Hyperoxia exposure in newborn mice, 95% O2
× 7 days beginning at birth, then 60% O2
× 14 days, impairs alveolar formation, measured as depressed alveolar surface density and volume density at P21. This was prevented in hEC-SOD TG mice (1
). The present studies were undertaken to elucidate the mechanisms by which hEC-SOD overexpression preserves lung development. We found that 95% O2
exposure profoundly impairs alveolar and bronchiolar epithelial proliferation at P3, measured by either BrdU uptake or Ki67 expression, when lung development begins the transition between saccular and alveolar phases in the mouse. Hyperoxia-impaired epithelial proliferation persisted at P5 and P7, although the magnitude of impairment was diminished. TG hEC-SOD overexpression in alveolar and, to a lesser extent, bronchiolar epithelium significantly preserved alveolar and bronchiolar epithelial proliferation at P3 in pups exposed to hyperoxia.
We found that the labeling indexes for BrdU uptake and Ki67 showed parallel trends, although the absolute values were not identical. At P7, for example, BrdU uptake in alveolar epithelium was actually lower in 95% O2
-exposed TG mice than in WT littermates, but there were no such differences in Ki67 labeling. In contrast, we found that air-exposed TG mice demonstrated more BrdU uptake in alveolar epithelial cells than air-exposed WT mice, but no differences in Ki67 were observed. We do not know whether this difference in proliferation is maladaptive, and no overt differences in lung morphology have been reported in EC-SOD TG adult mice (10
), although this has not been studied in detail.
Ki67 is expressed in cycling cells, maximally expressed during M-phase, whereas BrdU uptake may occur during DNA repair in DNA-damaged, non-cycling cells (6
), which would be expected during hyperoxia (23
). Ki67 and BrdU may therefore not identify identical cell populations, with Ki67 being the more specific marker of proliferation.
We attribute these observed effects to TG hEC-SOD (15
). Native mouse EC-SOD expression in newborns, in whole lung or by immunohistochemistry, was unaffected by 95% O2
exposure or by genotype at P3. Mouse EC-SOD expression was restricted to alveolar and bronchiolar epithelium and vascular smooth muscle. Extracellular expression was less widespread than intracellular expression in the newborn mouse, as previously described in newborn rabbits and rats (13
). TG hEC-SOD expression was overwhelmingly intracellular, and this was unaffected by hyperoxia. We did not measure activity levels of antioxidant enzymes in lungs of neonatal mice at P3, but we previously reported that EC-SOD activity in TG neonatal mice at P7 was ~2.5-fold the activity observed in WT mice (1
). TG hEC-SOD did not affect native antioxidant enzyme expression or activity in adult hyperoxiaexposed mice (10
There were no effects of hyperoxia or genotype on the other SOD isoforms, CuZn-SOD or Mn-SOD, determined by Western blotting. Hyperoxia has previously been reported to induce some antioxidant systems in newborns, both premature and term newborns (12
), but this is species specific with relatively modest increases in total SOD activity (<20%) observed in 2-day-old mice exposed to 95% O2
The differences in the protective effects of hEC-SOD over-expression on proliferation (alveolar > bronchiolar) may be due to the relatively greater expression of hSP-C promoterdriven hEC-SOD expression in type II cells. Because we found mouse EC-SOD expressed at relatively high levels in bronchiolar epithelium at P3, there may be less additional EC-SOD activity conferred by TG hEC-SOD, although this was not directly tested.
The relationship of oxidative stress to cellular proliferation during lung development is complex. Survival after hyperoxia in in vitro and in vivo models requires the arrest of cell cycling to permit repair of hyperoxia-induced DNA damage, which is associated with induction of the cyclin-dependent kinase inhibitor p21cip/waf
). DNA nicking (TUNEL) was induced in hyperoxia-exposed WT animals, but not in EC-SOD TG animals at P3. Although TUNEL may also identify dividing and apoptotic cells, we previously found that hyperoxia-induced TUNEL in newborn lung seldom identified apoptotic cells (5
). We found that hyperoxia increased p21cip/waf
mRNA in whole lung from WT animals at P3, which displayed the most significant proliferation arrest. This increase was partly prevented in EC-SOD TG animals at P3. Hyperoxia did not induce p53 mRNA, as was expected, since its effects on p53 are mediated predominantly through protein stability rather than transcription (14
). Effects on p21cip/waf
and p53 mRNA at the whole lung level are limited by the choice(s) of reference mRNAs that might themselves be affected. In our studies, there was little variation in GAPDH (or L32 cyclin, data not shown) mRNA signal. Because we studied mRNA in whole lung, we were unable to determine cell specificity of effects on p21cip/waf
expression. Proliferation arrest at a critical period of lung development may impair the appropriate patterning of type II differentiation and type I cell formation during postnatal lung development.
