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1.  Regulation of T Helper Cell Subsets by Cyclooxygenases and Their Metabolites 
Cyclooxygenases and their metabolites are important regulators of inflammatory responses and play critical roles in regulating the differentiation of T helper cell subsets in inflammatory diseases. In this review, we highlight new information on regulation of T helper cell subsets by cyclooxygenases and their metabolites. Prostanoids influence cytokine production on both antigen presenting cells and T cells to regulate the differentiation of naïve CD4+ T cells to Th1, Th2 and Th17 cell phenotypes. Cyclooxygenases and PGE2 generally exacerbate Th2 and Th17 phenotypes, while suppressing Th1 differentiation. Thus, cycloxygenases may play a critical role in diseases that involve immune cell dysfunction. Targeting of cyclooxygenases and their eicosanoid products may represent a new approach for treatment of inflammatory diseases, tumors and autoimmune disorders.
PMCID: PMC3620713  PMID: 23201570
Cyclooxygenases; T helper cells; Prostanoids; PGE2
2.  Cyclooxygenase-2 Inhibits T Helper Cell Type 9 Differentiation during Allergic Lung Inflammation via Down-regulation of IL-17RB 
Rationale: Helper CD4+ T cell subsets, including IL-9– and IL-10–producing T helper cell type 9 (Th9) cells, exist under certain inflammatory conditions. Cyclooxygenase (COX)-1 and COX-2 play important roles in allergic lung inflammation and asthma. It is unknown whether COX-derived eicosanoids regulate Th9 cells during allergic lung inflammation.
Objectives: To determine the role of COX metabolites in regulating Th9 cell differentiation and function during allergic lung inflammation.
Methods: COX-1−/−, COX-2−/−, and wild-type (WT) mice were studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of Th9 differentiation using flow cytometry, cytokine assays, confocal microscopy, real-time PCR, and immunoblotting. In addition, the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins (PGs), selective inhibitors, and siRNA knockdown.
Measurements and Main Results: Experimental endpoints were not different between COX-1−/− and WT mice; however, the percentage of IL-9+ CD4+ T cells was increased in lung, bronchoalveolar lavage fluid, lymph nodes, and blood of allergic COX-2−/− mice relative to WT. Bronchoalveolar lavage fluid IL-9 and IL-10, serum IL-9, and lung IL-17RB levels were significantly increased in allergic COX-2−/− mice or in WT mice treated with COX-2 inhibitors. IL-9, IL-10, and IL-17RB expression in vivo was inhibited by PGD2 and PGE2, which also reduced Th9 cell differentiation of murine and human naive CD4+ T cells in vitro. Inhibition of protein kinase A significantly increased Th9 cell differentiation of naive CD4+ T cells isolated from WT mice in vitro.
Conclusions: COX-2–derived PGD2 and PGE2 regulate Th9 cell differentiation by suppressing IL-17RB expression via a protein kinase A–dependent mechanism.
PMCID: PMC3707371  PMID: 23449692
T helper cell type 9 cells; cyclooxygenase 2; asthma; prostaglandins; IL-17RB
3.  Immunochemical quantification of cynomolgus CYP2J2, CYP4A, and CYP4F enzymes in liver and small intestine 
An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies.In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11, and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4, and CYP3A5, previously quantified.In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11, and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2%, 14.4%, 8.0%, 2.7%, and 0.3% of total immunoquantified CYP1-4 proteins, respectively.In small intestines, CYP2J2, CYP4F2/3, CYP4F11, and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9%, 36.4%, 2.4%, and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed.Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation.This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11, and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates.
PMCID: PMC4294552  PMID: 25138712
Cynomolgus monkey; cytochrome P450; expression; liver; small intestine
4.  Cyclooxygenase-2 Regulates Th17 Cell Differentiation during Allergic Lung Inflammation 
Rationale: Th17 cells comprise a distinct lineage of proinflammatory T helper cells that are major contributors to allergic responses. It is unknown whether cyclooxygenase (COX)-derived eicosanoids regulate Th17 cells during allergic lung inflammation.
Objectives: To determine the role of COX metabolites in regulating Th17 cell differentiation and function during allergic lung inflammation.
Methods: COX-1−/−, COX-2−/−, and wild-type mice were studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of Th17 differentiation using flow cytometry, cytokine assays, confocal microscopy, real-time polymerase chain reaction, and immunoblotting. In addition, the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins (PGs), selective inhibitors, and siRNA knockdown.
