Arlene Bulger

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

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.

Endothelin-A receptor blockade prevents and partially reverses neonatal hypoxic pulmonary vascular remodeling

Hypoxia-induced pulmonary vascular remodeling (HPVR) may lead to persistent pulmonary hypertension of the newborn or cor pulmonale. Endothelin-1 (ET-1), via endothelin-A (ETA) receptor activation, mediates hypoxic pulmonary vasoconstriction. Our objectives were to develop a newborn mouse model of HPVR and to test the hypothesis that ETA blockade would prevent and reverse HPVR in this model. C57BL/6 mice (n = 64) were exposed to 12% oxygen (HYP group) or room air (RA group) from birth to 2 wk of age. The mice were injected intraperitoneally daily with either BQ-610 (ETA blocker) or vehicle (cottonseed oil) from birth (prevention study) or from 6 d of age (reversal study). HPVR was assessed histologically by pulmonary vascular morphometry by an examiner masked to study group, and by measurement of the right ventricle to left ventricle (RV/LV) thickness ratio. Hypoxia increased medial wall thickness (%WT) in pulmonary arteries <100 μm in diameter and RV/LV thickness ratio. BQ-610 prevented the hypoxia-induced increase in %WT and RV/LV thickness ratio when given from birth, and later therapy partially reversed the hypoxia-induced increase in %WT but not RV/LV thickness ratio. These data show that in the newborn mouse model, chronic hypoxia leads to HPVR that can be completely prevented and partially reversed by ETA blockade. These results indicate that ET-1, acting via ETA receptors, is a mechanism of pathophysiologic significance underlying neonatal HPVR. Development of this newborn mouse model of HPVR facilitates investigation of mechanisms underlying this important and severe disease entity in human infants.

Loss of Thy-1 inhibits alveolar development in the newborn mouse lung

Transforming growth factor (TGF)-beta mediates hypoxia-induced inhibition of alveolar development in the newborn lung. TGF-beta is regulated primarily at the level of activation of latent TGF-beta. Fibroblasts expressing Thy-1 (CD90) inhibit TGF-beta activation. We hypothesized that loss of Thy-1 due to hypoxia may be a mechanism by which hypoxia increases TGF-beta activation and that animals deficient in Thy-1 will simulate the effects of hypoxia on lung development. To determine if loss of Thy-1 occurred during hypoxia, non-transgenic (C57BL/6) wild-type (WT) mice exposed to hypoxia were evaluated for Thy-1 mRNA and protein. To determine if Thy-1 deficiency simulated hypoxia, WT and Thy-1 null (Thy-1(-/-)) mice were exposed to air or hypoxia from birth to 2 wk, the critical period of lung development, and lung histology, function, parameters related to TGF-beta signaling, and extracellular matrix protein content were measured. To test if the phenotype in Thy-1(-/-) mice was due to excessive TGF-beta signaling, measurements were also performed in Thy-1(-/-) mice administered TGF-beta neutralizing antibody (1D11). We observed that hypoxia reduced Thy-1 mRNA and Thy-1 staining in WT mice. Thy-1(-/-) mice had impaired alveolarization, increased TGF-beta signaling, reduced lung epithelial and endothelial cell proliferation but increased fibroblast proliferation, and increased collagen and elastin. Lung compliance was lower, and tissue but not airway resistance was higher in Thy-1(-/-) mice at 2 wk. Thy-1(-/-) mice given 1D11 had improved alveolar development and lung function. These data support the hypothesis that hypoxia, by reducing Thy-1, increases TGF-beta activation, and thereby inhibits normal alveolar development.

