Transforming growth factor-beta signaling mediates hypoxia-induced pulmonary arterial remodeling and inhibition of alveolar development in newborn mouse lung

Abstract

Hypoxia causes abnormal neonatal pulmonary artery remodeling (PAR) and inhibition of alveolar development (IAD). Transforming growth factor (TGF)-beta is an important regulator of lung development and repair from injury. We tested the hypothesis that inhibition of TGF-beta signaling attenuates hypoxia-induced PAR and IAD. Mice with an inducible dominant-negative mutation of the TGF-beta type II receptor (DNTGFbetaRII) and nontransgenic wild-type (WT) mice were exposed to hypoxia (12% O(2)) or air from birth to 14 days of age. Expression of DNTGFbetaRII was induced by 20 microg/g ZnSO(4) given intraperitoneally daily from birth. PAR, IAD, cell proliferation, and expression of extracellular matrix (ECM) proteins were assessed. In WT mice, hypoxia led to thicker, more muscularized resistance pulmonary arteries and impaired alveolarization, accompanied by increases in active TGF-beta and phosphorylated Smad2. Hypoxia-induced PAR and IAD were greatly attenuated in DNTGFbetaRII mice given ZnSO(4) compared with WT control mice and DNTGFbetaRII mice not given ZnSO(4). The stimulatory effects of hypoxic exposure on pulmonary arterial cell proliferation and lung ECM proteins were abrogated in DNTGFbetaRII mice given ZnSO(4). These data support the conclusion that TGF-beta plays an important role in hypoxia-induced pulmonary vascular adaptation and IAD in the newborn animal model.

Fig. 1.

Hypoxic exposure increases active transforming growth factor (TGF)-β in newborn lung. A and B: representative photomicrographs of mouse lung sections from wild-type (WT) mice exposed to air (A) or hypoxia (B) for 14 days and stained with an antibody recognizing active TGF-β1 (red) (×400). C: levels of active TGF-β1 measured by ELISA from lung extracts of mice exposed to air or hypoxia from birth to 14 days of age (n = 6 mice/group; means ± SE) *P < 0.05 vs. air.

Fig. 2.

Administration of ZnSO₄ (zinc) but not 0.9% saline induces mRNA expression of DNTGFβRII in lungs of mouse pups, as demonstrated by RT-PCR. Each lane represents a sample from a different mouse pup.

Fig. 3.

Phosphorylated (p)Smad2 immunohistochemical staining and quantitation by Western blots in DNTGFβRII mouse pups at 14 days of age. A–D: representative photomicrographs of a resistance pulmonary artery (PA) and a bronchus (Br) from DNTGFβRII mouse pups given either saline (A, B) or zinc (C, D) during 14 days of air (A, C) or hypoxia (B, D) exposure (×400; calibration bars = 50 μm). Nuclear staining of pSmad2, indicating presence of TGF-β signaling, was increased in hypoxia-exposed DNTGFβRII mouse pups given saline (B) and reduced in DNTGFβRII pups that received zinc (C, D). E: representative Western blots of lung homogenates for pSmad2, Smad2, and β-tubulin from DNTGFβRII mouse pups given either saline or zinc during 14 days of air or hypoxia exposure. F and G: pSmad2-to-β-tubulin ratio (F) and pSmad2-to-Smad2 ratio (G) in Western blots quantitated by densitometry (means ± SE; n = 6 mice/group). *P < 0.05 vs. corresponding air; #P < 0.05 vs. corresponding saline.

Fig. 4.

Pulmonary arterial remodeling and right (RV)-to-left ventricle (LV) ratios in DNTGFβRII mouse pups at 14 days of age. A–D: representative photomicrographs of a resistance pulmonary artery (PA) and a bronchus (Br) from DNTGFβRII mouse pups given either saline (A, B) or zinc (C, D) during 14 days of air (A, C) or hypoxia (B, D) exposure (×400; calibration bars = 50 μm). In mouse pups given saline, pulmonary arterial wall thickness (arrows) is increased by hypoxia (B) compared with air (A). Administration of zinc significantly attenuates the hypoxia-induced increase in wall thickness (D) but does not change wall thickness of air-exposed animals (C). E and F: wall thickness (%) of pulmonary arteries (E) and RV-to-LV thickness ratio (F) at 14 days of age in DNTGFβRII mouse pups given either saline or zinc while being exposed to air or hypoxia (means ± SE; n = 6 mice/group). *P < 0.05 vs. corresponding air; #P < 0.05 vs. corresponding saline.