Measurements and Main Results: Th17 cell differentiation in lung, lymph nodes, and bronchoalveolar lavage fluid was significantly lower in COX-2−/− mice after ovalbumin sensitization and exposure in vivo. In vitro studies revealed significantly impaired Th17 cell differentiation of COX-2−/− naive CD4+ T cells with decreased Stat3 phosphorylation and RORγt expression. Synthetic PGF2α and PGI2 enhanced Th17 cell differentiation of COX-2−/− CD4+ T cells in vitro. The selective COX-2 inhibitor, NS-398, and PGF2α receptor and PGI2 receptor siRNA knockdown significantly decreased Th17 cell differentiation in vitro. Administration of synthetic PGs restored accumulation of Th17 cells in lungs of allergic COX-2−/− mice in vivo.
Conclusions: COX-2 is a critical regulator of Th17 cell differentiation during allergic lung inflammation via autocrine signaling of PGI2 and PGF2α through their respective cell surface receptors.
PMCID: PMC3172888  PMID: 21474648
Th17 cell; COX-2; asthma; prostaglandins; IL-17
5.  Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells 
•We examined epoxygenase product formation and regulation in endothelial cells.•The epoxygenase CYP2J2 is an LPS (TLR-4) inducible enzyme in endothelial cells.•The endothelial cell line EA.Hy926 synthesises epoxygenase products.•Inhibition of endothelial epoxygenases increases TNFα secretion.•Soluble epoxide hydrolase inhibitors reduce inflammation-induced TNFα and NFκB.
The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.
PMCID: PMC3991391  PMID: 24631907
AA, arachidonic acid; DHA, docosahexaenoic acid; DHET, dihydroxy eicosatrienoic acid; DHOME, dihydroxy-octadecenoic acid; DiHDPA, dihydroxy-docosapentaenoic acid; EET, epoxyeicosatrienoic acid; EPA, eicosapentaenoic acid; EPOME, epoxy-octadecenoic acid; HETE, hydroxyeicosatetraenoic acid; LA, linoleic acid; sEH, soluble epoxide hydrolase; Endothelial; TNFα; Epoxygenase; Soluble epoxide hydrolase
6.  Ventilation Defects Observed with Hyperpolarized 3He Magnetic Resonance Imaging in a Mouse Model of Acute Lung Injury 
Regions of diminished ventilation are often evident during functional pulmonary imaging studies, including hyperpolarized gas magnetic resonance imaging (MRI), positron emission tomography, and computed tomography (CT). The objective of this study was to characterize the hypointense regions observed via 3He MRI in a murine model of acute lung injury. LPS at doses ranging from 15–50 μg was intratracheally administered to C57BL/6 mice under anesthesia. Four hours after exposure to either LPS or saline vehicle, mice were imaged via hyperpolarized 3He MRI. All images were evaluated to identify regions of hypointense signals. Lungs were then characterized by conventional histology, or used to obtain tissue samples from regions of normal and hypointense 3He signals and analyzed for cytokine content. The characterization of 3He MRI images identified three distinct types of hypointense patterns: persistent defects, atelectatic defects, and dorsal lucencies. Persistent defects were associated with the administration of LPS. The number of persistent defects depended on the dose of LPS, with a significant increase in mean number of defects in 30–50-μg LPS-dosed mice versus saline-treated control mice. Atelectatic defects predominated in LPS-dosed mice under conditions of low-volume ventilation, and could be reversed with deep inspiration. Dorsal lucencies were present in nearly all mice studied, regardless of the experimental conditions, including control animals that did not receive LPS. A comparison of 3He MRI with histopathology did not identify tissue abnormalities in regions of low 3He signal, with the exception of a single region of atelectasis in one mouse. Furthermore, no statistically significant differences were evident in concentrations of IL-1β, IL-6, macrophage inflammatory protein (MIP)-1α, MIP-2, chemokine (C-X-C motif) ligand 1 (KC), TNFα, and monocyte chemotactic protein (MCP)-1 between hypointense and normally ventilated lung regions in LPS-dosed mice. Thus, this study defines the anatomic, functional, and biochemical characteristics of ventilation defects associated with the administration of LPS in a murine model of acute lung injury.
