Timothy D. Le Cras

Inhibition of VEGF receptors causes lung cell apoptosis and emphysema

Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolar structures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abundantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blocker SU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitor SU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed by angiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, although we observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decrease in lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the complex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH(2)-DCB prevented SU5416-induced septal cell apoptosis and emphysema development. These findings suggest that VEGF receptor signaling is required for maintenance of the alveolar structures and, further, that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema.

Diesel exhaust particle induction of IL-17A contributes to severe asthma

BACKGROUND IL-17A has been implicated in severe forms of asthma. However, the factors that promote IL-17A production during the pathogenesis of severe asthma remain undefined. Diesel exhaust particles (DEPs) are a major component of traffic-related air pollution and are implicated in asthma pathogenesis and exacerbation. OBJECTIVE We sought to determine the mechanism by which DEP exposure affects asthma severity using human and mouse studies. METHODS BALB/c mice were challenged with DEPs with or without house dust mite (HDM) extract. Airway inflammation and function, bronchoalveolar lavage fluid cytokine levels, and flow cytometry of lung T cells were assessed. The effect of DEP exposure on the frequency of asthma symptoms and serum cytokine levels was determined in children with allergic asthma. RESULTS In mice exposure to DEPs alone did not induce asthma. DEP and HDM coexposure markedly enhanced airway hyperresponsiveness compared with HDM exposure alone and generated a mixed T(H)2 and T(H)17 response, including IL-13(+)IL-17A(+) double-producing T cells. IL-17A neutralization prevented DEP-induced exacerbation of airway hyperresponsiveness. Among 235 high DEP-exposed children with allergic asthma, 32.2% had more frequent asthma symptoms over a 12-month period compared with only 14.2% in the low DEP-exposed group (P = .002). Additionally, high DEP-exposed children with allergic asthma had nearly 6 times higher serum IL-17A levels compared with low DEP-exposed children. CONCLUSIONS Expansion of T(H)17 cells contributes to DEP-mediated exacerbation of allergic asthma. Neutralization of IL-17A might be a useful potential therapeutic strategy to counteract the asthma-promoting effects of traffic-related air pollution, especially in highly exposed patients with severe allergic asthma.

Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma.

Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.

Rapamycin Prevents Transforming Growth Factor-α–Induced Pulmonary Fibrosis

Transforming growth factor (TGF)-alpha is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. Overexpression of TGF-alpha in transgenic mice causes progressive and severe pulmonary fibrosis; however, the intracellular signaling pathways downstream of EGFR mediating this response are unknown. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we observed increased PCNA protein and phosphorylation of Akt and p70S6K in whole lung homogenates in association with induction of TGF-alpha. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over a 7-week period. Daily administration of rapamycin prevented accumulation of total lung collagen, weight loss, and changes in pulmonary mechanics. Treatment of mice with rapamycin 4 weeks after the induction of TGF-alpha prevented additional weight loss, increases in total collagen, and changes in pulmonary mechanics. Rapamycin prevented further increases in established pulmonary fibrosis induced by EGFR activation. This study demonstrates that mammalian target of rapamycin (mTOR) is a major effector of EGFR-induced pulmonary fibrosis, providing support for further studies to determine the role of mTOR in the pathogenesis and treatment of pulmonary fibrosis.

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-α-induced pulmonary fibrosis

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.

Abnormal lung growth and the development of pulmonary hypertension in the Fawn-Hooded rat

