Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors.

Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or ß (PI5P4Kα and ß) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and ß in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K ß and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A(-/-), PIP4K2B(+/-), and TP53(-/-) mice were viable and had a dramatic reduction in tumor formation compared to TP53(-/-) littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.

PARK2 Depletion Connects Energy and Oxidative Stress to PI3K/Akt Activation via PTEN S-Nitrosylation.

PARK2 is a gene implicated in disease states with opposing responses in cell fate determination, yet its contribution in pro-survival signaling is largely unknown. Here we show that PARK2 is altered in over a third of all human cancers, and its depletion results in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors. PARK2 depletion contributes to AMPK-mediated activation of endothelial nitric oxide synthase (eNOS), enhanced levels of reactive oxygen species, and a concomitant increase in oxidized nitric oxide levels, thereby promoting the inhibition of PTEN by S-nitrosylation and ubiquitination. Notably, AMPK activation alone is sufficient to induce PTEN S-nitrosylation in the absence of PARK2 depletion. Park2 loss and Pten loss also display striking cooperativity to promote tumorigenesis in vivo. Together, our findings reveal an important missing mechanism that might account for PTEN suppression in PARK2-deficient tumors, and they highlight the importance of PTEN S-nitrosylation in supporting cell survival and proliferation under conditions of energy deprivation.

A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.

The functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury-repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance1. Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type that we call the 'pulmonary ionocyte', which co-expresses FOXI1, multiple subunits of the vacuolar-type H+-ATPase (V-ATPase) and CFTR, the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium.

Deletion of the gene Pip4k2c, a novel phosphatidylinositol kinase, results in hyperactivation of the immune system.

Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three enzymes PI5P4Kα, PI5P4Kß, and PI5P4Kγ, and these enzymes have been implicated in metabolic control, growth control, and a variety of stress responses. Here, we show that mice with germline deletion of type 2 phosphatidylinositol-5-phosphate 4-kinase gamma (Pip4k2c), the gene encoding PI5P4Kγ, appear normal in regard to growth and viability but have increased inflammation and T-cell activation as they age. Immune cell infiltrates increased in Pip4k2c(-/-) mouse tissues. Also, there was an increase in proinflammatory cytokines, including IFNγ, interleukin 12, and interleukin 2 in plasma of Pip4k2c(-/-) mice. Pip4k2c(-/-) mice had an increase in T-helper-cell populations and a decrease in regulatory T-cell populations with increased proliferation of T cells. Interestingly, mammalian target of rapamycin complex 1 (mTORC1) signaling was hyperactivated in several tissues from Pip4k2c(-/-) mice and treating Pip4k2c(-/-) mice with rapamycin reduced the inflammatory phenotype, resulting in a decrease in mTORC1 signaling in tissues and a decrease in proinflammatory cytokines in plasma. These results indicate that PI5P4Kγ plays a role in the regulation of the immune system via mTORC1 signaling.

Identification of CDCP1 as a hypoxia-inducible factor 2α (HIF-2α) target gene that is associated with survival in clear cell renal cell carcinoma patients.

CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that is highly expressed in stem cells and frequently overexpressed and tyrosine-phosphorylated in cancer. CDCP1 promotes cancer cell metastasis. However, the mechanisms that regulate CDCP1 are not well-defined. Here we show that hypoxia induces CDCP1 expression and tyrosine phosphorylation in hypoxia-inducible factor (HIF)-2α-, but not HIF-1α-, dependent fashion. shRNA knockdown of CDCP1 impairs cancer cell migration under hypoxic conditions, whereas overexpression of HIF-2α promotes the growth of tumor xenografts in association with enhanced CDCP1 expression and tyrosine phosphorylation. Immunohistochemistry analysis of tissue microarray samples from tumors of patients with clear cell renal cell carcinoma shows that increased CDCP1 expression correlates with decreased overall survival. Together, these data support a critical role for CDCP1 as a unique HIF-2α target gene involved in the regulation of cancer metastasis, and suggest that CDCP1 is a biomarker and potential therapeutic target for metastatic cancers.

Hyperoxia and interferon-γ-induced injury in developing lungs occur via cyclooxygenase-2 and the endoplasmic reticulum stress-dependent pathway.

