Aruni Jha

Airway mesenchymal cell death by mevalonate cascade inhibition: integration of autophagy, unfolded protein response and apoptosis focusing on Bcl2 family proteins.

HMG-CoA reductase, the proximal rate-limiting enzyme in the mevalonate pathway, is inhibited by statins. Beyond their cholesterol lowering impact, statins have pleiotropic effects and their use is linked to improved lung health. We have shown that mevalonate cascade inhibition induces apoptosis and autophagy in cultured human airway mesenchymal cells. Here, we show that simvastatin also induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in these cells. We tested whether coordination of ER stress, autophagy and apoptosis determines survival or demise of human lung mesenchymal cells exposed to statin. We observed that simvastatin exposure activates UPR (activated transcription factor 4, activated transcription factor 6 and IRE1α) and caspase-4 in primary human airway fibroblasts and smooth muscle cells. Exogenous mevalonate inhibited apoptosis, autophagy and UPR, but exogenous cholesterol was without impact, indicating that sterol intermediates are involved with mechanisms mediating statin effects. Caspase-4 inhibition decreased simvastatin-induced apoptosis, whereas inhibition of autophagy by ATG7 or ATG3 knockdown significantly increased cell death. In BAX(-/-)/BAK(-/-) murine embryonic fibroblasts, simvastatin-triggered apoptotic and UPR events were abrogated, but autophagy flux was increased leading to cell death via necrosis. Our data indicate that mevalonate cascade inhibition, likely associated with depletion of sterol intermediates, can lead to cell death via coordinated apoptosis, autophagy, and ER stress. The interplay between these pathways appears to be principally regulated by autophagy and Bcl-2-family pro-apoptotic proteins. These findings uncover multiple mechanisms of action of statins that could contribute to refining the use of such agent in treatment of lung disease.

Models to study airway smooth muscle contraction in vivo, ex vivo and in vitro : Implications in understanding asthma

Asthma is a chronic obstructive airway disease characterised by airway hyperresponsiveness (AHR) and airway wall remodelling. The effector of airway narrowing is the contraction of airway smooth muscle (ASM), yet the question of whether an inherent or acquired dysfunction in ASM contractile function plays a significant role in the disease pathophysiology remains contentious. The difficulty in determining the role of ASM lies in limitations with the models used to assess contraction. In vivo models provide a fully integrated physiological response but ASM contraction cannot be directly measured. Ex vivo and in vitro models can provide more direct assessment of ASM contraction but the loss of factors that may modulate ASM responsiveness and AHR, including interaction between multiple cell types and disruption of the mechanical environment, precludes a complete understanding of the disease process. In this review we detail key advantages of common in vivo, ex vivo and in vitro models of ASM contraction, as well as emerging tissue engineered models of ASM and whole airways. We also highlight important findings from each model with respect to the pathophysiology of asthma.

High-mobility group box 1 promotes extracellular matrix synthesis and wound repair in human bronchial epithelial cells

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) protein that binds Toll-like receptors (e.g., TLR4) and the receptor for advanced glycated end products (RAGE). The direct effects of HMGB1 on airway structural cells are not fully known. As epithelial cell responses are fundamental drivers of asthma, including abnormal repair-restitution linked to changes in extracellular matrix (ECM) synthesis, we tested the hypothesis that HMGB1 promotes bronchial epithelial cell wound repair via TLR4 and/or RAGE signaling that regulates ECM (fibronectin and the γ2-chain of laminin-5) and integrin protein abundance. To assess impact of HMGB1 we used molecular and pharmacological inhibitors of RAGE or TLR4 signaling in scratch wound, immunofluorescence, and immunoblotting assays to assess wound repair, ECM synthesis, and phosphorylation of intracellular signaling. HMGB1 increased wound closure, and this effect was attenuated by blocking RAGE and TLR4 signaling. HMGB1-induced fibronectin and laminin-5 (γ2 chain) was diminished by blocking RAGE and/or blunting TLR4 signaling. Similarly, induction of α3-integrin receptor for fibronectin and laminin-5 was also diminished by blocking TLR4 signaling and RAGE. Lastly, rapid and/or sustained phosphorylation of SMAD2, ERK1/2, and JNK signaling modulated HMGB1-induced wound closure. Our findings suggest a role for HMGB1 in human airway epithelial cell repair and restitution via multiple pathways mediated by TLR4 and RAGE that underpin increased ECM synthesis and modulation of cell-matrix adhesion.

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.

Novel drug targets for asthma and COPD: Lessons learned from in vitro and in vivo models

Asthma and chronic obstructive pulmonary disease (COPD) are highly prevalent respiratory diseases characterized by airway inflammation, airway obstruction and airway hyperresponsiveness. Whilst current therapies, such as β-agonists and glucocorticoids, may be effective at reducing symptoms, they do not reduce disease progression. Thus, there is a need to identify new therapeutic targets. In this review, we summarize the potential of novel targets or tools, including anti-inflammatories, phosphodiesterase inhibitors, kinase inhibitors, transient receptor potential channels, vitamin D and protease inhibitors, for the treatment of asthma and COPD.

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.

Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness

We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity.

A role for transient receptor potential ankyrin 1 cation channel (TRPA1) in airway hyper-responsiveness?

Airway smooth muscle (ASM) contraction controls the airway caliber. Airway narrowing is exaggerated in obstructive lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD). The mechanism by which ASM tone is dysregulated in disease is not clearly understood. Recent research on ion channels, particularly transient receptor potential cation channel, subfamily A, member 1 (TRPA1), is uncovering new understanding of altered airway function. TRPA1, a member of the TRP channel superfamily, is a chemo-sensitive cation channel that can be activated by a variety of external and internal stimuli, leading to the influx of Ca(2+). Functional TRPA1 channels have been identified in neuronal and non-neuronal tissues of the lung, including ASM. In the airways, these channels can regulate the release of mediators that are markers of airway inflammation in asthma and COPD. For, example, TRPA1 controls cigarette-smoke-induced inflammatory mediator release and Ca(2+) mobilization in vitro and in vivo, a response tied to disease pathology in COPD. Recent work has revealed that pharmacological or genetic inhibition of TRPA1 inhibits the allergen-induced airway inflammation in vitro and airway hyper-responsiveness (AHR) in vivo. Collectively, it appears that TRPA1 channels may be determinants of ASM contractility and local inflammation control, positioning them as part of novel mechanisms that control (patho)physiological function of airways and ASM.

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 β-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.

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

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.