We have previously shown that this model of hyperoxia-impaired neonatal lung development, continued to P21, ultimately leads to impaired alveolization and that this is significantly prevented in TG hEC-SOD mice (1
). This led us to conclude that preservation of alveolar and bronchiolar epithelial proliferation in TG hEC-SOD mice is likely adaptive, accompanied by safe repair of hyperoxia-induced DNA damage, although we did not evaluate this directly.
Alveolar formation depends on orderly differentiation of type II → type I cells. We found that hyperoxia consistently impaired apical expression of T1α in WT mice at P3, but not TG mice (). T1α is normally expressed on apical surfaces of type I cells, but hyperoxia-exposed WT mice demonstrated qualitatively less apical labeling than TG mice at P3. By day 7
, there were no apparent differences in localization. We did not observe consistent effects of hyperoxia on T1α in whole lung homogenates, in either WT or TG mice, at P3. Hyperoxia induced T1α expression in whole lung from both WT and TG mice at P7, consistent with earlier observations of T1α expression in adult mice (7
The precise function of T1α in type I homeostasis is not known, but gene deletion leads to failure of normal alveolar formation and death from respiratory failure at birth (29
). In vitro studies suggest that T1α may be necessary to maintenance of the flattened attenuated shape of the type I cell (31
). In vitro studies show that injury to type II cells may lead to differentiation or transdifferentiation to intermediate cell types before type I cells are formed (20
). Lack of apical labeling in the hyperoxia-exposed WT animals at P3 may represent this phenomenon.
EC-SOD overexpression in type II cells may also affect direct and indirect regulation of T1α expression by oxygen or reactive oxygen species. Cao and colleagues (7
) have shown that hyperoxia induces increased T1α expression in adult mice through effects on nuclear transcription factors Sp1 and Sp3 binding to DNA, but the identity of the molecular interaction between oxygen, reactive oxygen species, or intermediates, and Sp1/Sp3 binding and downstream effects on the T1α promoter is unknown. Hyperoxia effects may be indirectly mediated through thyroid transcription factor-1 (34
), which acts on the T1α promoter (30
). The relative abundance of oxygen-containing moieties will necessarily differ in the EC-SOD TG newborn mice and will likely differ in their WT littermates since newborns of many species are better able to induce enzymatic antioxidant expression during hyperoxia than adults (3
). These differences may explain the delay in the hyperoxia-induced increase of T1α in newborn WT mice. Alternatively, impaired, ectopic T1α expression during hyperoxia may be due to altered protein trafficking, which we have not directly tested.
Preservation of proliferation in alveolar epithelium from 95% O2
impairment at P3 by type II cell-targeted hEC-SOD overexpression may partly explain the protection of alveolar development against hyperoxia that we previously reported (1
). Formation of alveoli depends in part on differentiation of type II cells to type I cells as the lung enters the alveolar phase of development (20
) as well as type II cell-produced paracrine and autocrine factors, such as vascular endothelial growth factor, necessary for pulmonary capillary endothelial integrity (18
). The initial hyperoxia-induced arrest of type II cell proliferation, followed by a maladaptive type II cell hyperplasia that fails to yield normal alveolization, is also observed in the baboon model of BPD (25
). Failure to form normal alveoli may result in part from inadequate type I cell differentiation, which, in turn, could result from redox-sensitive changes to transcription factors that control expression of type I cell-specific antigen, necessary for type I cell differentiation (34
In summary, hEC-SOD overexpression driven by the human SP-C promoter partly preserves type II alveolar epithelial and bronchiolar epithelial proliferation during severe hyperoxic stress at P3, during the transition from the saccular to alveolar phases of normal murine lung development. This effect was accompanied by protection against DNA damage, a known target of oxidative stress, partly blocking induction of p21cip/waf mRNA expression. Apical T1α expression, thought to be necessary to alveolar formation, was preserved in hyperoxia-exposed EC-SOD TG mice at P3 but was impaired in WT littermates. We speculate that protection of alveolar type II cells from superoxide accumulation avoids biomolecular damage and maladaptive arrest of proliferation, protecting differentiation to type I epithelium.