We noted a marked increase in cyclooxygenase-2 (Cox2) and the activation of the endoplasmic reticulum (ER) stress pathway in newborn murine lung on exposure to hyperoxia and IFN-γ. We sought to evaluate Cox2-mediated ER stress pathway activation in hyperoxia-induced and IFN-γ-mediated injury in developing lungs. We applied in vivo genetic gain-of-function and genetic/chemical inhibition, as well as in vitro loss-of-function genetic strategies. Hyperoxia-induced and IFN-γ-mediated impaired alveolarization was rescued by Cox2 inhibition, using celecoxib. The use of small interfering RNA against the ER stress pathway mediator, the C/EBP homologous protein (CHOP; also known as growth arrest and DNA damage-inducible gene 153/GADD153), alleviated cell death in alveolar epithelial cells as well as in hyperoxia-induced and IFN-γ-mediated murine models of bronchopulmonary dysplasia (BPD). In addition, CHOP siRNA also restored alveolarization in the in vivo models. Furthermore, as evidence of clinical relevance, we show increased concentrations of Cox2 and ER stress pathway mediators in human lungs with BPD. Cox2, via CHOP, may significantly contribute to the final common pathway of hyperoxia-induced and IFN-γ-mediated injury in developing lungs and human BPD.

Small molecular modulation of macrophage migration inhibitory factor in the hyperoxia-induced mouse model of bronchopulmonary dysplasia.

BACKGROUND: The role and mechanism of action of MIF in bronchopulmonary dysplasia (BPD) are not known. We hypothesized that increased MIF signaling would ameliorate the pulmonary phenotype of BPD in the mouse lung. METHODS: We studied newborn wild type (WT), MIF knockout (MIFKO), and lung MIF transgenic (MIFTG) mice in room air and a BPD model, and examined the effects of administering a small molecule MIF agonist and antagonist. Lung morphometry was performed and mRNA and protein expression of vascular mediators were analyzed. RESULTS: The pulmonary phenotype of MIFKO and MIFTG mice lungs in room air (RA) and BPD model were comparable to the WT-BPD mice at postnatal (PN) day 14. Vascular endothelial growth factor (VEGF)-A, -R1 and Angiopoietin (Ang)1 mRNA were decreased, and Ang2 increased in the WT-BPD, MIFKO-RA, MIFKO-BPD, MIFTG-RA and MIFTG-BPD mice lungs, compared to appropriate controls. The protein expression of Ang1 in the MIFKO-RA was similar to WT-RA, but decreased in MIFTG-RA, and decreased in all the BPD groups. Ang2 was increased in MIFKO-RA, MIFTG-RA and in all 3 BPD groups. Tie2 was increased in WT-BPD compared to WT-RA, but decreased in MIFKO- and MIFTG- RA and BPD groups. VEGFR1 was uniformly decreased in MIFKO-RA, MIFTG-RA and in all 3 BPD groups. VEGF-A had a similar expression across all RA and BPD groups. There was partial recovery of the pulmonary phenotype in the WT-BPD model treated with the MIF agonist, and in the MIFTG mice treated with the MIF antagonist. CONCLUSIONS: These data point to the careful regulatory balance exerted by MIF in the developing lung and response to hyperoxia and support the potential therapeutic value of small molecule MIF modulation in BPD.

Hyperoxia causes angiopoietin 2-mediated acute lung injury and necrotic cell death.

The angiogenic growth factor angiopoietin 2 (Ang2) destabilizes blood vessels, enhances vascular leak and induces vascular regression and endothelial cell apoptosis. We considered that Ang2 might be important in hyperoxic acute lung injury (ALI). Here we have characterized the responses in lungs induced by hyperoxia in wild-type and Ang2-/- mice or those given either recombinant Ang2 or short interfering RNA (siRNA) targeted to Ang2. During hyperoxia Ang2 expression is induced in lung epithelial cells, while hyperoxia-induced oxidant injury, cell death, inflammation, permeability alterations and mortality are ameliorated in Ang2-/- and siRNA-treated mice. Hyperoxia induces and activates the extrinsic and mitochondrial cell death pathways and activates initiator and effector caspases through Ang2-dependent pathways in vivo. Ang2 increases inflammation and cell death during hyperoxia in vivo and stimulates epithelial necrosis in hyperoxia in vitro. Ang2 in plasma and alveolar edema fluid is increased in adults with ALI and pulmonary edema. Tracheal Ang2 is also increased in neonates that develop bronchopulmonary dysplasia. Ang2 is thus a mediator of epithelial necrosis with an important role in hyperoxic ALI and pulmonary edema.