The Fawn-Hooded rat (FHR) strain develops accelerated and severe pulmonary hypertension when exposed to slight decreases in alveolar PO(2). We recently observed that adult FHR lungs showed a striking pattern of disrupted alveolarization and hypothesized that abnormalities in lung growth in the perinatal period predisposes the FHR to the subsequent development of pulmonary hypertension. We found a reduction in lung weight in the fetus and 1-day- and 1-wk-old FHR compared with a normal rat strain (Sprague-Dawley). Alveolarization was reduced in infant and adult FHR lungs. In situ hybridization showed similar patterns of expression of two epithelial markers, surfactant protein C and 10-kDa Clara cell secretory protein, suggesting that the FHR lung is not characterized by global delays in epithelial maturation. Barium-gelatin angiograms demonstrated reduced background arterial filling and density in adult FHR lungs. Perinatal treatment of FHR with supplemental oxygen increased alveolarization and reduced the subsequent development of right ventricular hypertrophy in adult FHR. We conclude that the FHR strain is characterized by lung hypoplasia with reduced alveolarization and increased risk for developing pulmonary hypertension. We speculate that altered oxygen sensing may cause impaired lung alveolar and vascular growth in the FHR.The Fawn-Hooded rat (FHR) strain develops accelerated and severe pulmonary hypertension when exposed to slight decreases in alveolar[Formula: see text]. We recently observed that adult FHR lungs showed a striking pattern of disrupted alveolarization and hypothesized that abnormalities in lung growth in the perinatal period predisposes the FHR to the subsequent development of pulmonary hypertension. We found a reduction in lung weight in the fetus and 1-day- and 1-wk-old FHR compared with a normal rat strain (Sprague-Dawley). Alveolarization was reduced in infant and adult FHR lungs. In situ hybridization showed similar patterns of expression of two epithelial markers, surfactant protein C and 10-kDa Clara cell secretory protein, suggesting that the FHR lung is not characterized by global delays in epithelial maturation. Barium-gelatin angiograms demonstrated reduced background arterial filling and density in adult FHR lungs. Perinatal treatment of FHR with supplemental oxygen increased alveolarization and reduced the subsequent development of right ventricular hypertrophy in adult FHR. We conclude that the FHR strain is characterized by lung hypoplasia with reduced alveolarization and increased risk for developing pulmonary hypertension. We speculate that altered oxygen sensing may cause impaired lung alveolar and vascular growth in the FHR.

GP130-STAT3 regulates epithelial cell migration and is required for repair of the bronchiolar epithelium.

Following injury, bronchiolar cells undergo rapid squamous metaplasia, followed by proliferation and re-establishment of the complex columnar epithelium that is characteristic of the normal airway. Mechanisms that regulate the repair of bronchiolar epithelium are of considerable relevance for understanding the pathogenesis of both acute and chronic lung diseases associated with airway remodeling. This study was designed to identify the role of the GP130-STAT3 signaling pathway during repair of the bronchiolar epithelium. STAT3 (signal transducer and activator of transcription 3) and GP130 (glycoprotein 130) were each selectively deleted from the pulmonary epithelial cells of transgenic mice in vivo, producing Stat3(Delta/Delta) and Gp130(Delta/Delta) mice, respectively. Airway injury was induced in adult mice by administration of naphthalene, a toxicant of nonciliated respiratory epithelial cells (Clara cells). Nuclear STAT3 staining was induced in bronchiolar epithelial cells following naphthalene-mediated injury in control (Stat3(flox/flox)) mice. Whereas nearly complete repair of the bronchiolar epithelium was observed in control mice within 13 days, restoration of cell shape, cell density, and the pattern of ciliated and nonciliated cells did not occur in the peripheral bronchioles of either Stat3(Delta/Delta) or Gp130(Delta/Delta) mice. Expression of dominant-negative STAT3 inhibited airway epithelial cell migration during repair in vitro; wild-type STAT3 expression activated such migration. In the present study, we show that GP130-STAT3 signaling functions in a cell-autonomous manner to restore cell shape and numbers required for repair of the bronchiolar epithelium following injury.

Increased lung preproET-1 and decreased ETB-receptor gene expression in fetal pulmonary hypertension