Fig. 5.

Pulmonary arterial muscularization as evaluated by α-smooth muscle actin (α-SMA) staining in DNTGFβRII mouse pups at 14 days of age. A–H: representative photomicrographs of lung at low power (×100; A, C, E, G) and a resistance pulmonary artery (arrows) in high-power images (×400; B, D, F, H) from DNTGFβRII mouse pups given either saline (A–D) or zinc (E–H) during 14 days of air (A, B, E, F) or hypoxia (C, D, G, H) exposure (calibration bars = 250 μm in ×100 images and 50 μm in ×400 images). In mouse pups given saline, hypoxia increases α-SMA staining diffusely in the lung (C) and in resistance pulmonary arteries (D) compared with air (A, B). Small pulmonary arterioles (arrowhead) are also muscularized in the hypoxia-saline group (D). Administration of zinc significantly attenuates the hypoxia-induced increase in α-SMA staining in the lung (G) and in pulmonary arteries (H) but does not change staining in air-exposed animals (E, F). I–K: quantitation of muscularization of pulmonary arteries of all pulmonary arteries 20–150 μm (I), smaller (20–50 μm) pulmonary arteries (J), and larger (51–150 μm) pulmonary arteries (K) in DNTGFβRII mouse pups given either saline or zinc while being exposed to air or hypoxia demonstrates that hypoxia increases α-SMA staining of resistance pulmonary arteries, which is attenuated in animals given zinc (means ± SE; n = 80 arteries/group). *P < 0.05 vs. corresponding air; #P < 0.05 vs. corresponding saline.

Fig. 6.

Alveolar development in DNTGFβRII mouse pups at 14 days of age. A–D: representative photomicrographs of hematoxylin and eosin-stained sections of lungs from DNTGFβRII mouse pups given either saline (A, B) or zinc (C, D) during 14 days of air (A, C) or hypoxia (B, D) exposure (×100; calibration bars = 250 μm). In mouse pups given saline, alveolar size is larger in hypoxia-exposed (B) compared with air-exposed (A) mice, indicating delay in septation. Administration of zinc significantly attenuates the hypoxia-induced increase in alveolar size (D) but does not change alveoli of air-exposed animals (C). E and F: mean linear intercept (E) and radial alveolar count (F) at 14 days of age in DNTGFβRII mouse pups given either saline or zinc while being exposed to air or hypoxia (means ± SE; n = 6 mice/group). *P < 0.05 vs. corresponding air; #P < 0.05 vs. corresponding saline.

Fig. 7.

Cell proliferation in DNTGFβRII mouse pups at 14 days of age as determined by Ki67 staining. A–D: representative photomicrographs of Ki67-stained sections of lungs from DNTGFβRII mouse pups given either saline (A, B) or zinc (C, D) during 14 days of air (A, C) or hypoxia (B, D) exposure (×400; calibration bars = 50 μm). In mouse pups given saline, increased numbers of stained nuclei are seen in walls of pulmonary arteries (PA) in hypoxia-exposed (B) compared with air-exposed (A) mice, indicating increased vascular cell proliferation. Administration of zinc significantly attenuates the hypoxia-induced increase in vascular cell proliferation (D). E and F: quantitation of Ki67 staining in pulmonary arterial wall, expressed as number of Ki67⁺ nuclei per 1,000 μm² of vessel wall (E) and quantitation of Ki67 staining in alveoli, expressed as number of Ki67⁺ nuclei per 50,000 μm² of lung cross-sectional area (F) at 14 days of age in DNTGFβRII mouse pups given either saline or zinc while being exposed to air or hypoxia (means ± SE; n = 6 mice/group). *P < 0.05 vs. corresponding air; #P < 0.05 vs. corresponding saline.

Fig. 8.

Collagen, tropoelastin, lysyl oxidase, tenascin-C, and fibronectin mRNA levels measured by real-time quantitative RT-PCR in homogenized lungs from mice exposed to air or hypoxia from birth to 7 days (n = 6 mice/group; means ± SE). *P < 0.05 vs. corresponding air at same time point; #P < 0.05 vs. corresponding saline.

Fig. 9.

Collagen 1, c-fos, α-myosin heavy chain (MHC), and β-MHC mRNA levels measured by real-time quantitative RT-PCR in homogenized RV of mice exposed to air or hypoxia from birth to 7 days (n = 12 mice/group; means ± SE). *P < 0.05 vs. corresponding air at same time point; #P < 0.05 vs. corresponding saline.