Signaling through the hypoxia inducible factor (HIF)-VEGF-VEGF receptor system (VEGF signaling system) leads to angiogenesis and epithelial cell proliferation and is a key mechanism regulating alveolarization in lungs of newborn rats. Hyperoxia exposure (>95% O2 days 4-14) arrests lung alveolarization and may do so through suppression of the VEGF signaling system. Lung tissue mRNA levels of HIF-2alpha and VEGF increased from days 4-14 in normoxic animals, but hyperoxia suppressed these increases. Levels of HIF-2alpha and VEGF mRNA were correlated in the air but not the O2-treated group, suggesting that the low levels of HIF-2alpha observed at high O2 concentrations are not stimulating VEGF expression. VEGF164 protein levels increased with developmental age, and with hyperoxia to day 9, but continuing hyperoxia decreased levels by day 12. VEGFR1 and VEGFR2 mRNA expression also increased in air-exposed animals, and these, too, were significantly decreased by hyperoxia by day 9 and day 12, respectively. Receptor protein levels did not increase with development; however, O2 did decrease protein to less than air values. Hyperoxic suppression of VEGF signaling from days 9-14 may be one mechanism by which alveolarization is arrested.

We have shown that injury to alveolar epithelial type I cells may account, in part, for damage to the air-blood barrier of the lung in a rat model of Staphylococcus aureus pneumonia. We have also shown that alpha-toxin is an important cause of damage to the air-blood barrier; however, our data suggest that the toxin is not acting directly on alveolar type I cells.

ABTRACT Matrix metalloproteinases (MMP) are likely effectors of normal lung development, especially branching morphogenesis, angiogenesis, and extracellular matrix degradation. Because hyperoxia exposure (Ͼ95% O 2 ) from d 4 to 14 in newborn rat pups leads to arrest of alveolarization and mimics newborn chronic lung disease, we tested whether hyperoxia altered MMP-2 and -9 mRNA, protein, and enzymatic activity, and the mRNA and protein expression of the endogenous tissue inhibitor of MMP, TIMP-1. No changes due to hyperoxia exposure were observed in MMP-2 mRNA or pro-enzyme (72 kD) protein levels between d 6 and 14, although the overall protein mass and zymographic activity of the active (68 kD) enzyme were diminished (p Ͻ 0.05, ANOVA). However, hyperoxia significantly decreased levels of MMP-9 mRNA and pro-MMP-9 protein and diminished overall MMP-9 pro-enzyme activity.TIMP-1 mRNA was not elevated by hyperoxia until d 14, but protein levels were significantly (p Ͻ 0.001) elevated by hyperoxia from d 9 to 14. To estimate the potential of MMP inhibition to arrest alveolarization, administration of doxycycline (20 mg/ kg, twice daily by gavage), a pan-MMP proteolysis inhibitor, arrested lung alveolarization. We conclude that hyperoxia decreases MMP-9 mRNA, protein, and activity and elevates TIMP-1 protein, and these changes have the potential to contribute to the arrest of normal lung development. Lung alveoli are formed when immature saccules subdivide into functional gas-exchange units through formation of secondary septa (1, 2). The process of septation involves budding from the primary septum (saccular wall), formation of a double-capillary network, elongation of septa, coalescence of vessels to form a single capillary layer, and thinning of the septal walls (1, 2). Exposure of neonatal rats to hyperoxia during alveolarization interferes with the process of septation; alveolar number and internal surface area are decreased and the parenchymal airspace is enlarged (3-5). These parameters persist up to d 40 (6). Hyperoxia during the neonatal period also alters lung connective tissue, alters elastic fiber structure and concentration (7), and causes an inflammatory reaction characterized by interalveolar edema and proteinosis (3,5,8). Chronic inflammation of the neonatal lung leads to fibrosis and thickening of the septa (3, 5).MMP are a group of proteases that exist as pro-enzymes and are cleaved by other MMP to active forms that have several specialized functions, including extracellular matrix turnover. Some of their functions regulate processes associated with development, such as branching morphogenesis and angiogenesis as well as inflammatory processes and wound healing (9). MMP-2 and MMP-9, also called gelatinases-A and -B, respectively, cleave gelatin, type IV and V collagen, and elastin. Types IV, V, and VII collagens are associated with basement membranes (10). MMP-2 and MMP-9 exhibit increased gelatinolytic activities as the lung develops (11), and during the postnatal lung growth stage both MMP-2 and MM...

In this study, the authors examined in newborn rat lung tissues the release of leukotriene B(4) (LTB(4)) from tissue explants in vitro, the protein expression of the LT-synthesizing enzyme, 5-lipoxygenase (5-LO), and its activating protein (FLAP), and the effects of in vivo hyperoxic exposure (>95% O(2)) on these parameters. Basal LTB(4) output increased from 0.98 ng/mgDNA/30 min at day 1 to 3.3 ng/mgDNA/30 min at day 28 (P <.05). Exposure of rat pups to >95% O(2) from days 1 to 7 and 60% O(2) from days 7 to 28 stimulated a 1.6-fold (P <.05) increase in LTB(4) output, compared to normoxic pups (to 1.6 ng/mgDNA/30 min) by day 1 and on day 7. The calcium ionophore, A23187, caused an increase in LTB(4) output from both exposure groups, but LTB(4) output was consistently greater (P <.05) from hyperoxia-exposed pups. Western immunoblotting of lung tissue showed that 5-LO and FLAP protein mass increased (P <.05) from days 4 to 14. Hyperoxia exposure increased the mass of both proteins (P <.05). Immunohistochemistry localized 5-LO and FLAP mainly to alveolar macrophages on day 14, but some staining was evident in parenchymal tissue. These data show that hyperoxia increases LTB(4) output, as well as protein levels of 5-LO and FLAP, in newborn rat lungs during early postnatal life. Elevated LTB(4) may contribute to the etiology of newborn lung disease.