Endothelin (ET)-1, a potent vasoconstrictor and smooth muscle mitogen, is produced from its precursor, preproET-1, by endothelin-converting enzyme (ECE)-1 activity. ET-1 may bind to two receptors, ETA and ETB, that mediate vasoconstriction and vasodilation in the ovine fetal lung, respectively. ET-1 contributes to high pulmonary vascular resistance in experimental perinatal pulmonary hypertension induced by ligation of the ductus arteriosus in the fetal lamb. Physiological studies in this model have demonstrated enhanced ETA- and diminished ETB-receptor activities and a threefold increase in lung immunoreactive ET-1 protein content. We hypothesized that increased ET production and an imbalance in receptor expression would favor vasoconstriction and smooth muscle cell hypertrophy in pulmonary hypertension and may be partially due to alterations in gene expression. To test this hypothesis, we studied lung mRNA expression of preproET-1, ECE-1, and the ETA and ETB receptors in normal and hypertensive fetal lambs. Total RNA was isolated from whole lung tissue in normal late-gestation fetuses (135 ± 3 days; 147 days = term) and from animals with pulmonary hypertension after ductus arteriosus ligation for 8 days (134 ± 4 days). Ductus arteriosus ligation increased right ventricular hypertrophy [control 0.56 ± 0.02 vs. hypertension 0.85 ± 0.05; right ventricle/(left ventricle + septum); P < 0.05]. Northern blot analysis was performed using cDNA probes and was normalized to the signal for 18S rRNA. We found a 71 ± 24% increase in steady-state preproET-1 mRNA ( P < 0.05) and a 62 ± 5% decrease in ETB mRNA ( P < 0.05) expression in ductus arteriosus ligation. ECE-1 and ETA-receptor mRNA expression did not change. We conclude that chronic intrauterine pulmonary hypertension after ductus arteriosus ligation increases steady-state preproET-1 mRNA and decreases ETB-receptor mRNA without changing ECE-1 mRNA or ETA-receptor mRNA expression. These findings suggest that increased ET-1 production and decreased ETB-receptor expression may contribute to increased vasoconstrictor tone in this experimental model of neonatal pulmonary hypertension.

Vascular endothelial growth factor-A induces prenatal neovascularization and alters bronchial development in mice.

Pulmonary vascular development requires precise temporal and spatial expression of vascular endothelial growth factor-A (VEGF-A). Diminished expression of VEGF-A in preterm infants may contribute to the pathophysiology of respiratory distress syndrome. Because exogenous replacement of VEGF-A has been proposed as a therapeutic for respiratory distress syndrome, we used conditional activation of VEGF-A in bronchial epithelial cells to assess the effects of increase of VEGF-A on lung morphogenesis and survival in the developing mouse. Increased expression of VEGF-A in late stages of gestation was lethal at birth. Although born alive, the pups remained cyanotic and failed to establish respiration. Vascular and epithelial morphology of the main bronchus and primary and secondary bronchi were altered with neovascularization of the mucosal folds and partial obstruction of the conducting airways. Erythrocytes were observed in the pulmonary interstitium and in intra-alveolar spaces, indicating vascular leak. Increased diameter of pulmonary arteries and angioectatic structures were observed in VEGF-expressing mice. Bronchial expression of VEGF-A alters late-stage morphogenesis of conducting airways and primary bronchial arteries and causes respiratory failure at birth.

Rapamycin Attenuates Airway Hyperreactivity, Goblet Cells, and IgE in Experimental Allergic Asthma

The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues, promotes cell growth/differentiation, and regulates immune responses. Although inhibition of mTOR with rapamycin has potent immunosuppressive activity, mixed effects have been reported in OVA-induced models of allergic asthma. We investigated the impact of two rapamycin treatment protocols on the major characteristics of allergic asthma induced by the clinically relevant allergen, house dust mite (HDM). In protocol 1, BALB/c mice were exposed to 10 intranasal HDM doses over a period of 24 d and treated with rapamycin simultaneously during the sensitization/exposure period. In protocol 2, rapamycin was administered after the mice had been sensitized to HDM (i.p. injection) and prior to initiation of two intranasal HDM challenges over 4 d. Airway hyperreactivity (AHR), IgE, inflammatory cells, cytokines, leukotrienes, goblet cells, and activated T cells were assessed. In protocol 1, rapamycin blocked HDM-induced increases in AHR, inflammatory cell counts, and IgE, as well as attenuated goblet cell metaplasia. In protocol 2, rapamycin blocked increases in AHR, IgE, and T cell activation and reduced goblet cell metaplasia, but it had no effect on inflammatory cell counts. Increases in IL-13 and leukotrienes were also blocked by rapamycin, although increases in IL-4 were unaffected. These data demonstrated that rapamycin can inhibit cardinal features of allergic asthma, including increases in AHR, IgE, and goblet cells, most likely as a result of its ability to reduce the production of two key mediators of asthma: IL-13 and leukotrienes. These findings highlight the importance of the mTOR pathway in allergic airway disease.