Increased hyperoxia-induced lung injury in nitric oxide synthase 2 null mice is mediated via angiopoietin 2.

Supplemental oxygen is frequently prescribed. However, prolonged exposure to high concentrations of oxygen causes hyperoxic acute lung injury (HALI), which manifests as acute respiratory distress syndrome in adults and leads to bronchopulmonary dysplasia in newborns (NBs). Nitric oxide (NO), NO synthases (NOSs), and angiopoietin (Ang) 2 have been implicated in the pathogenesis of HALI. However, the mechanisms of the contributions of NOS/NO and the relationship(s) between NOS/NO and Ang2 have not been addressed. In addition, the relevance of these moieties in adults and NBs has not been evaluated. To address these issues, we compared the responses in hyperoxia of wild-type (NOS [+/+]) and NOS null (-/-) young adult and NB mice. When compared with NOS2(+/+) adult controls, NOS2(-/-) animals manifest exaggerated alveolar-capillary protein leak and premature death. These responses were associated with enhanced levels of structural cell death, enhanced expression of proapoptotic regulatory proteins, and Ang2. Importantly, silencing RNA knockdown of Ang2 decreased the levels of cell death and the expression of proapoptotic mediators. These effects were at least partially NOS2 specific, and were development dependent, because survival was similar in adult NOS3(+/+) and NOS3(-/-) mice and NB NOS2(+/+) and NOS2(-/-) mice, respectively. These studies demonstrate that NOS2 plays an important protective role in HALI in adult animals. They also demonstrate that this response is mediated, at least in part, by the ability of NOS2 to inhibit hyperoxia-induced Ang2 production and thereby decrease Ang2-induced tissue injury.

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice.

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 µM and a peak-plateau signal at 500 µM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.

A role for matrix metalloproteinase 9 in IFNγ-mediated injury in developing lungs: relevance to bronchopulmonary dysplasia.

We noted a marked increase in IFNγ mRNA in newborn (NB) murine lungs after exposure to hyperoxia. We sought to evaluate the role of IFNγ in lung injury in newborns. Using a unique triple-transgenic (TTG), IFNγ-overexpressing, lung-targeted, externally regulatable NB murine model, we describe a lung phenotype of impaired alveolarization, resembling human bronchopulmonary dysplasia (BPD). IFNγ-mediated abnormal lung architecture was associated with increased cell death and the upregulation of cell death pathway mediators caspases 3, 6, 8, and 9, and angiopoietin 2. Moreover, an increase was evident in cathepsins B, H, K, L, and S, and in matrix metalloproteinases (MMPs) 2, 9, 12, and 14. The IFNγ-mediated abnormal lung architecture was found to be MMP9-dependent, as indicated by the rescue of the IFNγ-induced pulmonary phenotype and survival during hyperoxia with a concomitant partial deficiency of MMP9. This result was concomitant with a decrease in caspases 3, 6, 8, and 9 and angiopoietin 2, but an increase in the expression of angiopoietin 1. In addition, NB IFNγ TTG mice exhibited significantly decreased survival during hyperoxia, compared with littermate controls. Furthermore, as evidence of clinical relevance, we show increased concentrations of the downstream targets of IFNγ chemokine (C-X-C motif) ligands (CXCL10 and CXCL11) in baboon and human lungs with BPD. IFNγ and its downstream targets may contribute significantly to the final common pathway of hyperoxia-induced injury in the developing lung and in human BPD.

A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-ß1-mediated injury in the developing murine lung.

