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2.  Wnt Coreceptor Lrp5 Is a Driver of Idiopathic Pulmonary Fibrosis 
Rationale: Wnt/β-catenin signaling has been implicated in lung fibrosis, but how this occurs and whether expression changes in Wnt pathway components predict disease progression is unknown.
Objectives: To determine whether the Wnt coreceptor Lrp5 drives pulmonary fibrosis in mice and is predictive of disease severity in humans.
Methods: We examined mice with impaired Wnt signaling caused by loss of the Wnt coreceptor Lrp5 in models of lung fibrosis induced by bleomycin or an adenovirus encoding an active form of transforming growth factor (TGF)-β. We also analyzed gene expression in peripheral blood mononuclear cells (PBMC) from patients with idiopathic pulmonary fibrosis (IPF).
Measurements and Main Results: In patients with IPF, analysis of peripheral blood mononuclear cells revealed that elevation of positive regulators, Lrp5 and 6, was independently associated with disease progression. LRP5 was also associated with disease severity at presentation in an additional cohort of patients with IPF. Lrp5 null mice were protected against bleomycin-induced pulmonary fibrosis, an effect that was phenocopied by direct inhibition of β-catenin signaling by the small molecular inhibitor of β-catenin responsive transcription. Transplantation of Lrp5 null bone marrow cells into wild-type mice did not limit fibrosis. Instead, Lrp5 loss was associated with reduced TGF-β production by alveolar type 2 cells and leukocytes. Consistent with a role of Lrp5 in the activation of TGF-β, Lrp5 null mice were not protected against lung fibrosis induced by TGF-β.
Conclusions: We show that the Wnt coreceptor, Lrp5, is a genetic driver of lung fibrosis in mice and a marker of disease progression and severity in humans with IPF. Evidence that TGF-β signaling can override a loss in Lrp5 has implications for patient selection and timing of Wnt pathway inhibitors in lung fibrosis.
PMCID: PMC4226053  PMID: 24921217
lung fibrosis; Wnt/β-catenin signaling; peripheral blood mononuclear cell
6.  Leptin Promotes Fibroproliferative Acute Respiratory Distress Syndrome by Inhibiting Peroxisome Proliferator–activated Receptor-γ 
Rationale: Diabetic patients have a lower incidence of acute respiratory distress syndrome (ARDS), and those who develop ARDS are less likely to die. The mechanisms that underlie this protection are unknown.
Objectives: To determine whether leptin resistance, a feature of diabetes, prevents fibroproliferation after lung injury.
Methods: We examined lung injury and fibroproliferation after the intratracheal instillation of bleomycin in wild-type and leptin-resistant (db/db) diabetic mice. We examined the effect of leptin on transforming growth factor (TGF)-β1–mediated transcription in primary normal human lung fibroblasts. Bronchoalveolar lavage fluid (BAL) samples from patients with ARDS and ventilated control subjects were obtained for measurement of leptin and active TGF-β1 levels.
Measurements and Main Results: Diabetic mice (db/db) were resistant to lung fibrosis. The db/db mice had higher levels of peroxisome proliferator–activated receptor-γ (PPARγ), an inhibitor of the transcriptional response to TGF-β1, a cytokine critical in the pathogenesis of fibroproliferative ARDS. In normal human lung fibroblasts, leptin augmented the transcription of profibrotic genes in response to TGF-β1 through a mechanism that required PPARγ. In patients with ARDS, BAL leptin levels were elevated and correlated with TGF-β1 levels. Overall, there was no significant relationship between BAL leptin levels and clinical outcomes; however, in nonobese patients, higher BAL leptin levels were associated with fewer intensive care unit– and ventilator-free days and higher mortality.
Conclusions: Leptin signaling is required for bleomycin-induced lung fibrosis. Leptin augments TGF-β1 signaling in lung fibroblasts by inhibiting PPARγ. These findings provide a mechanism for the observed protection against ARDS observed in diabetic patients.
PMCID: PMC3266063  PMID: 21317313
acute lung injury; fibrosis; lung; diabetes mellitus
7.  Epithelial Cell Death Is an Important Contributor to Oxidant-mediated Acute Lung Injury 
Rationale: Acute lung injury and the acute respiratory distress syndrome are characterized by increased lung oxidant stress and apoptotic cell death. The contribution of epithelial cell apoptosis to the development of lung injury is unknown.
Objectives: To determine whether oxidant-mediated activation of the intrinsic or extrinsic apoptotic pathway contributes to the development of acute lung injury.
Methods: Exposure of tissue-specific or global knockout mice or cells lacking critical components of the apoptotic pathway to hyperoxia, a well-established mouse model of oxidant-induced lung injury, for measurement of cell death, lung injury, and survival.
Measurements and Main Results: We found that the overexpression of SOD2 prevents hyperoxia-induced BAX activation and cell death in primary alveolar epithelial cells and prolongs the survival of mice exposed to hyperoxia. The conditional loss of BAX and BAK in the lung epithelium prevented hyperoxia-induced cell death in alveolar epithelial cells, ameliorated hyperoxia-induced lung injury, and prolonged survival in mice. By contrast, Cyclophilin D–deficient mice were not protected from hyperoxia, indicating that opening of the mitochondrial permeability transition pore is dispensable for hyperoxia-induced lung injury. Mice globally deficient in the BH3-only proteins BIM, BID, PUMA, or NOXA, which are proximal upstream regulators of BAX and BAK, were not protected against hyperoxia-induced lung injury suggesting redundancy of these proteins in the activation of BAX or BAK.
Conclusions: Mitochondrial oxidant generation initiates BAX- or BAK-dependent alveolar epithelial cell death, which contributes to hyperoxia-induced lung injury.
