Role of matrix metalloproteinase-2 in newborn mouse lungs under hypoxic conditions

Abstract

Hypoxia impairs normal neonatal pulmonary artery remodeling and alveolar development. Matrix metalloproteinase-2 (MMP-2), which regulates collagen breakdown, is important during development. Our objective was to test the hypothesis that hypoxia attenuates the normal postnatal increase in MMP-2 and evaluate alveolar development and pulmonary arterial remodeling in Mmp2 mice. C57BL/6 wild-type (WT), Mmp2, Mmp2, and MMP-inhibited (with doxycycline) mice were exposed to hypoxia (12% O2) or air from birth to 2 wk of age. Pulmonary arterial remodeling, alveolar development, and vascular collagen and elastin were evaluated. MMP-2 was estimated by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemistry, and zymography. We observed that 1) in WT mice, hypoxia led to thicker-walled pulmonary arteries and impaired alveolarization, accompanied by decreased MMP-2 and increased tissue inhibitor of metalloproteinases-2 (TIMP-2); 2) Mmp2 mice in air had thicker-walled arteries, impaired alveolarization, and increased perivascular collagen and elastin compared with WT; 3) hypoxia further inhibited alveolarization but did not alter arterial thickening in Mmp2 mice. Mmp2 and MMP-inhibited mice also had thicker-walled arteries than WT in air, but alveolarization was not different. We conclude that hypoxia reduces the postnatal MMP-2 increase in the lung, which may contribute to abnormal pulmonary arterial remodeling and impaired alveolarization.

Figure 1

MMP-2 in the developing postnatal WT mouse lung and the effects of hypoxia (12% O₂). (A, B) Immunohistochemical staining for proMMP-2 (brown) in lung sections from 14 d mice exposed to (A) air or (B) hypoxia (magnification, ×400; calibration bars = 50 μm). (C) Pro-MMP-2 staining quantitation as percent of tissue area (n = 6 mice/gp; mean ± SE; *p < 0.001). (D) Zymogram of lung extracts from mice exposed to air or hypoxia for 1, 3, and 14 d from birth. (E) Total MMP-2 measured by ELISA from lung extracts of mice exposed to air or hypoxia (Hyp) from 1 d for up to 14 d (n = 6 mice/gp; mean ± SE, *p < 0.05 vs air at same time point). (F) Active MMP-2 measured by ELISA from same samples as E (n = 6 mice/gp; mean ± SE, *p < 0.05 vs air at same time point). (G) Mmp2 mRNA by competitive real-time PCR in homogenized lungs from mice exposed to air or hypoxia from 1 d for up to 14 d (n = 6 mice/gp; mean ± SE, *p < 0.05 vs air at same time point).

Figure 2

TIMP-2 in the developing postnatal WT mouse lung and the effects of hypoxia. (A) Reverse zymogram for TIMPs in lung extracts from 14 d mice exposed to air or hypoxia from birth. (B) Densitometric quantitation of reverse zymograms for TIMP-2 from lung extracts of mice exposed to air or hypoxia (Hyp) from 1 d up to 14 d (n = 6 mice/gp; mean ± SE, *p < 0.05 vs air at same time point). (C) Timp2 mRNA by competitive real-time PCR in homogenized lungs from mice exposed to air or hypoxia from 1 d up to 14 d (n = 6 mice/gp; mean ± SE, *p < 0.05 vs air at same time point).

Figure 3

MMP-2 and alveolar development in WT and Mmp2^−/− mice. (A–D) Representative photomicrographs of mouse lungs from WT (A, B) and Mmp2^−/− mice (C, D) after 14 d of air (A, C) or hypoxia (B, D) exposure (magnification, ×100; calibration bars = 250 μm). Alveoli are small and well developed with many secondary crests in WT air mice (A), large with fewer secondary crests in WT hypoxia mice (B) and Mmp2^−/− air (C) mice, and are much larger with minimal septation in Mmp2^−/− hypoxic mice (D). (E) MLI and (F) RAC at 14 d of age in WT (+/+) mice given either vehicle or doxycycline (Dox), Mmp2^+/−, and Mmp2^−/− mice after air and hypoxic (Hyp) exposure (mean ± SE; n = 6 mice/gp; *p < 0.05 vs corresponding WT controls, §p < 0.05 vs corresponding air). (G) MLI in WT and Mmp2^−/− mice at 1 d, 14 d, and 8 –12 wk of age (mean ± SE; n = 6 mice/gp; *p < 0.05 vs WT at same time point).

Figure 4

MMP-2 and pulmonary arterial remodeling in WT and Mmp2^−/− mice. (A–D) Representative photomicrographs of mouse lung pulmonary arteries from WT (A, B) and Mmp2^−/− mice (C, D) after 14 d of air (A, C) or hypoxic (B, D) exposure (magnification, ×400; calibration bars = 50 μm; PA, pulmonary artery; Br, bronchus). The wall thickness of the pulmonary artery is less in the WT air mice (A) than in WT hypoxia (B), Mmp2^−/− air (C), or Mmp2^−/− hypoxic mice (D). (E) Wall thickness (%) of pulmonary arteries and (F) right ventricle/left ventricle (RV/LV) thickness ratio at 14 d of age in WT mice given either vehicle or doxycycline (Dox), Mmp2^+/−, and Mmp2^−/− mice after air and hypoxic exposure (mean ± SE; n = 6 mice/gp; *p < 0.05 vs corresponding WT controls, §p < 0.05 vs corresponding air). (G) Wall thickness (%) in air exposed WT and Mmp2^−/− mice at 1 d, 14 d, and 8 –12 wk of age (mean ± SE; n = 120 vessels/gp; *p < 0.05 vs WT at same time point).

Figure 5

Perivascular collagen in 14 d WT and Mmp2^−/− mice. (A–D) Representative photomicrographs of picric acid-Sirius red stained sections (collagen is stained red) of pulmonary arteries from WT (A, B) and Mmp2^−/− mice (C, D) after 14 d of air (A, C) or hypoxic (B, D) exposure (magnification, ×400; calibration bars = 50 μm; PA, pulmonary artery; Br, bronchus). Collagen in the perivascular area is least in the WT air mice (A), increased in WT hypoxia (B), and further increased in Mmp2^−/− air (C) and in Mmp2^−/− hypoxic mice (D). (E) Collagen area measured by quantitative image analysis at 14 d of age in WT and Mmp2^−/− mice after air and hypoxic exposure (mean ± SE; n = 6 mice/gp; *p < 0.05 vs corresponding WT controls, §p < 0.05 vs corresponding air).

Figure 6

Vascular elastin in 14 d WT and Mmp2^−/− mice. (A–D) Representative photomicrographs of elastic tissue stained sections (elastin is stained black) of pulmonary arteries from WT (A, B) and Mmp2^−/− mice (C, D) after 14 d of air (A, C) or hypoxia (B, D) exposure (magnification, ×400; calibration bars = 50 μm; PA, pulmonary artery; Br, bronchus). Elastin is least in the WT air mice (A), increased in WT hypoxia (B), further increased in Mmp2^−/− air (C), and is maximal in Mmp2^−/− hypoxic mice (D). (E) Elastin area measured by quantitative image analysis at 14 d of age in WT and Mmp2^−/− mice after air and hypoxic exposure (mean ± SE; n = 6 mice/gp; *p < 0.05 vs corresponding WT controls, §p < 0.05 vs corresponding air).