Novel DNA methylation changes in mouse lungs associated with chronic smoking.

Smoking is a potent cause of asthma exacerbations, chronic obstructive pulmonary disease (COPD) and many other health defects, and changes in DNA methylation (DNAm) have been identified as a potential link between smoking and these health outcomes. However, most studies of smoking and DNAm have been done using blood and other easily accessible tissues in humans, while evidence from more directly affected tissues such as the lungs is lacking. Here, we identified DNAm patterns in the lungs that are altered by smoking. We used an established mouse model to measure the effects of chronic smoke exposure first on lung phenotype immediately after smoking and then after a period of smoking cessation. Next, we determined whether our mouse model recapitulates previous DNAm patterns observed in smoking humans, specifically measuring DNAm at a candidate gene responsive to cigarette smoke, Cyp1a1. Finally, we carried out epigenome-wide DNAm analyses using the newly released Illumina mouse methylation microarrays. Our results recapitulate some of the phenotypes and DNAm patterns observed in human studies but reveal 32 differentially methylated genes specific to the lungs which have not been previously associated with smoking. The affected genes are associated with nicotine dependency, tumorigenesis and metastasis, immune cell dysfunction, lung function decline, and COPD. This research emphasizes the need to study CS-mediated DNAm signatures in directly affected tissues like the lungs, to fully understand mechanisms underlying CS-mediated health outcomes.

Early-life exposure to cigarette smoke primes lung function and DNA methylation changes at Cyp1a1 upon exposure later in life.

Prenatal and early-life exposure to cigarette smoke (CS) has repeatedly been shown to induce stable, long-term changes in DNA methylation (DNAm) in offspring. It has been hypothesized that these changes might be functionally related to the known outcomes of prenatal and early-life CS exposure, which include impaired lung development, altered lung function, and increased risk of asthma and wheeze. However, to date, few studies have examined DNAm changes induced by prenatal CS in tissues of the lung, and even fewer have attempted to examine the specific influences of prenatal versus early postnatal exposures. Here, we have established a mouse model of CS exposure which isolates the effects of prenatal and early postnatal CS exposures in early life. We have used this model to measure the effects of prenatal and/or postnatal CS exposures on lung function and immune cell infiltration as well as DNAm and expression of Cyp1a1, a candidate gene previously observed to demonstrate DNAm differences on CS exposure in humans. Our study revealed that exposure to CS prenatally and in the early postnatal period causes long-lasting differences in offspring lung function, gene expression, and lung Cyp1a1 DNAm, which wane over time but are reestablished on reexposure to CS in adulthood. This study creates a testable mouse model that can be used to investigate the effects of prenatal and early postnatal CS exposures and will contribute to the design of intervention strategies to mediate these detrimental effects.NEW & NOTEWORTHY Here, we isolated effects of prenatal from early postnatal cigarette smoke and showed that exposure to cigarette smoke early in life causes changes in offspring DNA methylation at Cyp1a1 that last through early adulthood but not into late adulthood. We also showed that smoking in adulthood reestablished these DNA methylation patterns at Cyp1a1, suggesting that a mechanism other than DNA methylation results in long-term memory associated with early-life cigarette smoke exposures at this gene.

MicroRNA-200b deficiency is not sufficient to increase susceptibility to allergen-induced airway inflammation and dysfunction in mice.

MicroRNA-200b (miR-200b) has emerged as a therapeutic option for reducing inflammation and airway dysfunction in asthma. miR-200b belongs to a family of miRNAs that regulate epithelial-to-mesenchymal (EMT) transition and IL-33 abundance. In asthma, miR-200b abundance is reduced in the airways and is correlated with disease severity. In addition, prophylactic treatment with a miR-200b mimetic reduces airway inflammation and airway dysfunction in a mouse model. However, it is unclear whether miR-200b deficiency is sufficient to drive airway dysfunction and airway inflammation in asthma. Here, we show that male and female mice deficient in miR-200b do not display heightened airway inflammation or alterations in lung function that are characteristic of asthma. Following sensitization with house dust mite (HDM), female miR-200b knockout (KO) mice have elevated total lung resistance and male miR-200b KO have increased airway resistance. However, neither male nor female miR-200b mice display any changes in methacholine sensitivity or responsiveness and do not have enhanced HDM-induced airway inflammation. Collectively, these findings suggest that loss of miR-200b does not drive airway inflammation and airway dysfunction in mice. Thus, although treatment with exogenous miR-200b may ameliorate inflammation in asthma, deficiency of miR-200b is not likely driving pathobiology in asthma.NEW & NOTEWORTHY MicroRNA-200b regulates the abundance of key asthma-related genes. However, loss of miR-200b does not potentiate allergic asthma in a mouse model, suggesting that miR-200b deficiency may not be sufficient to drive of asthma pathogenesis.

