Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia

Abstract

Transgenic (TG) human (h) extracellular superoxide dismutase (EC-SOD) targeted to type II cells protects postnatal newborn mouse lung development against hyperoxia by unknown mechanisms. Because alveolar development depends on timely proliferation of type II epithelium and differentiation to type I epithelium, we measured proliferation in bronchiolar and alveolar (surfactant protein C-positive) epithelium in air and 95% O2-exposed wild-type (WT) and TG hEC-SOD newborn mice at postnatal days 3, 5, and 7 (P3-P7), traversing the transition from saccular to alveolar stages. We found that TG hEC-SOD ameliorated the 95% O2-impaired bromodeoxyuridine uptake in alveolar and bronchiolar epithelium at P3, but not at P5 and P7, when overall epithelial proliferation rates were lower in air-exposed WT mice. Mouse EC-, CuZn-, and Mn-SOD expression were unaffected by hyperoxia or genotype. TG mice had less DNA damage than 95% O2-exposed WT mice at P3, measured by TdT-mediated dUTP nick end labeling (P < 0.05). Hyperoxia induced cell-cycle inhibitory protein p21cip/waf mRNA at P3, WT > TG, P = 0.06. 95% O2 impaired apical expression of type I cell alpha protein (T1alpha) in WT but not in TG mice at P3 and increased T1alpha in WT and TG mice at P7. Reducing the 95% O2-induced impairment of epithelial proliferation at a critical window of lung development was associated with protection against DNA damage and preservation of apical T1alpha expression at P3.

Fig. 1

Effect of hyperoxia and transgenic (TG) extracellular superoxide dismutase (EC-SOD) on alveolar and bronchiolar epithelial proliferation at postnatal days 3, 5, and 7 (P3, P5, and P7). Bromodeoxyuridine (BrdU) labeling indexes in cells lining distal air sacs (A) or bronchioles (B); means are 6 pups/group + SE. *P < 0.05 for indicated comparisons. Ki67 labeling indexes for cells lining air sacs (C) or bronchioles (D). E: BrdU labeling indexes for type II [surfactant protein C (SP-C)-positive cells], 5 pups/group, means + SE. *P < 0.05 wild type (WT) vs. TG. F: SP-C-immunostained (brown) cells are shown by black arrows; BrdU-labeled (purple) cells are shown by white arrows.

Fig. 2

Effect of hyperoxia and TG EC-SOD on mouse (m) and human (h) EC-SOD expression. A: representative immunoblots detecting human and mouse EC-SOD, CuZn-SOD, and Mn-SOD. B: mean hEC-SOD or mEC-SOD mRNA real-time PCR signals at crossing point of amplification, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal + SE, 8 pups/condition. *P < 0.05 vs. WT. C: mEC-SOD expression (A), airway, ×200; alveolar epithelium (B), arrows, ×1,000; and vascular smooth muscle (C) in small artery (black arrows) and large artery (white arrow), ×200. D shows control with no primary antibody, ×200.

Fig. 3

Effect of hyperoxia and TG EC-SOD on p21^cip/waf and p53 expression. A: RNase protection assay autofluorogram detecting p21, p53, and GAPDH mRNA in air- and 95% O₂-exposed animals, WT and TG, quantified in graph. B: values are pixels for p21 or p53, each normalized to corresponding GAPDH signal in each lane, mean of 8 pups/group + SE.

Fig. 4

Effect of hyperoxia and TG EC-SOD on DNA nicking [TdT-mediated dUTP nick end labeling (TUNEL)] at P3. Top: representative photomicrographs (×200 magnification) for each condition. Bottom: mean TUNEL intensity in pixels normalized to nuclear area (4′,6-diamidino-2-phenylindole perimeter), 5 pups/group + SE. *P < 0.05 vs. air WT, **P < 0.05 vs. O₂ WT.

Fig. 5

Effect of hyperoxia and TG EC-SOD on type I cell α protein (T1α) shown by Western blot. Effect of air, 95% O₂, and TG EC-SOD on T1α and β-actin in whole lung homogenates at P3 and P7.

Fig. 6

Effect of hyperoxia and TG EC-SOD on T1α shown by immunohistochemistry. T1α expression at P3 (A–D) in air (A, WT; B, TG) and 95% O₂-exposed mice (C, WT; D, TG) and P7 (E–H) in air (E, WT; F, TG) and 95% O₂-exposed mice (G, WT; H, TG); final magnification, ×200. C, inset: magnification at ×400 shows lack of apical T1α staining in 95% O₂-exposed WT mice at P3.