Autophagy and the unfolded protein response promote profibrotic effects of TGF-ß1 in human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-ß1 (TGF-ß1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3ßII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3ßII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-ß1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-ß1 induced the accumulation of LC3ßII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-ß1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-ß1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-ß1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-ß1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.

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.

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.

NMDA receptors mediate contractile responses in human airway smooth muscle cells.

Human airway smooth muscle (HASM) exhibits enhanced contractility in asthma. Inflammation is associated with airway hypercontractility, but factors that underpin these features are not fully elucidated. Glutamate toxicity associated with increased plasma glutamate concentrations was observed in airway inflammation, suggesting that multisubunit glutamate receptors, N-methyl-d-aspartate receptors (NMDA-R) contribute to airway hyperreactivity. We tested the hypothesis that HASM expresses NMDA-R subunits that can form functional receptors to mediate contractile responses to specific extracellular ligands. In cultured HASM cells, we measured NMDA-R subunit mRNA and protein abundance by quantitative PCR, immunoblotting, flow cytometry, and epifluorescence immunocytochemistry. We measured mRNA for a number of NMDA-R subunits, including the obligatory NR1 subunit, which we confirmed to be present as a protein. In vitro and ex vivo functional NMDA-R activation in HASM cells was measured using intracellular calcium flux (fura-2 AM), collagen gel contraction assays, and murine thin-cut lung slices (TCLS). NMDA, a pharmacological glutamate analog, induced cytosolic calcium mobilization in cultured HASM cells. We detected three different temporal patterns of calcium response, suggesting the presence of heterogeneous myocyte subpopulations. NMDA-R activation also induced airway contraction in murine TCLS and soft collagen gels seeded with HASM cells. Responses in cells, lung slices, and collagen gels were mediated by NMDA-R, as they could be blocked by (2R)-amino-5-phosphonopentanoate, a specific NMDA-R inhibitor. In summary, we reveal the presence of NMDA-R in HASM that mediate contractile responses via glutamatergic mechanisms. These findings suggest that accumulation of glutamate-like ligands for NMDA-R associated with airway inflammation contributes directly to airway hyperreactivity.

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.

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.

Jejunostomy tube feeding in patients undergoing esophagectomy.

BACKGROUND: Surgical jejunostomy tubes are a routine part of elective esophagectomies in patients with carcinomas and provide a route for nutritional support in those who experience complications. We wished to determine how frequently oral intake is delayed and the amount of nutrition delivered via the jejunostomy tube. METHODS: We reviewed the charts of all adults undergoing esophagectomy for carcinoma between January 2000 and June 2008. We determined the proportion of patients unable to resume oral nutrition after 8 days and the amount of nutrition delivered in each of the 8 days. RESULTS: In all, 111 patients underwent elective esophagectomy for carcinoma, and 103 had a jejunostomy tube placed. The mean age was 67 ± 10.8 years. The median time to oral intake was 7 (interquartile range 7-11) days. Seventy-four (67%) patients resumed oral intake within 8 days. The mean nutrition delivered by jejunostomy within the first 8 days as a percentage of the target was 45.6% (95% confidence interval 41.2%-49.9%). Six (5.4%) patients experienced complications attributable solely to the jejunostomy tube; 3 (2.9%) required surgery. Forty (38.8%) patients had abdominal issues serious enough to warrant delaying the progression of feeding. CONCLUSION: Two-thirds of patients undergoing elective esophagectomy were tolerating oral intake by the end of the eighth postoperative day, and less than half of the target nutrition was delivered over the first 8 days. We now selectively place surgical jejunostomy tubes in patients undergoing elective esophagectomies.