The clinical & economic burden of treatment-resistant depression in the Gulf-Cooperation Council: The Kingdom of Saudi Arabia, Kuwait and the United Arab Emirates.

OBJECTIVE: The current analysis assessed the economic and clinical burden of treatment-Resistant Depression (TRD) imposed on the Kingdom of Saudi Arabia (KSA), Kuwait and United Arab Emirates (UAE) from the societal perspective. METHODS: A Microsoft Excel® based Markov model was developed to estimate the overall burden of disease imposed by TRD across KSA, Kuwait and UAE. Data for the models' adaptation were retrieved from literature and validated by country-specific key opinion leaders. The cycle length and time horizon used in the model were 4 weeks and 1 year, respectively. RESULTS: The study results estimated that at the end of 1-year time horizon, overall burden imposed by TRD was 3994, 982 and 670 million USD in KSA, Kuwait, and UAE, respectively. This can be attributed to the high cost incurred due to non-responsive health state (ranging from 44% to 47%). The productivity loss was either the greatest or second greatest component of TRD's burden in the countries of interest (ranging from 32% to 43%). CONCLUSIONS: TRD represents a large clinical and economic burden on both individual patients and society. Hence, noval and innovative treatments are especially required for the management of TRD patients.

PlexinD1 Deficiency in Lung Interstitial Macrophages Exacerbates House Dust Mite-Induced Allergic Asthma.

Interstitial macrophages (IMs) are key regulators of allergic inflammation. We previously showed that the absence of semaphorin 3E (Sema3E) exacerbates asthma features in both acute and chronic asthma models. However, it has not been studied whether Sema3E, via its receptor plexinD1, regulates IM function in allergic asthma. Therefore, we investigated the role of plexinD1 deficiency on IMs in allergic asthma. We found that the absence of plexinD1 in IMs increased airway hyperresponsiveness, airway leukocyte numbers, allergen-specific IgE, goblet cell hyperplasia, and Th2/Th17 cytokine response in the house dust mite (HDM)-induced allergic asthma model. Muc5ac, Muc5b, and α-SMA genes were increased in mice with Plxnd1-deficient IMs compared with wild-type mice. Furthermore, plexinD1-deficient bone marrow-derived macrophages displayed reduced IL-10 mRNA expression, at both the baseline and following HDM challenge, compared with their wild-type counterpart mice. Our data suggest that Sema3E/plexinD1 signaling in IMs is a critical pathway that modulates airway inflammation, airway resistance, and tissue remodeling in the HDM murine model of allergic asthma. Reduced IL-10 expression by plexinD1-deficient macrophages may account for these enhanced allergic asthma features.

Maternal diabetes promotes offspring lung dysfunction and inflammation in a sex-dependent manner.

Exposure to maternal diabetes is increasingly recognized as a risk factor for chronic respiratory disease in children. It is currently unclear; however, whether maternal diabetes affects the lung health of male and female offspring equally. This study characterizes the sex-specific impact of a murine model of diet-induced gestational diabetes (GDM) on offspring lung function and airway inflammation. Female adult mice are fed a high-fat (45% kcal) diet for 6 wk prior to mating. Control offspring are from mothers fed a low-fat (10% kcal) diet. Offspring were weaned and fed a chow diet until 10 wk of age, at which point lung function was measured and lung lavage was collected. Male, but not female, offspring exposed to GDM had increased lung compliance and reduced lung resistance at baseline. Female offspring exposed to GDM displayed increased methacholine reactivity and elevated levels of proinflammatory cytokines [e.g., interleukin (IL)-1ß, IL-5, and CXCL1] in lung lavage. Female GDM offspring also displayed elevated abundance of matrix metalloproteinases (MMP) within their airways, namely, MMP-3 and MMP-8. These results indicate disparate effects of maternal diabetes on lung health and airway inflammation of male and female offspring exposed to GDM. Female mice may be at greater risk of inflammatory lung conditions, such as asthma, whereas male offspring display changes that more closely align with models of chronic obstructive pulmonary disease. In conclusion, there are important sex-based differences in the impact of maternal diabetes on offspring lung health that could signal differences in future disease risk.

