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).