CONTEXTE: Des cathéters de jéjunostomie chirurgicale sont d'emblée posés lors des oesophagectomies non urgentes chez les patients atteints de cancer et procurent une voie d'administration du soutien nutritionnel chez les patients qui présentent des complications. Nous avons voulu déterminer la fréquence à laquelle la prise orale est retardée et la quantité de solution pour nutrition parentérale administrée par le cathéter de jéjunostomie. MÉTHODES: Nous avons analysé les dossiers de tous les adultes soumis à une oesophagectomie pour un cancer entre janvier 2000 et juin 2008. Nous avons calculé la proportion de patients incapables de recommencer à se nourrir par la bouche après 8 jours et la quantité de solution administrée à chacun des 8 jours. RÉSULTATS: En tout, 111 patients ont subi une oesophagectomie non urgente pour un cancer et on a posé un cathéter de jéjunostomie à 103 d'entre eux. L'âge moyen était de 67 ± 10,8 ans. L'intervalle médian avant le début des prises orales a été de 7 jours (fourchette interquartile de 7­11). Soixante-quatorze patients (67 %) ont recommencé à s'alimenter par la bouche en l'espace de 8 jours. La quantité moyenne de solution pour nutrition parentérale administrée par jéjunostomie au cours des 8 premiers jours en pourcentage de l'objectif cible a été de 45,6 % (intervalle de confiance [IC] de 95 %, 41,2 %­49,9 %). Six patients (5,4 %) ont présenté des complications attribuables uniquement au cathéter de jéjunostomie; 3 (2,9 %) ont eu besoin d'une chirurgie. Quarante patients (38,8 %) ont présenté des symptômes abdominaux suffi - samment graves pour retarder la progression de l'alimentation. CONCLUSION: Les deux tiers des patients soumis à une oesophagectomie non urgente toléraient la prise orale à la fin du huitième jour postopératoire et moins de la moitié de la nutrition cible a été administrée au cours des 8 premiers jours. Nous plaçons maintenant des cathéters de jéjunostomie chirurgicale de façon sélective chez les patients qui subissent des oesophagectomies non urgentes.

Laminin drives survival signals to promote a contractile smooth muscle phenotype and airway hyperreactivity.

Increased airway smooth muscle (ASM) mass is believed to underlie the relatively fixed airway hyperresponsiveness (AHR) in asthma. Developments of therapeutic approaches to reverse airway remodeling are impeded by our lack of insight on the mechanisms behind the increase in mass of contractile ASM cells. Increased expression of laminin, an extracellular matrix protein, is associated with asthma. Our studies investigate the role of laminin-induced ASM survival signals in the development of increased ASM and AHR. Antagonizing laminin integrin binding using the laminin-selective competing peptide, YIGSR, and mimicking laminin with exogenous α2-chain laminin, we show that laminin is both necessary and sufficient to induce ASM cell survival, concomitant with the induction of ASM contractile phenotype. Using siRNA, we show that the laminin-binding integrin α7ß1 mediates this process. Moreover, in laminin-211-deficient mice, allergen-induced AHR was not observed. Notably, ASM cells from asthmatic airways express a higher abundance of intracellular cell survival proteins, consistent with a role for reduced rates of cell apoptosis in development of ASM hyperplasia. Targeting the laminin-integrin α7ß1 signaling pathway may offer new avenues for the development of therapies to reduce the increase in mass of contractile phenotype ASM cells that underlie AHR in asthma.

Leiomyoma of the trachea.

Primary tracheal tumors are rare. Approximately 1% of them are leiomyoma. Given the rarity of these lesions, optimal management has not been defined. Bronchoscopic, local surgical excision and partial tracheal resection have all been described. One report of recurrence after resection has been published. The incidence of recurrence following local excision is unknown. We report a case of an incidental tracheal leiomyoma diagnosed and treated with a combined approach.

Geranylgeranyl transferase 1 modulates autophagy and apoptosis in human airway smooth muscle.