Disrupting Tryptophan in the Central Hydrophobic Region Selectively Mitigates Immunomodulatory Activities of the Innate Defence Regulator Peptide IDR-1002.

Innate defense regulator (IDR) peptides show promise as immunomodulatory therapeutics. However, there is limited understanding of the relationship of IDR peptide sequence and/or structure with its immunomodulatory activity. We previously reported that an IDR peptide, IDR-1002, reduces airway hyperresponsiveness (AHR) and inflammation in a house dust mite (HDM)-challenged murine model of airway inflammation. Here, we examined the sequence-to-function relationship of IDR-1002 in HDM-challenged mice and human bronchial epithelial cells (HBEC). We demonstrated that the tryptophan (W8) in the central hydrophobic region of IDR-1002 is required for the peptide to (i) suppress the pro-inflammatory cytokine IL-33, and induce anti-inflammatory mediators IL-1RA and stanniocalcin-1 in HBEC, and (ii) reduce IL-33 abundance, and eosinophil and neutrophil infiltration, in the lungs of HDM-challenged mice, without affecting the capacity to improve AHR, suggesting multimodal activity in vivo. Findings from this study can be used to design IDR peptides with targeted impact on immunomodulation and pathophysiology in respiratory diseases.

Integrating Proteomes for Lung Tissues and Lavage Reveals Pathways That Link Responses in Allergen-Challenged Mice.

To capture interplay between biological pathways, we analyzed the proteome from matched lung tissues and bronchoalveolar lavage fluid (BALF) of individual allergen-naïve and house dust mite (HDM)-challenged BALB/c mice, a model of allergic asthma. Unbiased label-free liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis quantified 2675 proteins from tissues and BALF of allergen-naïve and HDM-exposed mice. In comparing the four datasets, we found significantly greater diversity in proteins between lung tissues and BALF than in the changes induced by HDM challenge. The biological pathways enriched after allergen exposure were compartment-dependent. Lung tissues featured innate immune responses and oxidative stress, while BALF most strongly revealed changes in metabolism. We combined lung tissues and BALF proteomes, which principally highlighted oxidation reduction (redox) pathways, a finding influenced chiefly by the lung tissue dataset. Integrating lung and BALF proteomes also uncovered new proteins and biological pathways that may mediate lung tissue and BALF interactions after allergen challenge, for example, B-cell receptor signaling. We demonstrate that enhanced insight is fostered when different biological compartments from the lung are investigated in parallel. Integration of proteomes from lung tissues and BALF compartments reveals new information about protein networks in response to environmental challenge and interaction between intracellular and extracellular processes.

Allergen inhalation generates pro-inflammatory oxidised phosphatidylcholine associated with airway dysfunction.

Oxidised phosphatidylcholines (OxPCs) are produced under conditions of elevated oxidative stress and can contribute to human disease pathobiology. However, their role in allergic asthma is unexplored. The aim of this study was to characterise the OxPC profile in the airways after allergen challenge of people with airway hyperresponsiveness (AHR) or mild asthma. The capacity of OxPCs to contribute to pathobiology associated with asthma was also to be determined.Using bronchoalveolar lavage fluid from two human cohorts, OxPC species were quantified using ultra-high performance liquid chromatography-tandem mass spectrometry. Murine thin-cut lung slices were used to measure airway narrowing caused by OxPCs. Human airway smooth muscle (HASM) cells were exposed to OxPCs to assess concentration-associated changes in inflammatory phenotype and activation of signalling networks.OxPC profiles in the airways were different between people with and without AHR and correlated with methacholine responsiveness. Exposing patients with mild asthma to allergens produced unique OxPC signatures that associated with the severity of the late asthma response. OxPCs dose-dependently induced 15% airway narrowing in murine thin-cut lung slices. In HASM cells, OxPCs dose-dependently increased the biosynthesis of cyclooxygenase-2, interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor and the production of oxylipins via protein kinase C-dependent pathways.Data from human cohorts and primary HASM cell culture show that OxPCs are present in the airways, increase after allergen challenge and correlate with metrics of airway dysfunction. Furthermore, OxPCs may contribute to asthma pathobiology by promoting airway narrowing and inducing a pro-inflammatory phenotype and contraction of airway smooth muscle. OxPCs represent a potential novel target for treating oxidative stress-associated pathobiology in asthma.