PMCID: PMC3095984  PMID: 20595465
ventilation defect; lipopolysaccharide; lung inflammation; magnetic resonance imaging; cytokines
7.  Allergy-related outcomes in relation to serum IgE: Results from the National Health and Nutrition Examination Survey 2005–2006 
The National Health and Nutrition Examination Survey (NHANES) 2005–2006 was the first population-based study to investigate levels of serum total and allergen-specific immunoglobulin E (IgE) in the general US population.
We estimated prevalence of allergy-related outcomes and examined relationships between serum IgE levels and these outcomes in a representative sample of the US population.
Data for this cross-sectional analysis were obtained from the NHANES 2005–2006. Study subjects aged 6 years and older (N=8086) had blood taken for measurement of total IgE and 19 specific IgEs against common aeroallergens, including Alternaria alternata, Aspergillus fumigatus, Bermuda grass, birch, oak, ragweed, Russian thistle, rye grass, cat dander, cockroach, dog dander, dust mite (Dermatophagoides farinae and D. pteronyssinus), mouse and rat urine proteins; and selected foods (egg white, cow’s milk, peanut, and shrimp). Serum samples were analyzed for total and allergen-specific IgEs using the Pharmacia CAP System. Information on allergy-related outcomes and demographics was collected by questionnaire.
In the NHANES 2005–2006, 6.6% reported current hay fever and 23.5% suffered from current allergies. Allergy-related outcomes increased with increasing total IgE (adjusted ORs for a 10-fold increase in total IgE =1.86, 95% CI:1.44–2.41 for hay fever and 1.64, 95% CI: 1.41–1.91 for allergies). Elevated levels of plant-, pet-, and mold-specific IgEs contributed independently to allergy-related symptoms. The greatest increase in odds was observed for hay fever and plant-specific IgEs (adjusted OR=4.75, 95% CI:3.83–5.88).
In the US population, self-reported allergy symptoms are most consistently associated with elevated levels of plant-, pet-, and mold-specific IgEs.
PMCID: PMC3108140  PMID: 21320720
allergen; allergy; allergic sensitization; serum IgE
8.  Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice 
Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.
PMCID: PMC3248288  PMID: 22182838
9.  Gene Delivery of Cytochrome P450 Epoxygenase Ameliorates Monocrotaline-Induced Pulmonary Artery Hypertension in Rats 
Pulmonary arterial hypertension (PAH) is a life-threatening disease that leads to progressive pulmonary hypertension, right heart failure, and death. Endothelial dysfunction and inflammation were implicated in the pathogenesis of PAH. Epoxyeicosatrienoic acids (EETs), products of the cytochrome P450 epoxygenase metabolism of arachidonic acid, are potent vasodilators that possess anti-inflammatory and other protective properties in endothelial cells. We investigated whether gene delivery with the human cytochrome P450 epoxygenase 2J2 (CYP2J2) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Significant pulmonary hypertension developed 3 weeks after the administration of MCT, but gene therapy with CYP2J2 significantly attenuated the development of pulmonary hypertension and pulmonary vascular remodeling, without causing changes in systemic arterial pressure or heart rate. These effects were associated with increased pulmonary endothelial NO synthase (eNOS) expression and its activity, inhibition of inflammation in the lungs, and transforming growth factor (TGF)-β/type II bone morphogenetic protein receptor (BMPRII)-drosophila mothers against decapentaplegic proteins (Smads) signaling. Collectively, these data suggest that gene therapy with CYP2J2 may have potential as a novel therapeutic approach to this progressive and oftentimes lethal disorder.
PMCID: PMC2993093  PMID: 20118222
arachidonic acids; cytochrome P450 epoxygenase; gene therapy; monocrotaline
10.  Roles of the epoxygenase CYP2J2 in the endothelium 
Prostaglandins & other lipid mediators  2013;107:10.1016/j.prostaglandins.2013.02.003.
Cytochrome p450 (CYP)2J2 is an epoxygenase enzyme that metabolises arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs are inactivated by soluble epoxide hydrolase (sEH), which converts them in to their corresponding dihydroxyeicosatrienoic acids (DHETs). CYP2J2 is highly expressed in cardiovascular tissue including the heart and vascular endothelial cells. CYP2J2 and the EETs it produces have been shown to have a diverse range of effects on the vasculature, including the regulation of inflammation, vascular tone, cellular proliferation, angiogenesis, and metabolism. This review will examine these established and emerging roles of CYP2J2 in the biology of vascular endothelial cells.