PMCID: PMC3086743  PMID: 20959557
cell death; epithelium; Bcl-2 proteins; acute respiratory distress syndrome
9.  Electroporation-mediated Gene Transfer of the Na+,K+-ATPase Rescues Endotoxin-induced Lung Injury 
Rationale: Acute lung injury and acute respiratory distress syndrome are common clinical syndromes resulting largely from the accumulation of and inability to clear pulmonary edema, due to injury to the alveolar epithelium. Gene therapy may represent an important alternative for the treatment and prevention of these diseases by restoring alveolar epithelial function. We have recently developed an electroporation strategy to transfer genes to the lungs of mice, with high efficiency and low inflammation.
Objectives: We asked whether electroporation-mediated transfer of genes encoding subunits of the Na+,K+-ATPase could protect from LPS-induced lung injury or be used to treat already injured lungs by up-regulating mechanisms of pulmonary edema clearance.
Methods: Plasmids were delivered to the lungs of mice using transthoracic electroporation. Lung injury was induced by intratracheal administration of LPS (4 mg/kg body weight). Biochemical, cellular, and physiologic measurements were taken to assess gene transfer and lung injury.
Measurements and Main Results: Improvements in wet-to-dry ratios, pulmonary effusions, bronchoalveolar lavage protein levels and cellularity, alveolar fluid clearance, and respiratory mechanics were seen after delivery of plasmids expressing Na+,K+-ATPase subunits, but not control plasmids, in LPS-injured lungs. Delivery of plasmids expressing Na+,K+-ATPase subunits both protected from subsequent lung injury and partially reversed existing lung injury by these measures.
Conclusions: These results demonstrate that electroporation can be used effectively in healthy and injured lungs to facilitate gene delivery and expression. To our knowledge, this is the first successful use of gene delivery to treat existing lung injury, and may have future clinical potential.
PMCID: PMC1994223  PMID: 17556717
acute lung injury; alveolar fluid clearance; electroporation; gene therapy; pulmonary edema
10.  Leptin Resistance Protects Mice from Hyperoxia-induced Acute Lung Injury 
Rationale: Human data suggest that the incidence of acute lung injury is reduced in patients with type II diabetes mellitus. However, the mechanisms by which diabetes confers protection from lung injury are unknown.
Objectives: To determine whether leptin resistance, which is seen in humans with diabetes, protects mice from hyperoxic lung injury.
Methods: Wild-type (leptin responsive) and db/db (leptin resistant) mice were used in these studies. Mice were exposed to hyperoxia (100% O2) for 84 hours to induce lung injury and up to 168 hours for survival studies. Alveolar fluid clearance was measured in vivo.
Measurements and Main Results: Lung leptin levels were increased both in wild-type and leptin receptor–defective db/db mice after hyperoxia. Hyperoxia-induced lung injury was decreased in db/db compared with wild-type mice. Hyperoxia increased lung permeability in wild-type mice but not in db/db mice. Compared with wild-type control animals, db/db mice were resistant to hyperoxia-induced mortality (lethal dose for 50% of mice, 152 vs. 108 h). Intratracheal instillation of leptin at a dose that was observed in the bronchoalveolar lavage fluid during hyperoxia caused lung injury in wild-type but not in db/db mice. Intratracheal pretreatment with a leptin receptor inhibitor attenuated leptin-induced lung edema. The hyperoxia-induced release of proinflammatory cytokines was attenuated in db/db mice. Despite resistance to lung injury, db/db mice had diminished alveolar fluid clearance and reduced Na,K-ATPase function compared with wild-type mice.
Conclusions: These results indicate that leptin can induce and that resistance to leptin attenuates hyperoxia-induced lung injury and hyperoxia-induced inflammatory cytokines in the lung.
PMCID: PMC1899284  PMID: 17185651
alveolar fluid clearance; pulmonary edema; Na,K-ATPase; diabetes mellitus; oxygen
11.  p53 Mediates Particulate Matter–induced Alveolar Epithelial Cell Mitochondria-regulated Apoptosis 
Rationale: Exposure to particulate matter (PM) causes lung cancer by mechanisms that are unknown, but p53 dysfunction is implicated.
Objective: We determined whether p53 is required for PM-induced apoptosis in both human and rodent alveolar type (AT) 2 cells.
Methods: A well-characterized form of urban PM was used to determine whether it induces mitochondrial dysfunction (mitochondrial membrane potential change [ΔΨm] and caspase-9 activation), p53 protein and mRNA expression, and apoptosis (DNA fragmentation and annexin V staining) in vitro using A549 cells and primary isolated human and rat AT2 cells. The role of p53 was assessed using inhibitors of p53-dependent transcription, pifithrin-α, and a genetic approach (overexpressing E6 or dominant negative p53). In mice, the in vivo effects of PM causing p53 expression and apoptosis were assessed 72 h after a single PM intratracheal instillation.
Measurements and Main Results: PM-induced apoptosis in A549 cells was characterized by increased p53 mRNA and protein expression, mitochondrial translocation of Bax and p53, a reduction in ΔΨm, and caspase-9 activation, and these effects were blocked by inhibiting p53-dependent transcription. Similar findings were noted in primary isolated human and rat AT2 cells. A549-ρ° cells that are incapable of mitochondrial reactive oxygen species production were protected against PM-induced ΔΨm, p53 expression, and apoptosis. In mice, PM induced p53 expression and apoptosis at the bronchoalveolar duct junctions.
Conclusions: These data suggest a novel interaction between p53 and the mitochondria in mediating PM-induced apoptosis that is relevant to the pathogenesis of lung cancer from air pollution.
PMCID: PMC2648105  PMID: 16946128
apoptosis; mitochondria; p53; particulate matter; reactive oxygen species

Results 1-11 (11)