Geranylgeranyl transferase 1 (GGT1) is involved in the posttranslational prenylation of signaling proteins, such as small GTPases. We have shown that blocking the formation of isoprenoids with statins regulates survival of human lung mesenchymal cells; thus, we tested the hypothesis that GGT1 may specifically modulate programmed cell death pathways in these cells. To this end, human airway smooth muscle (HASM) cells were treated with the selective GGT1 inhibitor GGTi-298. Apoptosis was seen using assays for cellular DNA content and caspase activation. Induction of autophagy was observed using transmission electron microscopy, immunoblotting for LC3 lipidation and Atg5-12 complex content, and confocal microscopy to detect formation of lysosome-localized LC3 punctae. Notably, GGT1 inhibition induced expression of p53-dependent proteins, p53 upregulated modulator of apoptosis (Noxa), and damage-regulated autophagy modulator (DRAM), this was inhibited by the p53 transcriptional activation inhibitor cyclic-pifithrin-α. Inhibition of autophagy with bafilomycin-A1 or short-hairpin RNA silencing of Atg7 substantially augmented GGTi-298-induced apoptosis. Overall, we demonstrate for the first time that pharmacological inhibition of GGT1 induces simultaneous p53-dependent apoptosis and autophagy in HASM. Moreover, autophagy regulates apoptosis induction. Thus, our findings identify GGT1 as a key regulator of HASM cell viability.

Simvastatin inhibits TGFß1-induced fibronectin in human airway fibroblasts.

BACKGROUND: Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-ß1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFß1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects. METHODS: We used simvastatin (1-15 µM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 µM) and farnesyl transferase (FT; FTI-277, 10 µM) were used to determine whether GGT1 and FT contribute to TGFß1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 µM) or farnesylpyrophosphate (30 µM). RESULTS: Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFß1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFß1-induced signaling. Asthmatic fibroblasts exhibited greater TGFß1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin. CONCLUSIONS: We conclude that TGFß1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.

Src mediates cytokine-stimulated gene expression in airway myocytes through ERK MAPK.

The p38 and extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) participate in cytokine-stimulated inflammatory gene expression in airway smooth muscle cells. The following study was undertaken to determine whether Src tyrosine kinases are signaling intermediaries upstream of cytokine-stimulated MAPK activation and gene expression. Treating human airway myocytes with interleukin (IL)-1ß, tumor necrosis factor (TNF) α and interferon (IFN) γ caused a rapid 1.8-fold increase in Src family tyrosine kinase activity within 1 minute that remained 2.3 to 2.7 fold above basal conditions for 15 minutes. This activity was blocked by addition of 30 µM PP1, a pyrimidine inhibitor specific for Src family tyrosine kinases, in immune-complex assays to confirm that this stimulus activates Src tyrosine kinase. Addition of PP1 also blocked cytokine-stimulated expression of IL-1ß, IL-6 and IL-8, while decreasing phosphorylation of ERK, but not p38 MAPK. Since this inflammatory stimulus may activate additional inflammatory signaling pathways downstream of Src, we tested the effects of PP1 on phosphorylation of signal transducers and activators of transcription (STAT). PP1 had no effect on cytokine-stimulated STAT 1 or STAT 3 phosphorylation. These results demonstrate that Src tyrosine kinases participate in the regulation of IL-1ß, IL-6 and IL-8 expression and that these effects of Src are mediated through activation of ERK MAPK and not p38 MAPK or STAT1/STAT3 phosphorylation.

Mevalonate cascade regulation of airway mesenchymal cell autophagy and apoptosis: a dual role for p53.

Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM) and human airway fibroblasts (HAF), autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3) and immunoblotting (LC3 lipidation and Atg12-5 complex formation). Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA), NOXA, and damage-regulated autophagy modulator (DRAM). Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin-α and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis) and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy). Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease.

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells.

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-ß(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-ß(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-ß(1). The failure by TGF-ß(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-ß(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.

The mevalonate cascade as a target to suppress extracellular matrix synthesis by human airway smooth muscle.