PMCID: PMC3711961  PMID: 23474289
endothelial; epoxygenase; inflammation; metabolism; angiogenesis; dilation
11.  Hormonal Influences on Lung Function and Response to Environmental Agents 
Numerous studies in humans and experimental animals have identified considerable sex differences in respiratory physiology and in the response of the lung to environmental agents. These differences appear to be mediated, at least in part, by sex hormones and their nuclear receptors. Moreover, animal models are increasingly used to study pathogenic mechanisms and test potential therapies for a variety of human lung diseases, many of which appear to be influenced by sex and sex hormones. In this article, data are summarized from studies of lung function and disease in which sex differences have been observed. Specific attention is paid to animal models of acute lung injury, nonallergic and allergic lung inflammation, and lung fibrosis. It is anticipated that continued investigation of the role of sex and sex hormones in animal models will provide valuable insight into the pathogenesis and potential treatments for a variety of acute and chronic human lung diseases.
PMCID: PMC3266051  PMID: 19934354
sex; sex hormones; respiratory mechanics; inflammation; airway
13.  Cytochrome P450 2C8 ω3-Long-Chain Polyunsaturated Fatty Acid Metabolites Increase Mouse Retinal Pathologic Neovascularization—Brief Report 
Regulation of angiogenesis is critical for many diseases. Specifically, pathological retinal neovascularization, a major cause of blindness, is suppressed with dietary ω3-long-chain polyunsaturated fatty acids (ω3LCPUFAs) through antiangiogenic metabolites of cyclooxygenase and lipoxygenase. Cytochrome P450 epoxygenases (CYP2C8) also metabolize LCPUFAs, producing bioactive epoxides, which are inactivated by soluble epoxide hydrolase (sEH) to transdihydrodiols. The effect of these enzymes and their metabolites on neovascularization is unknown.
Approach and Results
The mouse model of oxygen-induced retinopathy was used to investigate retinal neovascularization. We found that CYP2C (localized in wild-type monocytes/macrophages) is upregulated in oxygen-induced retinopathy, whereas sEH is suppressed, resulting in an increased retinal epoxide:diol ratio. With a ω3LCPUFA-enriched diet, retinal neovascularization increases in Tie2-driven human-CYP2C8–overexpressing mice (Tie2-CYP2C8-Tg), associated with increased plasma 19,20-epoxydocosapentaenoic acid and retinal epoxide:diol ratio. 19,20-Epoxydocosapentaenoic acids and the epoxide:diol ratio are decreased with overexpression of sEH (Tie2-sEH-Tg). Overexpression of CYP2C8 or sEH in mice does not change normal retinal vascular development compared with their wild-type littermate controls. The proangiogenic role in retina of CYP2C8 with both ω3LCPUFA and ω6LCPUFA and antiangiogenic role of sEH in ω3LCPUFA metabolism were corroborated in aortic ring assays.
Our results suggest that CYP2C ω3LCPUFA metabolites promote retinal pathological angiogenesis. CYP2C8 is part of a novel lipid metabolic pathway influencing retinal neovascularization.
PMCID: PMC4005334  PMID: 24458713
angiogenesis factor; cytochrome P450 CYP2C8 (human); pathologic neovascularization
14.  Indoor Allergens in School and Daycare Environments 
Most studies that have examined exposure to indoor allergens have focused on home environments. However, allergen exposures can be encountered in environments other than the home. For example, many children spend a large part of their time in schools and daycare facilities. Over the past two decades, a large number of studies have been conducted in school and daycare environments. However, the role of indoor exposures in allergy and asthma development or morbidity in these settings is not well characterized. The purpose of this review is to evaluate the importance of indoor allergen exposures in school and daycare settings. We summarize the key findings from recent scientific literature, describe exposure characteristics, discuss the role of these exposures in relation to asthma and allergy symptoms, and provide information on the effectiveness of published interventions.
PMCID: PMC2749571  PMID: 19577284
allergen; indoor; exposure; asthma; allergy; school; daycare
15.  Nanoparticle-Mediated Drug Delivery and Pulmonary Hypertension 
Hypertension  2009;53(5):751-753.
PMCID: PMC2694215  PMID: 19307468
16.  Cockroach Allergen Reduction by Cockroach Control Alone in Low-Income, Urban Homes-A Randomized Control Trial 
We previously reported significant reductions in cockroach allergen concentrations in urban homes by reducing cockroach infestations.