BACKGROUND: Transforming growth factor-beta 1 (TGF-ß1) has been implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung. In addition, the c-JunNH2-terminal kinase (JNK) pathway has been shown to have a role for TGF-ß1-mediated effects. We hypothesized that the JNK pathway is an important regulator of hyperoxia-induced pulmonary responses in the developing murine lung. RESULTS: We used cultured human lung epithelial cells, fetal rat lung fibroblasts and a neonatal TGF-ß1 transgenic mouse model. We demonstrate that hyperoxia inhibits cell proliferation, activates cell death mediators and causes cell death, and promotes myofibroblast transdifferentiation, in a dose-dependent manner. Except for fibroblast proliferation, the effects were mediated via the JNK pathway. In addition, since we observed increased expression of TGF-ß1 by epithelial cells on exposure to hyperoxia, we used a TGF-ß1 transgenic mouse model to determine the role of JNK activation in TGF-ß1 induced effects on lung development and on exposure to hyperoxia. We noted that, in this model, inhibition of JNK signaling significantly improved the spontaneously impaired alveolarization in room air and decreased mortality on exposure to hyperoxia. CONCLUSIONS: When viewed in combination, these studies demonstrate that hyperoxia-induced cell death, myofibroblast transdifferentiation, TGF-ß1- and hyperoxia-mediated pulmonary responses are mediated, at least in part, via signaling through the JNK pathway.

Dantrolene mitigates caerulein-induced pancreatitis in vivo in mice.

Acute pancreatitis is a painful, inflammatory disorder for which adequate treatments are lacking. An early, critical step in its development is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. This Ca(2+) release is modulated by the intracellular Ca(2+) channel the ryanodine receptor (RYR). We have previously shown that RYR inhibition reduces pathological intra-acinar protease activation, an early marker of pancreatitis. In this study, we examined whether pretreatment with the RYR inhibitor dantrolene attenuates the severity of caerulein-induced pancreatitis in mice. Immunofluorescent labeling for RYR from mouse pancreatic sections showed localization to the basolateral region of the acinar cell. After 1 h of caerulein hyperstimulation in vivo, dantrolene 1) reduced pancreatic trypsin activity by 59% (P < 0.05) and 2) mitigated early ultrastructural derangements within the acinar cell. Eight hours after pancreatitis induction, dantrolene reduced pancreatic trypsin activity and serum amylase by 61 and 32%, respectively (P < 0.05). At this later time point, overall histological severity of pancreatitis was reduced by 63% with dantrolene pretreatment (P < 0.05). TUNEL-positive cells were reduced by 58% (P < 0.05). These data suggest that the RYR plays an important role in mediating early acinar cell events during in vivo pancreatitis and contributes to disease severity. Blockade of Ca(2+) signals and particularly RYR-Ca(2+) may be useful as prophylactic treatment for this disease in high-risk settings for pancreatitis.

Developmental regulation of NO-mediated VEGF-induced effects in the lung.

Vascular endothelial growth factor (VEGF) is known to have a pivotal role in lung development and in a variety of pathologic conditions in the adult lung. Our earlier studies have shown that NO is a critical mediator of VEGF-induced vascular and extravascular effects in the adult murine lung. As significant differences have been reported in the cytokine responses in the adult versus the neonatal lung, we hypothesized that there may be significant differences in VEGF-induced alterations in the developing as opposed to the mature lung. Furthermore, nitric oxide (NO) mediation of these VEGF-induced effects may be developmentally regulated. Using a novel externally regulatable lung-targeted transgenic murine model, we found that VEGF-induced pulmonary hemorrhage was mediated by NO-dependent mechanisms in adults and newborns. VEGF enhanced surfactant production in adults as well as increased surfactant and lung development in newborns, via an NO-independent mechanism. While the enhanced survival in hyperoxia in the adult was partly NO-dependent, there was enhanced hyperoxia-induced lung injury in the newborn. In addition, human amniotic fluid VEGF levels correlated positively with surfactant phospholipids. Tracheal aspirate VEGF levels had an initial spike, followed by a decline, and then a subsequent rise, in human neonates with an outcome of bronchopulmonary dysplasia or death. Our data show that VEGF can have injurious as well as potentially beneficial developmental effects, of which some are NO dependent, others NO independent. This opens up the possibility of selective manipulation of any VEGF-based intervention using NO inhibitors for maximal potential clinical benefit.

TRRAP is a central regulator of human multiciliated cell formation.