Smooth muscle cells promote fibroproliferative airway remodeling in asthma, and transforming growth factor ß1 (TGFß1) is a key inductive signal. Statins are widely used to treat hyperlipidemia. Growing evidence indicates they also exert a positive impact on lung health, but the underlying mechanisms are unclear. We assessed the effects of 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase inhibition with simvastatin on the fibrotic function of primary cultured human airway smooth muscle cells. Simvastatin blocked de novo cholesterol synthesis, but total myocyte cholesterol content was unaffected. Simvastatin also abrogated TGFß1-induced collagen I and fibronectin expression, and prevented collagen I secretion. The depletion of mevalonate cascade intermediates downstream from HMG-CoA underpinned the effects of simvastatin, because co-incubation with mevalonate, geranylgeranylpyrophosphate, or farnesylpyrophosphate prevented the inhibition of matrix protein expression. We also showed that human airway myocytes express both geranylgeranyl transferase 1 (GGT1) and farnesyltransferase (FT), and the inhibition of GGT1 (GGTI inhibitor-286, 10 µM), but not FT (FTI inhibitor-277, 10 µM), mirrored the suppressive effects of simvastatin on collagen I and fibronectin expression and collagen I secretion. Moreover, simvastatin and GGTI-286 both prevented TGFß1-induced membrane association of RhoA, a downstream target of GGT1. Our findings suggest that simvastatin and GGTI-286 inhibit synthesis and secretion of extracellular matrix proteins by human airway smooth muscle cells by suppressing GGT1-mediated posttranslational modification of signaling molecules such as RhoA. These findings reveal mechanisms related to evidence for the positive impact of statins on pulmonary health.

beta-Dystroglycan binds caveolin-1 in smooth muscle: a functional role in caveolae distribution and Ca2+ release.

The dystrophin-glycoprotein complex (DGC) links the extracellular matrix and actin cytoskeleton. Caveolae form membrane arrays on smooth muscle cells; we investigated the mechanism for this organization. Caveolin-1 and beta-dystroglycan, the core transmembrane DGC subunit, colocalize in airway smooth muscle. Immunoprecipitation revealed the association of caveolin-1 with beta-dystroglycan. Disruption of actin filaments disordered caveolae arrays, reduced association of beta-dystroglycan and caveolin-1 to lipid rafts, and suppressed the sensitivity and responsiveness of methacholine-induced intracellular Ca2+ release. We generated novel human airway smooth muscle cell lines expressing shRNA to stably silence beta-dystroglycan expression. In these myocytes, caveolae arrays were disorganized, caveolae structural proteins caveolin-1 and PTRF/cavin were displaced, the signaling proteins PLCbeta1 and G(alphaq), which are required for receptor-mediated Ca2+ release, were absent from caveolae, and the sensitivity and responsiveness of methacholine-induced intracellular Ca2+ release, was diminished. These data reveal an interaction between caveolin-1 and beta-dystroglycan and demonstrate that this association, in concert with anchorage to the actin cytoskeleton, underpins the spatial organization and functional role of caveolae in receptor-mediated Ca2+ release, which is an essential initiator step in smooth muscle contraction.

Statin-triggered cell death in primary human lung mesenchymal cells involves p53-PUMA and release of Smac and Omi but not cytochrome c.