Determine the effectiveness of pest control performed by professional entomologists, compared to commercial companies, in reducing cockroach allergen.
This 3-arm randomized controlled trial enrolled 60 cockroach-infested homes in North Carolina. Homes were randomly assigned to a control group or one of two treatment groups. Treatment 1 had insecticide baits placed by entomologists from North Carolina State University. Treatment 2 received pest control from a randomly assigned commercial company. Vacuumed dust sampling and cockroach trapping were conducted at 0, 6 and 12 months. Dust samples were analyzed by ELISA.
In Treatment 1 homes, there were significant reductions in geometric mean trap counts compared to Control and Treatment 2 homes at 12 months. Relative to control, significant 12-month reductions in Bla g 1 were evident in Treatment 1 homes at all sampled sites, except bedroom floor. From baseline to month 12, geometric mean Bla g 1 concentrations (U/g) decreased from 64.2 to 5.6 in kitchen, 10.6 to 1.1 in living room, 10.7 to 1.9 in bedroom floor and 3.6 to 2.3 in bed. Treatment 2 homes showed no significant 12-month allergen reductions versus control.
Reductions in Bla g 1 in cockroach-infested homes can be achieved by reducing infestations; however, the magnitude of allergen reduction is dependent on the thoroughness and effectiveness of cockroach eradication efforts.
PMCID: PMC2804464  PMID: 17825893
cockroaches; cockroach allergen; Bla g 1; Bla g 2; indoor allergens; intervention trial
17.  The Cytochrome P450 Epoxygenase Pathway Regulates the Hepatic Inflammatory Response in Fatty Liver Disease 
PLoS ONE  2014;9(10):e110162.
Fatty liver disease is an emerging public health problem without effective therapies, and chronic hepatic inflammation is a key pathologic mediator in its progression. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory effects. Although promoting the effects of EETs elicits anti-inflammatory and protective effects in the cardiovascular system, the contribution of CYP-derived EETs to the regulation of fatty liver disease-associated inflammation and injury is unknown. Using the atherogenic diet model of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), our studies demonstrated that induction of fatty liver disease significantly and preferentially suppresses hepatic CYP epoxygenase expression and activity, and both hepatic and circulating levels of EETs in mice. Furthermore, mice with targeted disruption of Ephx2 (the gene encoding soluble epoxide hydrolase) exhibited restored hepatic and circulating EET levels and a significantly attenuated induction of hepatic inflammation and injury. Collectively, these data suggest that suppression of hepatic CYP-mediated EET biosynthesis is an important pathological consequence of fatty liver disease-associated inflammation, and that the CYP epoxygenase pathway is a central regulator of the hepatic inflammatory response in NAFLD/NASH. Future studies investigating the utility of therapeutic strategies that promote the effects of CYP-derived EETs in NAFLD/NASH are warranted.
PMCID: PMC4195706  PMID: 25310404
18.  Male Sex Hormones Exacerbate Lung Function Impairment after Bleomycin-Induced Pulmonary Fibrosis 
The roles of sex hormones as modulators of lung function and disease have received significant attention as differential sex responses to various lung insults have been recently reported. The present study used a bleomycin-induced pulmonary fibrosis model in C57BL/6 mice to examine potential sex differences in physiological and pathological outcomes. Endpoints measured included invasive lung function assessment, immunological response, lung collagen deposition, and a quantitative histological analysis of pulmonary fibrosis. Male mice had significantly higher basal static lung compliance than female mice (P < 0.05) and a more pronounced decline in static compliance after bleomycin administration when expressed as overall change or percentage of baseline change (P < 0.05). In contrast, there were no significant differences between the sexes in immune cell infiltration into the lung or in total lung collagen content after bleomycin. Total lung histopathology scores measured using the Ashcroft method did not differ between the sexes, while a quantitative histopathology scoring system designed to determine where within the lung the fibrosis occurred indicated a tendency toward more fibrosis immediately adjacent to airways in bleomycin-treated male versus female mice. Furthermore, castrated male mice exhibited a female-like response to bleomycin while female mice given exogenous androgen exhibited a male-like response. These data indicate that androgens play an exacerbating role in decreased lung function after bleomycin administration, and traditional measures of fibrosis may miss critical differences in lung function between the sexes. Sex differences should be carefully considered when designing and interpreting experimental models of pulmonary fibrosis in mice.
