CFTR dysfunction increases endoglin and TGF-ß signaling in airway epithelia.

Endoglin (ENG) regulates signaling by transforming growth factor-ß (TGF-ß), a genetic modifier of cystic fibrosis (CF) lung disease severity. We hypothesized that ENG mediates TGF-ß pathobiology in CF airway epithelia. Comparing CF and non-CF human lungs, we measured ENG by qPCR, immunoblotting and ELISA. In human bronchial epithelial cell lines (16HBE), we used CFTR siRNA knockdown and functional inhibition (CFTRINH -172) to connect loss of CFTR to ENG synthesis. Plasmid overexpression of ENG assessed the direct effect of ENG on TGF-ß transcription and signal amplification in 16HBE cells. We found ENG protein to be increased more than fivefold both in human CF bronchoalveolar fluid (BALF) and human CF lung homogenates. ENG transcripts were increased threefold in CF, with a twofold increase in TGF-ß signaling. CFTR knockdown in 16HBE cells tripled ENG transcription and doubled protein levels with corresponding increases in TGF-ß signaling. Plasmid overexpression of ENG alone nearly doubled TGF-ß1 mRNA and increased TGF-ß signaling in 16HBE cells. These experiments identify that loss of CFTR function increases ENG expression in CF epithelia and amplifies TGF-ß signaling. Targeting ENG may offer a novel therapeutic opportunity to address TGF-ß associated pathobiology in CF.

Hyperoxia Induces Intracellular Acidification in Neonatal Mouse Lung Fibroblasts: Real-Time Investigation Using Plasmonically Enhanced Raman Spectroscopy.

It is important to understand the molecular mechanisms underlying oxygen toxicity, which contributes to multiple human disorders. The archetype model of oxygen toxicity is neonatal lung injury induced by hyperoxia exposure. Here, we utilized plasmonically enhanced Raman spectroscopy (PERS) in combination with fluorescence and proteomic analysis to provide comprehensive information on hyperoxia-induced biomolecular modifications in neonatal mouse lung fibroblasts (nMLFs). During this study, we made the novel observation that hyperoxia induces intracellular acidification in nMLF, which we probed in real-time using label-free PERS. We found that intracellular acidification induces conformational modifications in proteins followed by significant changes in Raman vibrations corresponding to aromatic amino acids such as phenylalanine and tryptophan as well as cysteine moieties. Hyperoxia-induced intracellular pH changes and subsequent modifications in protein expression and associated post-translational modifications within the cells were further validated by fluorescence and proteomic analysis. These new insights may help identifying unique oxidant stress-induced mechanisms in disease processes and may guide the development of more efficient therapeutic strategies.

VARA attenuates hyperoxia-induced impaired alveolar development and lung function in newborn mice.

We have recently shown that a combination of vitamin A (VA) and retinoic acid (RA) in a 10:1 molar ratio (VARA) synergistically increases lung retinoid content in newborn rodents, more than either VA or RA alone in equimolar amounts. We hypothesized that the increase in lung retinoids would reduce oxidative stress and proinflammatory cytokines, resulting in attenuation of alveolar simplification and abnormal lung function in hyperoxia-exposed newborn mice. Newborn C57BL/6 mice were exposed to 85% O2 (hyperoxia) or air (normoxia) for 7 or 14 days from birth and given vehicle or VARA every other day. Lung retinol content was measured by HPLC, function was assessed by flexiVent, and development was evaluated by radial alveolar counts, mean linear intercept, and secondary septal crest density. Mediators of oxidative stress, inflammation, and alveolar development were evaluated in lung homogenates. We observed that VARA increased lung retinol stores and attenuated hyperoxia-induced alveolar simplification while increasing lung compliance and lowering resistance. VARA attenuated hyperoxia-induced increases in DNA damage and protein oxidation accompanied with a reduction in nuclear factor (erythroid-derived 2)-like 2 protein but did not alter malondialdehyde adducts, nitrotyrosine, or myeloperoxidase concentrations. Interferon-γ and macrophage inflammatory protein-2α mRNA and protein increased with hyperoxia, and this increase was attenuated by VARA. Our study suggests that the VARA combination may be a potential therapeutic strategy in conditions characterized by VA deficiency and hyperoxia-induced lung injury during lung development, such as bronchopulmonary dysplasia in preterm infants.

Transforming growth factor-ß regulates endothelin-1 signaling in the newborn mouse lung during hypoxia exposure.

We have previously shown that inhibition of transforming growth factor-ß (TGF-ß) signaling attenuates hypoxia-induced inhibition of alveolar development and abnormal pulmonary vascular remodeling in the newborn mice and that endothelin-A receptor (ETAR) antagonists prevent and reverse the vascular remodeling. The current study tested the hypothesis that inhibition of TGF-ß signaling attenuates endothelin-1 (ET-1) expression and thereby reduces effects of hypoxia on the newborn lung. C57BL/6 mice were exposed from birth to 2 wk of age to either air or hypoxia (12% O(2)) while being given either BQ610 (ETAR antagonist), BQ788 (ETBR antagonist), 1D11 (TGF-ß neutralizing antibody), or vehicle. Lung function and development and TGF-ß and ET-1 synthesis were assessed. Hypoxia inhibited alveolar development, decreased lung compliance, and increased lung resistance. These effects were associated with increased TGF-ß synthesis and signaling and increased ET-1 synthesis. BQ610 (but not BQ788) improved lung function, without altering alveolar development or increased TGF-ß signaling in hypoxia-exposed animals. Inhibition of TGF-ß signaling reduced ET-1 in vivo, which was confirmed in vitro in mouse pulmonary endothelial, fibroblast, and epithelial cells. ETAR blockade improves function but not development of the hypoxic newborn lung. Reduction of ET-1 via inhibition of TGF-ß signaling indicates that TGF-ß is upstream of ET-1 during hypoxia-induced signaling in the newborn lung.

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

Hypoxia enhances transforming growth factor-ß (TGF-ß) 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-ß 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-ß signaling, and collagen content were made. To determine whether excessive TGF-ß signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-ß-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-ß 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-ß signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-ß 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-ß signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-ß 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-beta1). 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.

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.