Characterization of immune responses and the lung transcriptome in a murine model of IL-33 challenge.

IL-33 induces airway inflammation and hyper-responsiveness in respiratory diseases. Although defined as a therapeutic target, there are limited studies that have comprehensively investigated IL-33-mediated responses in the lungs in vivo. In this study, we characterized immunological and physiological responses induced by intranasal IL-33 challenge, in a mouse model. We identified specific cytokines, IL-4, IL-5, IL-6, IL-10, IP-10 and MIP1-α, that are increased in bronchoalveolar lavage and lung tissues by IL-33. Using transcriptomics (RNA-Seq) we demonstrated that 2279 transcripts were up-regulated and 1378 downregulated (≥ 2-fold, p < 0.01) in lung tissues, in response to IL-33. Bioinformatic interrogation of the RNA-Seq data was used to predict biological pathways and upstream regulators involved in IL-33-mediated responses. We showed that the mRNA and protein of STAT4, a predicted upstream regulator of IL-33-induced transcripts, was significantly enhanced in the lungs following IL-33 challenge. Overall, this study provides specific IL-33-induced molecular targets and endpoints that can be used as a resource for in vivo studies, e.g. in preclinical murine models examining novel interventions to target downstream effects of IL-33.

The importance of reporting house dust mite endotoxin abundance: impact on the lung transcriptome.

The abundance of lipopolysaccharide (LPS) in house dust mite (HDM) preparations is broad and mirrors the variability seen in the homes of people with asthma. LPS in commercially available stocks ranges from 31 to 5,2000 endotoxin units. The influence of vastly different LPS loads on the mechanisms that define the immune and inflammatory phenotype of HDM-challenged mice has not been defined. This aim of the study was to understand the lung phenotype of mice challenged with HDM extract containing high or low levels of LPS. Female BALB/c mice were sensitized for 2 wk with commercial HDM extract containing either high (36,000 endotoxin units; HHDM) or low (615 endotoxin units; LHDM) levels of LPS. Lung phenotype was characterized by measuring lung function, total and differential cell counts, cytokine abundance, and the lung transcriptome by RNA-sequencing. LPS levels in HDM stocks used for preclinical asthma research in mice remain poorly reported. In 2019, only 14% of papers specified LPS concentration in HDM lots. Specific differences existed in airway responsiveness between mice challenged with HHDM or LHDM. HHDM- and LHDM-induced cytokine profiles of bronchial lavage were significantly different and the lung transcriptome was differentially enriched for genes involved in DNA damage repair or cilium movement, following HHDM or LHDM challenge, respectively. The abundance of LPS in commercially available HDM influences the phenotype of allergic airways inflammation in mice. Failure to report the level of LPS in HDM extracts used in animal models of airway disease will lead to inconsistency in reproducibility and reliability of published data.

Immunomodulatory innate defence regulator (IDR) peptide alleviates airway inflammation and hyper-responsiveness.