The multiciliated cell (MCC) is an evolutionarily conserved cell type, which in vertebrates functions to promote directional fluid flow across epithelial tissues. In the conducting airway, MCCs are generated by basal stem/progenitor cells and act in concert with secretory cells to perform mucociliary clearance to expel pathogens from the lung. Studies in multiple systems, including Xenopus laevis epidermis, murine trachea, and zebrafish kidney, have uncovered a transcriptional network that regulates multiple steps of multiciliogenesis, ultimately leading to an MCC with hundreds of motile cilia extended from their apical surface, which beat in a coordinated fashion. Here, we used a pool-based short hairpin RNA screening approach and identified TRRAP, an essential component of multiple histone acetyltransferase complexes, as a central regulator of MCC formation. Using a combination of immunofluorescence, signaling pathway modulation, and genomic approaches, we show that (a) TRRAP acts downstream of the Notch2-mediated basal progenitor cell fate decision and upstream of Multicilin to control MCC differentiation; and (b) TRRAP binds to the promoters and regulates the expression of a network of genes involved in MCC differentiation and function, including several genes associated with human ciliopathies.

Role of Nitric Oxide Isoforms in Vascular and Alveolar Development and Lung Injury in Vascular Endothelial Growth Factor Overexpressing Neonatal Mice Lungs.

BACKGROUND: The role of vascular endothelial growth factor (VEGF)-induced 3 different nitric oxide synthase (NOS) isoforms in lung development and injury in the newborn (NB) lung are not known. We hypothesized that VEGF-induced specific NOS pathways are critical regulators of lung development and injury. METHODOLOGY: We studied NB wild type (WT), lung epithelial cell-targeted VEGF165 doxycycline-inducible overexpressing transgenic (VEGFTG), VEGFTG treated with a NOS1 inhibitor (L-NIO), VEGFTG x NOS2-/- and VEGFTG x NOS3+/- mice in room air (RA) for 7 postnatal (PN) days. Lung morphometry (chord length), vascular markers (Ang1, Ang2, Notch2, vWF, CD31 and VE-cadherin), cell proliferation (Ki67), vascular permeability, injury and oxidative stress markers (hemosiderin, nitrotyrosine and 8-OHdG) were evaluated. RESULTS: VEGF overexpression in RA led to increased chord length and vascular markers at PN7, which were significantly decreased to control values in VEGFTG x NOS2-/- and VEGFTG x NOS3+/- lungs. However, we found no noticeable effect on chord length and vascular markers in the VEGFTG / NOS1 inhibited group. In the NB VEGFTG mouse model, we found VEGF-induced vascular permeability in the NB murine lung was partially dependent on NOS2 and NOS3-signaling pathways. In addition, the inhibition of NOS2 and NOS3 resulted in a significant decrease in VEGF-induced hemosiderin, nitrotyrosine- and 8-OHdG positive cells at PN7. NOS1 inhibition had no significant effect. CONCLUSION: Our data showed that the complete absence of NOS2 and partial deficiency of NOS3 confers protection against VEGF-induced pathologic lung vascular and alveolar developmental changes, as well as injury markers. Inhibition of NOS1 does not have any modulating role on VEGF-induced changes in the NB lung. Overall, our data suggests that there is a significant differential regulation in the NOS-mediated effects of VEGF overexpression in the developing mouse lung.

A critical regulatory role for macrophage migration inhibitory factor in hyperoxia-induced injury in the developing murine lung.

BACKGROUND: The role and mechanism of action of MIF in hyperoxia-induced acute lung injury (HALI) in the newborn lung are not known. We hypothesized that MIF is a critical regulatory molecule in HALI in the developing lung. METHODOLOGY: We studied newborn wild type (WT), MIF knockout (MIFKO), and MIF lung transgenic (MIFTG) mice in room air and hyperoxia exposure for 7 postnatal (PN) days. Lung morphometry was performed and mRNA and protein expression of vascular mediators were analyzed. RESULTS: MIF mRNA and protein expression were significantly increased in WT lungs at PN7 of hyperoxia exposure. The pattern of expression of Angiopoietin 2 protein (in MIFKO>WT>MIFTG) was similar to the mortality pattern (MIFKO>WT>MIFTG) in hyperoxia at PN7. In room air, MIFKO and MIFTG had modest but significant increases in chord length, compared to WT. This was associated with decreased expression of Angiopoietin 1 and Tie 2 proteins in the MIFKO and MIFTG, as compared to the WT control lungs in room air. However, on hyperoxia exposure, while the chord length was increased from their respective room air controls, there were no differences between the 3 genotypes. CONCLUSION: These data point to the potential roles of Angiopoietins 1, 2 and their receptor Tie2 in the MIF-regulated response in room air and upon hyperoxia exposure in the neonatal lung.

