Overexpression of EC-SOD protects against oxidative stress. The ratio of reduced to oxidized glutathione (GSH/GSSG) was measured in lung tissue using a glutathione assay kit (Cayman Chemical) in WT and EC-SOD TG mice exposed to 1 day of hypoxia. This time point was selected to test whether there was evidence for oxidative stress despite the small increase in EC-SOD activity levels observed at 1 day of hypoxia and test the impact of EC-SOD on the redox state of the lung. *Decreased GSH/GSSG vs. WT normoxic lungs; #increased GSH/GSSG vs. WT normoxic lungs. P < 0.05 by 1-way ANOVA; n = 4.

Lung EC-SOD immunolocalization in chronic hypoxia. A: immunolocalization of endogenous mouse EC-SOD in WT and EC-SOD TG mice exposed to 1 and 35 days of hypoxia using a polyclonal rabbit anti-mouse EC-SOD antibody (1:200) followed by a goat anti-rabbit secondary IgG antibody conjugated to HRP, developed with DAB (brown stain) and counterstained with hematoxylin (blue stain). B: immunolocalization of the human EC-SOD transgene in WT (absent staining) and EC-SOD TG mice exposed to 35 days of hypoxia using a polyclonal rabbit anti-human EC-SOD antibody (1:500). Bar represents 50 μm.

Overexpression of EC-SOD limits pulmonary vascular remodeling, as shown by cell proliferation, muscularization of small vessels, and collagen deposition. Cell proliferation was evaluated by Ki67 immunostaining with a rabbit monoclonal Ki67 antibody (1:200 Lab Vision, NeoMarkers) in lung sections of mice exposed to 0, 1, 3, or 35 days of hypoxia. A: the number of Ki67 positive nuclei in cells within the medial and intimal layer of similar sized pulmonary arteries (50–200 μm) was counted in 7–10 vessels for each lung by an investigator blinded to the experimental conditions. The average number of Ki67 positive cells in the pulmonary artery per animal was analyzed. *P < 0.05 by 1-way ANOVA vs. normoxic WT (n = 3–4). B: Ki67 staining in representative pulmonary arteries of WT and EC-SOD TG mice exposed to normoxia or 3 days of hypoxia (brown color with DAB detection system, arrow indicates Ki67 positive cell within vessel wall). Bar represents 50 μm. C: muscularization was evaluated by α-smooth muscle actin (α-SMA) staining (mouse monoclonal α-SMA antibody, 1:100, Clone 1A4, Sigma) and in small pulmonary vessels after 0, 1, 3, and 35 days of hypoxia. Small vessels (<50 μm) with α-SMA staining within >75% of the circumference of the vessel were counted in 10 peripheral high-powered fields (HPF; ×20 objective) by an investigator blinded to the experimental conditions. The average number of stained vessels in each HPF per animal was analyzed by 1-way ANOVA. *P < 0.05 vs. normoxic WT (n = 4–6). D: representative images of α-SMA immunostaining (brown color with DAB detection system) in small pulmonary vessels (arrow). Bar represents 50 μm. E: chronic hypoxic-induced pulmonary vascular remodeling was also evaluated by visualizing collagen deposition around similar sized pulmonary arteries (arrow) located at the junction of the terminal bronchiole and alveolar duct after staining with picrosirius red (red signal). Sections were counterstained with hematoxylin. A representative image was selected for each mouse strain at 35 days of hypoxia. The bar represents 50 μm.

Overexpression of EC-SOD prevented the hypoxic induction of the Egr-1-responsive gene target, tissue factor (TF), in the lung. A: TF mRNA was measured by semiquantitative RT-PCR in the lungs of mice exposed to hypobaric hypoxia (18,000 ft) for 1 day. Bands from a single representative blot are shown along with a bar graph that displays the relative densitometry of TF/β-actin standardized to WT normoxia. *P < 0.05 vs. normoxic WT by 1-way ANOVA (n = 4). B: TF protein from the lungs of mice exposed to hypobaric hypoxia for 1 day was compared by Western blot analysis. Bands from a single representative blot are shown along with a bar graph that displays the relative densitometry of TF/β-actin. *P < 0.05 vs. normoxic WT by 1-way ANOVA (n = 4–7).

Lung extracellular superoxide dismutase (EC-SOD) activity in chronic hypoxia. Lung EC-SOD was separated from intracellular SODs using a concavalin A column, and EC-SOD activity levels were measured using a SOD assay kit (Dojindo Molecular Technologies) after 0, 1, or 35 days of hypobaric (Hypo) hypoxia in wild-type (WT) mice (A) and mice overexpressing lung EC-SOD (B). EC-SOD activity was expressed as U/g tissue. *P < 0.05 vs. WT normoxic lungs compared by 1-way ANOVA; n = 4–5.

Lung EC-SOD protein expression in chronic hypoxia. Endogenous mouse EC-SOD (mEC-SOD) protein expression in lungs of WT and EC-SOD TG mice exposed to 1 day (A) and 35 days (B) of hypoxia was evaluated by Western blot analysis using a polyclonal rabbit anti-mouse EC-SOD antibody (1:1,000). C: Western blot analysis of human EC-SOD (hEC-SOD) transgene expression in mice exposed to hypoxia using a polyclonal rabbit anti-human EC-SOD antibody (1:5,000). Lanes from a representative blot are shown with the densitometric measurements of EC-SOD standardized to β-actin and analyzed by 1-way ANOVA. *P < 0.05 vs. normoxic WT lung; n = 4–7.

Overexpression of EC-SOD limited the development of chronic hypoxic pulmonary hypertension. Chronic hypoxic pulmonary hypertension was assessed by right ventricular systolic pressure (RVSP, mmHg) measurements via direct RV puncture in WT mice (white bars) and EC-SOD TG mice (black bars) exposed to 35 days of hypobaric hypoxia. *P < 0.05 vs. normoxic WT and #P < 0.05 vs. normoxic EC-SOD TG by 1-way ANOVA; n = 9–10.

Overexpression of EC-SOD prevented the hypoxic induction of lung Egr-1 mRNA and nuclear protein expression. A: Egr-1 mRNA was measured by semiquantitative RT-PCR in the lungs of mice exposed to hypobaric hypoxia (18,000 ft) for 1 day. Bands from a single representative blot are shown along with a bar graph that displays the relative densitometry of Egr-1/β-actin standardized to WT normoxia. *P < 0.05 vs. normoxic WT by 1-way ANOVA (n = 3–4). B: Egr-1 protein in nuclear extracts from the lungs of mice exposed to hypobaric hypoxia for 1 day was compared by Western blot analysis. Bands from a single representative blot are shown along with a bar graph that displays the relative densitometry of Egr-1 normalized to the TATA nuclear loading control. *P < 0.05 vs. normoxic by 1-way ANOVA (n = 4–7). C: the protein expression of the major corepressor of Egr-1, NAB2, was compared by Western blot analysis in nuclear protein extracts from the lungs of mice exposed to hypobaric hypoxia for 1 day. Bands from a single representative blot are shown.