PMCID: PMC2438447  PMID: 18276795
fibrosis; bleomycin; sex; respiratory mechanics
19.  Role of caveolae in shear stress-mediated endothelium-dependent dilation in coronary arteries 
Cardiovascular Research  2013;100(1):151-159.
Caveolae are membrane microdomains where important signalling pathways are assembled and molecular effects transduced. In this study, we hypothesized that shear stress-mediated vasodilation (SSD) of mouse small coronary arteries (MCA) is caveolae-dependent.
Methods and results
MCA (80–150 μm) isolated from wild-type (WT) and caveolin-1 null (Cav-1−/−) mice were subjected to physiological levels of shear stress (1–25 dynes/cm2) with and without pre-incubation of inhibitors of nitric oxide synthase (L-NAME), cyclooxygenase (indomethacin, INDO), or cytochrome P450 epoxygenase (SKF 525A). SSD was endothelium-dependent in WT and Cav-1−/− coronaries but that in Cav-1−/− was significantly diminished compared with WT. Pre-incubation with L-NAME, INDO, or SKF 525A significantly reduced SSD in WT but not in Cav-1−/− mice. Vessels from the soluble epoxide hydrolase null (Ephx2−/−) mice showed enhanced SSD, which was further augmented by the presence of arachidonic acid. In donor–detector-coupled vessel experiments, Cav-1−/− donor vessels produced diminished dilation in WT endothelium-denuded detector vessels compared with WT donor vessels. Shear stress elicited a robust intracellular Ca2+ increase in vascular endothelial cells isolated from WT but not those from Cav-1−/− mice.
Integrity of caveolae is critical for endothelium-dependent SSD in MCA. Cav-1−/− endothelium is deficient in shear stress-mediated generation of vasodilators including NO, prostaglandins, and epoxyeicosatrienoic acids. Caveolae plays a critical role in endothelial signal transduction from shear stress to vasodilator production and release.
PMCID: PMC3778958  PMID: 23787000
Caveolae; Shear stress; Coronary artery; Soluble epoxide hydrolase; Calcium
20.  Role of Cyclooxygenase-2 in Exacerbation of Allergen-Induced Airway Remodeling by Multiwalled Carbon Nanotubes 
The emergence of nanotechnology has produced a multitude of engineered nanomaterials such as carbon nanotubes (CNTs), and concerns have been raised about their effects on human health, especially for susceptible populations such as individuals with asthma. Multiwalled CNTs (MWCNTs) have been shown to exacerbate ovalbumin (OVA)–induced airway remodeling in mice. Moreover, cyclooxygenase-2 (COX-2) has been described as a protective factor in asthma. We postulated that COX-2–deficient (COX-2−/−) mice would be susceptible to MWCNT-induced exacerbations of allergen-induced airway remodeling, including airway inflammation, fibrosis, and mucus-cell metaplasia (i.e., the formation of goblet cells). Wild-type (WT) or COX-2−/− mice were sensitized to OVA to induce allergic airway inflammation before a single dose of MWCNTs (4 mg/kg) delivered to the lungs by oropharyngeal aspiration. MWCNTs significantly increased OVA-induced lung inflammation and mucus-cell metaplasia in COX-2−/− mice compared with WT mice. However, airway fibrosis after exposure to allergen and MWCNTs was no different between WT and COX-2−/− mice. Concentrations of certain prostanoids (prostaglandin D2 and thromboxane B2) were enhanced by OVA or MWCNTs in COX-2−/− mice. No differences in COX-1 mRNA concentrations were evident between WT and COX-2−/− mice treated with OVA and MWCNTs. Interestingly, MWCNTs significantly enhanced allergen-induced cytokines involved in Th2 (IL-13 and IL-5), Th1 (CXCL10), and Th17 (IL-17A) inflammatory responses in COX-2−/− mice, but not in WT mice. We conclude that exacerbations of allergen-induced airway inflammation and mucus-cell metaplasia by MWCNTs are enhanced by deficiencies in COX-2, and are associated with the activation of a mixed Th1/Th2/Th17 immune response.
