Lack of EC-SOD worsens alveolar and vascular development in a neonatal mouse model of bleomycin-induced bronchopulmonary dysplasia and pulmonary hypertension

Abstract

Background: Pulmonary hypertension (PH) worsens clinical outcomes in former preterm infants with bronchopulmonary dysplasia (BPD). Oxidant stress disrupts alveolar and vascular development in models of BPD. Bleomycin causes oxidative stress and induces BPD and PAH in neonatal rats. Disruption in the vascular endothelial growth factor (VEGF) and nitric oxide signaling pathways contributes to BPD. We hypothesized that loss of EC-SOD would worsen PAH associated with BPD in a neonatal mouse model of bleomycin-induced BPD by disrupting the VEGF/NO signaling pathway.

Methods: Neonatal wild-type mice (WT), and mice lacking EC-SOD (EC-SOD KO) received intraperitoneal bleomycin (2 units/kg) or phosphate-buffered saline (PBS) three times weekly and were evaluated at weeks 3 or 4.

Results: Lack of EC-SOD impaired alveolar development and resulted in PH (elevated right ventricular systolic pressures, right ventricular hypertrophy (RVH)), decreased vessel density, and increased small vessel muscularization. Exposure to bleomycin further impaired alveolar development, worsened RVH and vascular remodeling. Lack of EC-SOD and bleomycin treatment decreased lung total and phosphorylated VEGFR2 and eNOS protein expression.

Conclusion: EC-SOD is critical in preserving normal lung development and loss of EC-SOD results in disrupted alveolar development, PAH and vascular remodeling at baseline, which is further worsened with bleomycin and associated with decreased activation of VEGFR2.

Figure 1

Loss of EC-SOD impairs alveolar development at baseline and worsens bleomycin-induced BPD. Figure 1a-d. Representative image of pentachrome stain of lung sections from 4 week old WT and EC-SOD KO mice exposed to IP PBS or bleomycin. (a) WT PBS, (b) WT Bleo, (c) KO PBS, (d) KO Bleo, scale bar = 200 microns. Figure 1e-f. Morphometric analysis, radial alveolar counts (RAC), mean linear intercept (MLI), nodal point density (NPD), and surface area (SA) of WT and EC-SOD KO mice exposed to IP PBS or Bleomycin. *p < 0.05 for strain, **p < 0.01 for treatment by 2-way ANOVA, n=4–9.

Figure 2

Loss of EC-SOD causes pulmonary hypertension at baseline in neonatal mice, which is worsened by treatment with bleomycin. Figure 2a. RV/LV +S weight at 4 weeks in WT and EC-SOD KO mice following IP PBS or bleomycin treatment, *p < 0.05 for strain by 2 -way ANOVA. Figure 2b. RVSP by direct RV puncture at 4 weeks of age in WT and EC-SOD KO mice following IP PBS or bleomycin treatment, * p < 0.01 for strain by 2-way ANOVA. Figure 2c. LV/body weight at 4 weeks of age in WT and EC-SOD KO mice following IP PBS or bleomycin, * p < 0.05 for treatment by 2-way ANOVA. Figure 2d. RV/LV+S weight at 3 weeks in WT and EC-SOD KO mice following IP PBS or bleomycin treatment, * p = 0.06 for strain, ** p < 0.005 for treatment by 2-way ANOVA, n=3–8.

Figure 3

Pulmonary vascular density is decreased in neonatal mice lacking EC-SOD. Treatment with bleomycin further decreases vessel density. Figure 3a-d. Representative factor VIII staining in 4-week old WT and EC-SOD KO mice treated with IP PBS or bleomycin, (a) WT PBS, (b) WT Bleo, (c) KO PBS, (d) KO Bleo, scale bar = 200 microns. Figure 3e. Vessel density in WT and EC-SOD KO mice following IP PBS or bleomycin treatment, *p=0.1 for strain, **p < 0.0001 for treatment by 2-way ANOVA, n=3–5.

Figure 4

Muscularization of small vessels is increased in neonatal mice lacking EC-SOD and with bleomycin treatment. Figure 4a-d. Representative α-SMA staining in 4-week old WT and EC-SOD KO mice treated with IP PBS or bleomycin, (a) WT PBS, (b) WT Bleo, (c) KO PBS, (d) KO Bleo, scale bar = 200 microns. Figure 4e. Muscularization of small vessels (<30 microns) expressed as ratio of muscularized vessels/total number vessels, *p < 0.0001 for strain and **p <0.0001 for treatment by 2- way ANOVA, n=3–5.

Figure 5

Lack of EC-SOD and treatment with bleomycin decrease pulmonary expression of total and active VEGRFR2 and eNOS. Figure 5a. Western blot analysis for total VEGFR2, pVEGFR2, eNOS and β-actin. Figure 5b. Analysis of protein expression for total VEGFR2 standardized to β-actin *p < 0.05 for strain and **p < 0.05 for treatment by 2-way ANOVA. Figure 5c. Active phosphorylated VEGFR2 relative to β-actin, *p < 0.01 for treatment by 2-way ANOVA. Figure 5d. eNOS expression relative to β-actin, *p < 0.06 for strain, **p < 0.0001 for treatment by 2-way ANOVA, n=3–6.

Figure 6

No change in lung EC-SOD expression or activity in WT mice treated with IP bleomycin. Figure 6a. Western blot analysis for EC-SOD and β-actin. Figure 6b. No change in EC-SOD expression in WT mice treated with bleomycin. Figure 6c. No change in EC-SOD activity in WT mice treated with bleomycin, n=3–9.