BACKGROUND: Exacerbation in asthma is associated with decreased expression of specific host defence peptides (HDPs) in the lungs. We examined the effects of a synthetic derivative of HDP, innate defence regulator (IDR) peptide IDR-1002, in house dust mite (HDM)-challenged murine model of asthma, in interleukin (IL)-33-challenged mice and in human primary bronchial epithelial cells (PBECs). METHODS: IDR-1002 (6 mg/kg per mouse) was administered (subcutaneously) in HDM-challenged and/or IL-33-challenged BALB/c mice. Lung function analysis was performed with increasing dose of methacholine by flexiVent small animal ventilator, cell differentials in bronchoalveolar lavage performed by modified Wright-Giemsa staining, and cytokines monitored by MesoScale Discovery assay and ELISA. PBECs stimulated with tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), with or without IDR-1002, were analysed by western blots. RESULTS: IDR-1002 blunted HDM challenge-induced airway hyper-responsiveness (AHR), and lung leucocyte accumulation including that of eosinophils and neutrophils, in HDM-challenged mice. Concomitantly, IDR-1002 suppressed HDM-induced IL-33 in the lungs. IFN-γ/TNF-α-induced IL-33 production was abrogated by IDR-1002 in PBECs. Administration of IL-33 in HDM-challenged mice, or challenge with IL-33 alone, mitigated the ability of IDR-1002 to control leucocyte accumulation in the lungs, suggesting that the suppression of IL-33 is essential for the anti-inflammatory activity of IDR-1002. In contrast, the peptide significantly reduced either HDM, IL-33 or HDM+IL-33 co-challenge-induced AHR in vivo. CONCLUSION: This study demonstrates that an immunomodulatory IDR peptide controls the pathophysiology of asthma in a murine model. As IL-33 is implicated in steroid-refractory severe asthma, our findings on the effects of IDR-1002 may contribute to the development of novel therapies for steroid-refractory severe asthma.

Prophylactic benefits of systemically delivered simvastatin treatment in a house dust mite challenged murine model of allergic asthma.

BACKGROUND AND PURPOSE: Systemically delivered statins can blunt airway inflammation in ovalbumin-challenged mice. However, in asthma clinical trials the beneficial effects of introducing oral statins are not compelling. We have invetigated this discrepancy using a clinically relevant murine model of allergic asthma, and by including a prophylactic study arm. EXPERIMENTAL APPROACH: Adult mice were: 1) challenged with house dust mite (HDM) alone or with subcutaneous (s.c.) simvastatin for two weeks; or 2) also treated with simvastatin for one week prior to HDM challenge. We assayed lung function, inflammatory cell influx and cytokine profile, goblet cell abundance, and simvastatin concentration in serum, lung lavage and tissue. KEY RESULTS: Ultrahigh performance liquid chromatography-tandem mass spectrometry revealed that pharmacologically active simvastatin reached peak serum concentration after 8 h, but declined rapidly. Prophylactic treatment doubled peak serum simvastatin and repeated s.c. delivery established stable serum levels, but simvastatin was undetectable in the lungs. Both simvastatin treatment arms suppressed indices of HDM-induced airway inflammation and goblet cell hyperplasia, but this was significantly greater with prophylactic therapy, in particular, inhibition of neutrophil and eosinophil influx, and cytokine accumulation. Conversely, neither acute nor prophylactic delivery of simvastatin prevented HDM challenge-induced airway hyperreactivity. CONCLUSION AND IMPLICATIONS: Systemically administered simvastatin accumulates in the blood, but not in lung tissues, and reduces leukocyte influx and associated lung inflammation. Prophylactic therapy has the greatest anti-inflammatory effects, but as observed in human clinical trials, systemic simvastatin therapy does not prevent allergic airway hyperreactivity.

MicroRNA-200b regulates distal airway development by maintaining epithelial integrity.

miR-200b plays a role in epithelial-to-mesenchymal transition (EMT) in cancer. We recently reported abnormal expression of miR-200b in the context of human pulmonary hypoplasia in congenital diaphragmatic hernia (CDH). Smaller lung size, a lower number of airway generations, and a thicker mesenchyme characterize pulmonary hypoplasia in CDH. The aim of this study was to define the role of miR-200b during lung development. Here we show that miR-200b-/- mice have abnormal lung function due to dysfunctional surfactant, increased fibroblast-like cells and thicker mesenchyme in between the alveolar walls. We profiled the lung transcriptome in miR-200b-/- mice, and, using Gene Ontology analysis, we determined that the most affected biological processes include cell cycle, apoptosis and protein transport. Our results demonstrate that miR-200b regulates distal airway development through maintaining an epithelial cell phenotype. The lung abnormalities observed in miR-200b-/- mice recapitulate lung hypoplasia in CDH.

Biosignature for airway inflammation in a house dust mite-challenged murine model of allergic asthma.

