Disrupted postnatal lung development in heme oxygenase-1 deficient mice

Abstract

Background: Heme oxygenase (HO) degrades cellular heme to carbon monoxide, iron and biliverdin. The HO-1 isoform is both inducible and cyto-protective during oxidative stress, inflammation and lung injury. However, little is known about its precise role and function in lung development. We hypothesized that HO-1 is required for mouse postnatal lung alveolar development and that vascular expression of HO-1 is essential and protective during postnatal alveolar development.

Methods: Neonatal lung development in wildtype and HO-1 mutant mice was evaluated by histological and molecular methods. Furthermore, these newborn mice were treated with postnatal dexamethasone (Dex) till postnatal 14 days, and evaluated for lung development.

Results: Compared to wildtype littermates, HO-1 mutant mice exhibited disrupted lung alveolar structure including simplification, disorganization and reduced secondary crest formation. These defects in alveolar development were more pronounced when these mice were challenged with Dex treatment. Expression levels of both vascular endothelial and alveolar epithelial markers were also further decreased in HO-1 mutants after Dex treatment.

Conclusions: These experiments demonstrate that HO-1 is required in normal lung development and that HO-1 disruption and dexamethasone exposure are additive in the disruption of postnatal lung growth. We speculate that HO-1 is involved in postnatal lung development through modulation of pulmonary vascular development.

Figure 1

HO-1 homozygous mutant mice display disrupted alveolar development. A-D: H & E staining of lung sections at P10. A: Wildtype; B: HO-1 +/-; C, D: HO-1 -/-. In A and B, normal organized alveolar sac and formation of secondary septations are shown. In HO-1 homozygous mutants, alveolar development was disrupted at various severities. Panel C represents a mutant with mildly enlarged alveolar airspaces, and Panel D illustrates another mutant with more severe defects including a dramatically disorganized alveolar sac, missing septation, and a thickened interstitial region. E: Radial alveolar counts (RAC) of lung sections from wildtype (WT) and HO-1 -/- littermates at P10 (n = 4 in each group). _* P < 0.05 vs. WT. The HO-1 mutants demonstrated significant decreased RAC.

Figure 2

Dexamethasone treatment disrupts postnatal alveolar development. A, B: Representative H&E staining of mouse lung sections at P10. Wildtype newborn mice were injected daily with saline (Control in A) or 1 ug/pup dexamethasone (Dex, in B). Note the enlarged alveolar airspace and thinning of the alveolar wall in Dex-treated animals (B). C. Lung alveolar counts in control and Dex-treated mice. _* P < 0.05 vs. Control. D. HO protein levels in control and Dex-injected lungs. Upper panel: representative Western blot of P10 lungs from Control and Dex-injected mice with antibodies against HO-1, HO-2 and ß-actin (loading control). Samples from two animals for each group are shown. HO-1 protein levels were visibly decreased in Dex-injected samples, whereas HO-2 protein levels remained unchanged. Lower panel: densitometric values for HO-1 protein levels normalized with ß-actin, and expressed as ratio to control. _* P < 0.05 vs. Control.

Figure 3

Dexamethasone treatment exacerbates the alveolar defects in HO-1 mutant mice. A-F: Representative H&E staining of mouse lung sections at P14 at 5X and 20X magnifications. A-C: Wildtype; D-F: HO-1 -/-. A, D: lungs from untreated animals. B, C, E, F: lungs from Dex-treated animals (P3-P14, 0.25 ug/pup/day). In untreated group, lungs from HO-1-/- animals showed simplified, enlarged, and disorganized alveolar structure (A, D). Postnatal Dex treatment in wildtype animals resulted in alveolar simplification and loss of secondary septation (A, and B, C). Dex treatment in HO-1 -/- animals resulted in more dramatic disruption of the alveolar structure with larger alveolar space, thinning of the alveolar wall, and lack of secondary septation. V: pulmonary vasculature. A: airway. Arrowhead indicates the normal secondary septae. Arrows indicate the elongated and thinning of the alveolar wall. G: Quantification of alveolar development by RAC of the lung sections at P14. _* P < 0.05 vs. wildtype, † P < 0.05 vs. untreated group of same genotype. n = 3-4 for each group.

Figure 4

Expression of lung epithelial and vascular genes in wildtype and HO-1 -/- after dexamethasone treatment. Gene expression of surfactant protein (SP)-A, -B, -C, and D, as well as Flk-1 and Tie-2 was determined by realtime PCR and normalized to 18 S mRNA levels. Data represents relative quantification of mRNA to wildtype littermates at baseline (no treatment). _* P < 0.05 vs. WT, † P < 0.05 vs. untreated group of same genotype. n = 3-4 for each group.