PMCID: PMC3824045  PMID: 23642096
carbon nanotubes; nanoparticles; asthma; inflammation; COX-2
21.  Cyclooxygenase-2 Deficiency Exacerbates Bleomycin-Induced Lung Dysfunction but Not Fibrosis 
Cyclooxygenase (COX)-derived eicosanoids have been implicated in the pathogenesis of pulmonary fibrosis. Uncertainty regarding the influence of COX-2 on experimental pulmonary fibrosis prompted us to clarify the fibrotic and functional effects of intratracheal bleomycin administration in mice genetically deficient in COX-2. Further, the effects of airway-specific COX-1 overexpression on fibrotic and functional outcomes in wild-type and COX-2 knockout mice were assessed. Equivalent increases in airway cell influx, lung collagen content, and histopathologic evidence of fibrosis were observed in wild-type and COX-2 knockout mice 21 d after bleomycin treatment, suggesting that COX-2 deficiency did not alter the extent or severity of fibrosis in this model. However, bleomycin-induced alterations in respiratory mechanics were more severe in COX-2 knockout mice than in wild-type mice, as illustrated by a greater decrease in static compliance compared with genotype-matched, saline-treated control mice (26 ± 3% versus 11 ± 4% decreases for COX-2 knockout and wild-type mice, respectively; P < 0.05). The influence of COX-1 overexpression in airway Clara cells was also examined. Whereas the fibrotic effects of bleomycin were not altered in wild-type or COX-2 knockout mice overexpressing COX-1, the exaggerated lung function decrement in bleomycin-treated COX-2 knockout mice was prevented by COX-1 overexpression and coincided with decreased airway cysteinyl leukotriene levels. Collectively, these data suggest an important regulatory role for COX-2 in the maintenance of lung function in the setting of lung fibrosis, but not in the progression of the fibrotic process per se.
PMCID: PMC1994226  PMID: 17496151
cyclooxygenase; fibrosis; respiratory mechanics; prostaglandin; transgenic
22.  Exposure to multiple indoor allergens in US homes and relationship to asthma 
The National Survey of Lead and Allergens in Housing was the first population-based study to measure indoor allergen levels in US homes.
We characterized the overall burden to multiple allergens and examined whether elevated allergen levels were associated with occupants’ asthma status.
This cross-sectional study surveyed a nationally representative sample of 831 housing units in 75 different locations throughout the US. Information was collected by questionnaire and environmental assessments. Allergen concentrations in dust samples were assessed by immunoassays. The following cut points were used to define elevated allergen levels: 10 μg/g for Der p 1, Der f 1, and Can f 1; 8 μg/g for Fel d 1; 8 U/g Bla g 1; 1.6 μg/g for mouse urinary protein; and 7 μg/g for Alternaria antigens. Allergen burden was considered high when 4 or more allergens exceeded elevated levels in any of the sampling locations.
Exposure to multiple allergens was common in US homes. Of the surveyed homes, 51.5% had at least 6 detectable allergens and 45.8% had at least 3 allergens exceeding elevated levels. Occupants’ race, income, housing type, absence of children, and presence of smokers, pets, cockroaches, rodents and mold/moisture related problems were independent predictors of high allergen burden. Among atopics, high allergen burden increased the odds of having asthma symptoms (OR=1.81, 95% CI: 1.04-3.15).
Elevated allergen levels in the home are associated with asthma symptoms in allergic individuals.
Clinical implication
In allergic asthma, indoor allergen exposures play an important role in asthma exacerbations.
PMCID: PMC2376121  PMID: 18255132
allergen; indoor; exposure; asthma; allergy
23.  G-protein Pathway Suppressor 2 (GPS2) Interacts with the Regulatory Factor X4 Variant 3 (RFX4_v3) and Functions as a Transcriptional Co-activator* 
The Journal of biological chemistry  2008;283(13):8580-8590.
