had significant RVH as indicated by the increase in RV/LV+S ratio compared with room air control rats. H₂S significantly reduced RVH (n = 6 animals per group, *P<0.005 hyperoxia vs. other groups). (F) Treatment with GYY4137 significantly attenuated PDGF-induced proliferation (n = 6/group, *p<0.001).

Figure 5. H₂S rescues alveolarization after established O₂-induced lung injury.

(A) Representative H&E-stained lung sections of animals treated with GYY4137 from day P14–P24, after established lung injury, and harvested at P30. H₂S in O₂-exposed animals restored alveolar growth. (B) This is confirmed by the mean linear intercept (n = 5, *P<0.0001 hyperoxia vs. other groups, scale bar 65 µm).

Figure 6. GYY4137 treatment activates the PI3K pathway and decreases apoptosis in O₂-induced lung injury.

(A) Immunoblots show decreased P-Akt and Sirtuin1 expression in hyperoxic-exposed lungs. (B) Treatment with H₂S increased expression of P-Akt and Sirtuin1 expression in hyperoxic lungs (n = 3/group, *P<0.005). (C) In vivo H₂S decreases apoptosis in oxygen-exposed lungs with BPD. Immunoblots of total caspase-3 and actin are shown for the four experimental groups. (D) Hyperoxia-exposed lungs showed increased total caspase-3 expression, which was attenuated by in vivo H₂S treatment (n = 3/group, *P<0.005).

Figure 7. Rat lung epithelial cells (RLEs) exposed to hyperoxia decreased mitochondrial ΔΨm and increased mROS.

Representative confocal microscopy at high magnification (×100) of rat lung epithelial cells (RLEs) showing (A) decreased ΔΨm (TMRM) and (C) increased mROS production (MitoSOX) in hyperoxia (TMRM and MitoSOX are in red, merged with nuclear stain DAPI in blue). Hyperoxia exposed RLEs treated with H₂S have significantly increased ΔΨm (B) and decreased mROS (D) compared to hyperoxic control (n = 4 per group, *P<0.005 hyperoxia vs. other groups).