Role of nitric oxide in regulating neonatal porcine pulmonary artery smooth muscle cell proliferation

Nitric oxide (NO), which is known to inhibit systemic vascular smooth muscle cell proliferation, is used in the management of neonatal pulmonary hypertension. Our objectives were to determine: (1) if endogenous NO production by neonatal porcine pulmonary artery smooth muscle cells (PASMCs) varied with oxygen tension in vitro, and (2) the effect of exogenous NO and inducible NO synthase (iNOS) stimulators and inhibitors on PASMC proliferation and apoptosis. PASMCs were exposed to different conditions (varying PO2, NO donors and scavengers, iNOS stimulators and inhibitors) and proliferation, apoptosis, and cyclic guanosine 5′-monophosphate (cGMP) assessed. PASMCs proliferated best between 5 and 10% O2 but cGMP levels were similar at all oxygen levels. NO donors (S-nitroso-N-acetyl-penicillamine, NOC-12, NOC-18) inhibited PASMC proliferation in a dose-dependent manner with associated cGMP increases, while NO scavengers (carboxy-PTIO), iNOS stimulators (interleukin-1β, lipopolysaccharide), and iNOS inhibitors (aminoethylisothiourea) did not affect proliferation or cGMP. No changes in apoptosis were found at the concentrations of NO donors or iNOS stimulators used. These results suggest that while exogenous NO inhibits PASMC proliferation, endogenous NO may not regulate proliferation during changes in oxygen tension or cytokine levels. Endothelial derived and inhaled NO may attenuate smooth muscle hyperplasia and vascular remodeling. Inducible NOS in porcine PASMCs appears resistant to stimulation with interleukin-1β or lipopolysaccharide. The mechanisms underlying hypoxia-mediated changes in PASMC proliferation require investigation.

Role of Matrix Metalloproteinase-2 in Newborn Mouse Lungs under Hypoxic Conditions

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.

Hypoxia-induced inhibition of lung development is attenuated by the peroxisome proliferator-activated receptor-γ agonist rosiglitazone

Hypoxia enhances transforming growth factor-β (TGF-β) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-β signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-β signaling, and collagen content were made. To determine whether excessive TGF-β signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-β-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-β signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-β signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-β signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-β signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-β signaling and improving alveolar development.

Vitamin A and retinoic acid act synergistically to increase lung retinyl esters during normoxia and reduce hyperoxic lung injury in newborn mice.

We have shown that vitamin A (VA) and retinoic acid (RA) synergistically increase lung retinyl ester content in neonatal rats. To confirm whether this biochemical synergism attenuates early neonatal hyperoxic lung injury in mice, we exposed newborn C57BL/6 mice to 95% O2 or air from birth to 4 d. The agent [vehicle, VA, RA, or the combination vitamin A + retinoic acid (VARA)] was given orally daily. Lung and liver retinyl ester content was measured, and lung injury and development were evaluated. We observed that lung, but not liver, retinyl ester levels were increased more by VARA than by VA or RA alone. Hyperoxic lung injury was reduced by VA and RA, and more so by VARA. VARA attenuated the hyperoxia-induced increases in macrophage inflammatory protein (MIP)-2 mRNA and protein expression, but did not alter hyperoxia-induced effects on peptide growth factors (PDGF, VEGF, and TGF-β1). The 4-d exposure to hyperoxia or retinoids did not lead to observable differences in lung development. We conclude that the VARA combination has synergistic effects on lung retinyl ester concentrations and on the attenuation of hyperoxia-induced lung injury in newborn mice, possibly by modulation of inflammatory mediators.

Neurodevelopmental impairment following neonatal hyperoxia in the mouse.