The regulatory factor X4 variant 3 (RFX4_v3) is a brain-specific isoform of the transcription factor RFX4. Insertional mutagenesis in mice demonstrates that Rfx4_v3 is crucial for normal brain development. Many genes involved in critical processes during brain morphogenesis are dysregulated in Rfx4_v3 mutant brains. For example, Cx3cl1 is a CX3C-type chemokine that is abundant in brain and is a direct transcriptional target of RFX4_v3 through a specific promoter X-box (X-box 1), the responsive element for RFX proteins. To identify potential interacting partners for RFX4_v3, we performed yeast two-hybrid analysis. Nine candidate interactors were identified, including G-protein pathway suppressor 2 (GPS2). Indirect immunofluorescence demonstrated that GPS2 and RFX4_v3 co-localized to the nucleus. Both GPS2 and RFX4_v3 mRNAs were also present in most portions of the adult mouse brain, as well as in brains at different ages, suggesting that the two proteins could bind to each other. Co-immunoprecipitation assays indicated that physical interactions between GPS2 and RFX4_v3 did indeed occur. Furthermore, GPS2 was recruited to the Cx3cl1 promoter by RFX4_v3 and potentiated RFX4_v3 transactivation on this promoter through X-box 1, suggesting that the protein-protein interaction was functionally relevant. GPS2 bound to both the carboxyl-terminal region (amino acids 575-735) and the middle region (amino acids 250-574) of the RFX4_v3 protein. RFX4_v3 amino acids 1-574 stimulated the Cx3cl1 promoter to the similar extent as the full-length RFX4_v3 protein; however, deletion of the carboxyl-terminal region of RFX4_v3 impaired the co-activating abilities of GPS2. Based on these data, we conclude that GPS2 interacts with RFX4_v3 to modulate transactivation of genes involved in brain morphogenesis, including Cx3Cl1.
PMCID: PMC2365754  PMID: 18218630
24.  Asthma Cases Attributable to Atopy: Results from the Third National Health and Nutrition Examination Survey 
The percentage of asthma cases attributable to atopy is the subject of debate.
The objectives were to estimate the percentage of asthma cases in the U.S. population attributable to atopy and to examine associations between allergen-specific skin tests and asthma.
Data were obtained from NHANES III in which subjects aged 6–59 years were skin tested with 10 allergens. Atopy was defined as at least one positive allergen-specific test. Doctor-diagnosed current asthma was assessed by questionnaire.
In the U.S., 56.3% of the asthma cases were attributable to atopy, and that percentage was greater among males than females, among persons in the highest education category than in lower education categories, and among persons living in highly populated metropolitan areas than in all other areas. Each allergen-specific test was strongly associated with asthma before adjustment (odds ratios varied from 2.1 to 4.5); however, after adjustment by all the allergens, only tests to cat, Alternaria, white oak, and perennial rye were independently associated with asthma. Perennial rye was inversely associated with asthma. Of the 10 allergens, a positive response to cat accounted for the highest percentage of asthma cases (29.3%).
About half of the current asthma cases in the U.S. population represented by NHANES III were attributable to atopy. Some allergen-specific skin tests were not independently associated with asthma.
Clinical Implications
If atopy could be prevented or reversed, or its effect on asthma blocked, then a large percentage of asthma cases in the U.S. population could be prevented.
PMCID: PMC2291202  PMID: 17889931
allergens; allergic sensitization; allergy skin test; asthma; atopy; epidemiology; NHANES III; skin prick test; survey
25.  Gender Differences in Murine Airway Responsiveness and Lipopolysaccharide-Induced Inflammation1 
The roles of gender and sex hormones in lung function and disease are complex and not completely understood. The present study examined the influence of gender on lung function and respiratory mechanics in naive mice and on acute airway inflammation and hyperresponsiveness induced by intratracheal LPS administration. Basal lung function characteristics did not differ between naive males and females, but males demonstrated significantly greater airway responsiveness than females following aerosolized methacholine challenge as evidenced by increased respiratory system resistance and elastance (p < 0.05). Following LPS administration, males developed more severe hypothermia and greater airway hyperresponsiveness than females (p < 0.05). Inflammatory indices including bronchoalveolar lavage fluid total cells, neutrophils, and TNF-α content were greater in males than in females 6 h following LPS administration (p < 0.05), whereas whole-lung TLR-4 protein levels did not differ among treatment groups, suggesting that differential expression of TLR-4 before or after LPS exposure did not underlie the observed inflammatory outcomes. Gonadectomy decreased airway inflammation in males but did not alter inflammation in females, whereas administration of exogenous testosterone to intact females increased their inflammatory responses to levels observed in intact males. LPS-induced airway hyperresponsiveness was also decreased in castrated males and was increased in females administered exogenous testosterone. Collectively, these data indicate that airway responsiveness in naive mice is influenced by gender, and that male mice have exaggerated airway inflammatory and functional responses to LPS compared with females. These gender differences are mediated, at least in part, by effects of androgens.
PMCID: PMC2262913  PMID: 16785560

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