House dust mite (HDM) challenge is commonly used in murine models of allergic asthma for preclinical pathophysiological studies. However, few studies define objective readouts or biomarkers in this model. In this study we characterized immune responses and defined molecular markers that are specifically altered after HDM challenge. In this murine model, we used repeated HDM challenge for two weeks which induced hallmarks of allergic asthma seen in humans, including airway hyper-responsiveness (AHR) and elevated levels of circulating total and HDM-specific IgE and IgG1. Kinetic studies showed that at least 24 h after last HDM challenge results in significant AHR along with eosinophil infiltration in the lungs. Histologic assessment of lung revealed increased epithelial thickness and goblet cell hyperplasia, in the absence of airway wall collagen deposition, suggesting ongoing tissue repair concomitant with acute allergic lung inflammation. Thus, this model may be suitable to delineate airway inflammation processes that precede airway remodeling and development of fixed airway obstruction. We observed that a panel of cytokines e.g. IFN-γ, IL-1ß, IL-4, IL-5, IL-6, KC, TNF-α, IL-13, IL-33, MDC and TARC were elevated in lung tissue and bronchoalveolar fluid, indicating local lung inflammation. However, levels of these cytokines remained unchanged in serum, reflecting lack of systemic inflammation in this model. Based on these findings, we further monitored the expression of 84 selected genes in lung tissues by quantitative real-time PCR array, and identified 31 mRNAs that were significantly up-regulated in lung tissue from HDM-challenged mice. These included genes associated with human asthma (e.g. clca3, ear11, il-13, il-13ra2, il-10, il-21, arg1 and chia1) and leukocyte recruitment in the lungs (e.g. ccl11, ccl12 and ccl24). This study describes a biosignature to enable broad and systematic interrogation of molecular mechanisms and intervention strategies for airway inflammation pertinent to allergic asthma that precedes and possibly potentiates airway remodeling and fibrosis.

Protective effect of Salacia oblonga against tobacco smoke-induced DNA damage and cellular changes in pancreatic ß-cells.

CONTEXT: Tobacco smoking generates a tremendous amount of free radicals that induce oxidative stress (OS) in diabetics (pancreatic islet cells are defective). Salacia oblonga Wall. (Celastraceae) is a proven antioxidant and antidiabetic plant whose mechanism of action is yet to be explored. OBJECTIVE: The present study focuses on the protective ability of S. oblonga in tobacco smoke-induced oxidatively stressed pancreatic ß-cell line. MATERIALS AND METHODS: The RINm5f cell line was exposed to tobacco smoke concentrate (TSC) (0.5-10%, 24 h), plant extract (1-75 µg/ml, 3 h), and their combinations. Cell viability was determined through MTT assay. Microscopic analysis was carried out in unstained and nonyl acridine orange-stained cells. The effect of toxic doses of TSC on DNA integrity was analyzed through DNA fragmentation assay. The TSC-induced nitric oxide generation was determined spectrophototmetrically. The expression of anti-apoptotic protein Bcl-X under the above treatment conditions was carried out through RT-PCR. RESULTS: The LD50 dose for TSC was found to be 1% TSC. Salacia oblonga extracts (10 and 15 µg/ml) were found to be optimum safe doses that significantly increased cell viability and decreased the nitric oxide production in TSC-treated cells. Pre-treatment with plant extract suppressed apoptosis through probable increase in the expression of anti-apoptotic protein Bcl-X in TSC-treated cells. Thus, the overall efficiency of plant extract in recovering cellular damage was proven. DISCUSSION AND CONCLUSION: The results suggest that TSC-induced cellular alterations are related to rise in nitric oxide and Bcl-X mRNA expression and propose that S. oblonga may confer significant cytoprotection against OS-mediated injury in ß-cells.

Characterization of the dystrophin-glycoprotein complex in airway smooth muscle: role of δ-sarcoglycan in airway responsiveness.