Statins inhibit 3-hydroxy-3-methyl-glutarylcoenzyme CoA (HMG-CoA) reductase, the proximal enzyme for cholesterol biosynthesis. They exhibit pleiotropic effects and are linked to health benefits for diseases including cancer and lung disease. Understanding their mechanism of action could point to new therapies, thus we investigated the response of primary cultured human airway mesenchymal cells, which play an effector role in asthma and chronic obstructive lung disease (COPD), to simvastatin exposure. Simvastatin induced apoptosis involving caspase-9, -3 and -7, but not caspase-8 in airway smooth muscle cells and fibroblasts. HMG-CoA inhibition did not alter cellular cholesterol content but did abrogate de novo cholesterol synthesis. Pro-apoptotic effects were prevented by exogenous mevalonate, geranylgeranyl pyrophosphate and farnesyl pyrophosphate, downstream products of HMG-CoA. Simvastatin increased expression of Bax, oligomerization of Bax and Bak, and expression of BH3-only p53-dependent genes, PUMA and NOXA. Inhibition of p53 and silencing of p53 unregulated modulator of apoptosis (PUMA) expression partly counteracted simvastatin-induced cell death, suggesting a role for p53-independent mechanisms. Simvastatin did not induce mitochondrial release of cytochrome c, but did promote release of inhibitor of apoptosis (IAP) proteins, Smac and Omi. Simvastatin also inhibited mitochondrial fission with the loss of mitochondrial Drp1, an essential component of mitochondrial fission machinery. Thus, simvastatin activates novel apoptosis pathways in lung mesenchymal cells involving p53, IAP inhibitor release, and disruption of mitochondrial fission.

Effect of maintenance azithromycin on established bronchiolitis obliterans syndrome in lung transplant patients.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS), the main cause of late mortality following lung transplantation, is defined as an irreversible decline in forced expiratory volume in 1 s (FEV1). Previous studies using azithromycin for BOS in lung transplant patients have demonstrated a potential reversibility of the decline in FEV1. OBJECTIVES: To examine whether initiating azithromycin reverses decline in FEV1 in lung transplant recipients with established BOS of at least three months. METHODS: Pulmonary function tests were performed every three months in seven lung transplant recipients with established BOS of at least three months. FEV1 was recorded at six and three months before initiation, at time of initiation, and three, six, nine and 12 months postazithromycin initiation. The primary end point was change in FEV1. During the study, no immunosuppressive medication changes or acute rejection episodes occurred. RESULTS: Mean time from transplant to azithromycin initiation was 64 months (range 17 to 117 months). Mean time from BOS diagnosis to azithromycin initiation was 22 months (range three to 67 months). Rate of FEV1 decline from six months before azithromycin initiation, and rates of FEV1 increase from initiation to three and 12 months post-treatment initiation, were not statistically significant (P=0.32, P=0.16 and P=0.18, respectively). Following a trend toward improvement in the first three months after treatment initiation, FEV1 tended to stabilize. DISCUSSION: Although several studies address the possible benefit of maintenance azithromycin in lung transplant patients with BOS, the role of the drug remains unproven in these patients, and would best be addressed by a large randomized controlled trial.

Expression of the dystrophin-glycoprotein complex is a marker for human airway smooth muscle phenotype maturation.

Airway smooth muscle (ASM) cells may contribute to asthma pathogenesis through their capacity to switch between a synthetic/proliferative and a contractile phenotype. The multimeric dystrophin-glycoprotein complex (DGC) spans the sarcolemma, linking the actin cytoskeleton and extracellular matrix. The DGC is expressed in smooth muscle tissue, but its functional role is not fully established. We tested whether contractile phenotype maturation of human ASM is associated with accumulation of DGC proteins. We compared subconfluent, serum-fed cultures and confluent cultures subjected to serum deprivation, which express a contractile phenotype. Western blotting confirmed that beta-dystroglycan, beta-, delta-, and epsilon-sarcoglycan, and dystrophin abundance increased six- to eightfold in association with smooth muscle myosin heavy chain (smMHC) and calponin accumulation during 4-day serum deprivation. Immunocytochemistry showed that the accumulation of DGC subunits was specifically localized to a subset of cells that exhibit robust staining for smMHC. Laminin competing peptide (YIGSR, 1 microM) and phosphatidylinositol 3-kinase (PI3K) inhibitors (20 microM LY-294002 or 100 nM wortmannin) abrogated the accumulation of smMHC, calponin, and DGC proteins. These studies demonstrate that the accumulation of DGC is an integral feature for phenotype maturation of human ASM cells. This provides a strong rationale for future studies investigating the role of the DGC in ASM smooth muscle physiology in health and disease.