Increased hyperoxia-induced mortality and acute lung injury in IL-13 null mice.

IL-13 is a critical effector at sites of Th2 inflammation and remodeling. As a result, anti-IL-13-based therapies are being actively developed to treat a variety of diseases and disorders. However, the beneficial effects of endogenous IL-13 in the normal and diseased lung have not been adequately defined. We hypothesized that endogenous IL-13 is an important regulator of oxidant-induced lung injury and inflammation. To test this hypothesis, we compared the effects of 100% O(2) in mice with wild-type and null IL-13 loci. In this study, we demonstrate that hyperoxia significantly augments the expression of the components of the IL-13R, IL-13Ralpha1, and IL-4Ralpha. We also demonstrate that, in the absence of IL-13, hyperoxia-induced tissue inflammation is decreased. In contrast, in the IL-13 null mice, DNA injury, cell death, caspase expression, and activation and mortality are augmented. Interestingly, the levels of the cytoprotective cytokines vascular endothelial cell growth factor, IL-6, and IL-11 were decreased in the bronchoalveolar lavage fluid. These studies demonstrate that the expression of the IL-13R is augmented and that the endogenous IL-13-IL-13R pathway contributes to the induction of inflammation and the inhibition of injury in hyperoxic acute lung injury.

Developmental differences in the responses of IL-6 and IL-13 transgenic mice exposed to hyperoxia.

Our previous work has shown that adult mice with overexpression of IL-6 and IL-13 in the lung have enhanced survival in hyperoxia associated with reduced hyperoxia-induced lung injury and cell death. We hypothesized that there are developmental differences in these responses in the adult vs. the newborn (NB) animal, and these responses have clinical relevance in the human NB. We compared the responses to 100% O(2) of NB IL-6 and IL-13 transgenic mice with wild-type littermate controls by evaluating mortality, lung tissue TUNEL staining, and mRNA expression using RT-PCR. We used ELISA to measure IL-6 levels in tracheal aspirates from human neonates. Our results show that, in contrast to the cytoprotective effects in mature mice, IL-6 caused significantly increased mortality, DNA injury, caspases, cell death regulator and angiogenic factor expression in hyperoxia in the NB. Furthermore, tracheal aspirate levels of IL-6 were significantly increased in premature neonates with respiratory distress syndrome who had an adverse outcome (bronchopulmonary dysplasia/death). In contrast to the protective effects in adults, there was no survival advantage to the NB IL-13 mice in hyperoxia. These findings imply that caution should be exercised in extrapolating results from the adult to the NB.

Essential role of nitric oxide in VEGF-induced, asthma-like angiogenic, inflammatory, mucus, and physiologic responses in the lung.

VEGF, nitric oxide (NO), inflammation, and vascular- and extravascular remodeling coexist in asthma and other disorders. In these responses, VEGF regulates angiogenesis. VEGF also induces inflammation and remodeling. The mechanisms of the latter responses have not been defined, however. We hypothesized that VEGF-induces extravascular tissue responses via NO-dependent mechanisms. To evaluate this hypothesis, we compared the effects of transgenic VEGF165 in lungs from normal mice, mice treated with pan-NO synthase (NOS) or endothelial NOS (eNOS) inhibitors, and mice with null mutations of inducible NOS (iNOS) or eNOS. These studies demonstrate that VEGF selectively stimulates eNOS and iNOS. They also demonstrate that VEGF induces pulmonary alterations via NO-dependent and -independent mechanisms with angiogenesis, edema, mucus metaplasia, airway hyperresponsiveness, lymphocyte accumulation, dendritic cell hyperplasia and S-nitrosoglutathione reductase stimulation being NO-dependent and dendritic cell activation being NO-independent. Furthermore, they demonstrate that eNOS and iNOS both contribute to these responses. NO/NOS-based interventions may be therapeutic in VEGF-driven inflammation and remodeling.