The dystrophin-glycoprotein complex (DGC) is an integral part of caveolae microdomains, and its interaction with caveolin-1 is essential for the phenotype and functional properties of airway smooth muscle (ASM). The sarcoglycan complex provides stability to the dystroglycan complex, but its role in ASM contraction and lung physiology in not understood. We tested whether δ-sarcoglycan (δ-SG), through its interaction with the DGC, is a determinant of ASM contraction ex vivo and airway mechanics in vivo. We measured methacholine (MCh)-induced isometric contraction and airway mechanics in δ-SG KO and wild-type mice. Last, we performed immunoblotting and transmission electron microscopy to assess DGC protein expression and the ultrastructural features of tracheal smooth muscle. Our results reveal an age-dependent reduction in the MCh-induced tracheal isometric force and significant reduction in airway resistance at high concentrations of MCh (50.0 mg/mL) in δ-SG KO mice. The changes in contraction and lung function correlated with decreased caveolin-1 and ß-dystroglycan abundance, as well as an age-dependent loss of caveolae in the cell membrane of tracheal smooth muscle in δ-SG KO mice. Collectively, these results confirm and extend understanding of a functional role for the DGC in the contractile properties of ASM and demonstrate that this results in altered lung function in vivo.

Chronic exposure to perfluorinated compounds: Impact on airway hyperresponsiveness and inflammation.

Emerging epidemiological evidence reveals a link between lung disease and exposure to indoor pollutants such as perfluorinated compounds (PFCs). PFC exposure during critical developmental stages may increase asthma susceptibility. Thus, in a murine model, we tested the hypothesis that early life and continued exposure to two ubiquitous household PFCs, perfluorooctanoic acid (PFOA) and perflurooctanesulfonic acid (PFOS), can induce lung dysfunction that exacerbates allergen-induced airway hyperresponsiveness (AHR) and inflammation. Balb/c mice were exposed to PFOA or PFOS (4 mg/kg chow) from gestation day 2 to 12 wk of age by feeding pregnant and nursing dams, and weaned pups. Some pups were also sensitized and challenged with ovalbumin (OVA). We assessed lung function and inflammatory cell and cytokine expression in the lung and examined bronchial goblet cell number. PFOA, but not PFOS, without the OVA sensitization/challenge induced AHR concomitant with a 25-fold increase of lung macrophages. PFOA exposure did not affect OVA-induced lung inflammatory cell number. In contrast, PFOS exposure inhibited OVA-induced lung inflammation, decreasing total cell number in lung lavage by 68.7%. Interferon-γ mRNA in the lung was elevated in all PFC-exposed groups. Despite these effects, neither PFOA nor PFOS affected OVA-induced AHR. Our data do not reveal PFOA or PFOS exposure as a risk factor for more severe allergic asthma-like symptoms, but PFOA alone can induce airway inflammation and alter airway function.

Role of dystrophin in airway smooth muscle phenotype, contraction and lung function.

Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD) and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC) and calponin and reduced Ca2+ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3ß and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh) when compared to genetic control BL10ScSnJ mice (wild-type). In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM.

Simultaneous quantification of simvastatin and simvastatin hydroxy acid in blood serum at physiological pH by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS).

Simvastatin attenuates airway inflammation and hyperreactivity, hallmarks of asthma, in allergen-challenged mice. As such, it is under consideration as a novel therapeutic, thus it is important to quantify the levels of simvastatin and its pharmacologically active and interconvertible metabolite, simvastatin hydroxy acid, that can be attained in the body. Methods exist to measure the concentrations of these compounds in biological media; however they do not maintain a physiological pH, and as a result do not accurately measure the ratio of these two compounds that exists in vivo. We developed a new method to measure simvastatin and simvastatin hydroxy acid more accurately in serum from mice by ultra high performance liquid chromatography-tandem mass spectrometry. We minimized the time that the compounds were in aqueous solution, and buffered samples to a physiological pH value of 7.4. Limits of quantification (LOQ) were 0.16 ng mL(-1) extract (1.3 ng mL(-1) serum) for simvastatin, and 8.3 ng mL(-1) extract (66 ng mL(-1) serum) for simvastatin hydroxy acid, respectively. No interconversion was observed, based on spike-and-recovery experiments of solutions containing both compounds. The method was applied using biological samples from mice challenged with house dust mite extract and simultaneously treated with subcutaneous simvastatin injection. Simvastatin hydroxy acid concentrations became significantly increased after a 2 week pre-treatment regime, whereas simvastatin concentrations were below the LOQ for all serum samples.