Extremely premature infants are often exposed to supra-physiologic concentrations of oxygen, and frequently have hypoxemic episodes. These preterm infants are at high risk (~40%) for neurodevelopmental impairment (NDI) even in the absence of obvious intracranial pathology such as intraventricular hemorrhage or periventricular leukomalacia. The etiology for NDI has not been determined, and there are no animal models to simulate neurodevelopmental outcomes of prematurity. Our objectives were to develop and characterize a mouse model to determine long-term effects of chronic hypoxia or hyperoxia exposure on neurodevelopment. Newborn C57BL/6 mice were exposed to hypoxia (12% O(2)) or hyperoxia (85% O(2)) from postnatal days 1 to 14 and then returned to air. At 12-14 weeks of age, neurobehavioral assessment (Water Maze test, Novel Object Recognition test, Open Field test, Elevated Plus Maze, and Rotarod test) was performed, followed by MRI and brain histology. Neurobehavioral testing revealed that hyperoxia-exposed mice did poorly on the water maze and novel object recognition tests compared to air-exposed mice. MRI demonstrated smaller hippocampi in hyperoxia- and hypoxia-exposed mice with a greater reduction in hyperoxia-exposed mice, including a smaller cerebellum in hyperoxia-exposed mice. Brain histology showed reduced CA1 and CA3 and increased dentate gyral width in hippocampus. In conclusion, neonatal hyperoxia in mice leads to abnormal neurobehavior, primarily deficits in spatial and recognition memory, associated with smaller hippocampal sizes, similar to findings in ex-preterm infants. This animal model may be useful to determine mechanisms underlying developmental programming of NDI in preterm infants, and for evaluation of therapeutic strategies.

Endothelin-1 mediates hypoxia-induced increases in vascular collagen in the newborn mouse lung.

Endothelin-1 (ET-1) mediates hypoxia-mediated pulmonary vascular remodeling (HPVR), and endothelin-A receptor (ET-AR) blockade prevents HPVR in newborn mice. Our objective was to determine postnatal effects of chronic hypoxia and/or ET-AR blockade on lung ET-1, ET-AR, ET-BR, and vascular collagen and elastin. Newborn C57BL/6 mice (n = 6–8/gp) given either BQ610 (ET-AR blocker) or vehicle were exposed to air or hypoxia (12% O2) from birth for 1, 3, or 14 d. Lung ET-1 was assessed by ELISA, and ET-AR and ET-BR by immunohistochemistry. Vascular collagen and elastin were assessed by quantitative image analysis. ET-1, ET-AR, ET-BR, collagen I and III, and tropoelastin mRNA levels were assessed by real-time quantitative RT-PCR. We observed that: 1) hypoxia attenuated the normal postnatal decrease in ET-1 and collagen content; 2) ET-AR blockade reduced collagen independent of O2; 3) hypoxia increased elastin mRNA expression and attenuated the normal postnatal decrease in elastin content; and 4) BQ610 reduced elastin mRNA but not elastin content. We conclude that, in neonatal mice, hypoxia attenuates normal postnatal decreases in ET-1, vascular collagen, and elastin. ET-AR blockade reduces collagen fiber area but not mRNA, and does not decrease elastin despite reducing its expression.

Titanium oxide nanoparticle instillation induces inflammation and inhibits lung development in mice

Nanoparticles are used in an increasing number of biomedical, industrial, and food applications, but their safety profiles in developing organisms, including the human fetus and infant, have not been evaluated. Titanium oxide (TiO(2)) nanoparticles, which are commonly used in cosmetics, sunscreens, paints, and food, have been shown to induce emphysema and lung inflammation in adult mice. We hypothesized that exposure of newborn mice to TiO(2) would induce lung inflammation and inhibit lung development. C57BL/6 mice were exposed to TiO(2) (anatase; 8-10 nm) nanoparticles by intranasal instillation as a single dose on postnatal day 4 (P4) or as three doses on postnatal days 4, 7, and 10 (each dose = 1 μg/g body wt). Measurements of lung function (compliance and resistance), development (morphometry), inflammation (histology; multiplex analysis of bronchoalveolar lavage fluid for cytokines; PCR array and multiplex analysis of lung homogenates for cytokines) was performed on postnatal day 14. It was observed that a single dose of TiO(2) nanoparticles led to inflammatory cell influx, and multiple doses led to increased inflammation and inhibition of lung development without significant effects on lung function. Macrophages were noted to take up the TiO(2) nanoparticles, followed by polymorphonuclear infiltrate. Multiple cytokines and matrix metalloproteinase-9 were increased in lung homogenates, and VEGF was reduced. These results suggest that exposure of the developing lung to nanoparticles may lead to ineffective clearance by macrophages and persistent inflammation with resulting effects on lung development and may possibly impact the risk of respiratory disorders in later life.