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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-β1 (TGF-β1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3βII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3βII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-β1-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-β1 induced the accumulation of LC3βII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-β1-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-β1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-β1-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-β1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these 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.

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-β(1) (TGF-β(1)) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3βII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3βII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-β(1)-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-β(1) induced the accumulation of LC3βII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-β(1)-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-β(1) induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-β1-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-β(1) in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.

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. © 2014 Elsevier B.V.

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.

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)-beta 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-beta 1 induced profound increases in the contractile phenotype markers sm-a-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-beta 1. The failure by TGF-beta 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-beta 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.

Smooth muscle cells promote fibroproliferative airway remodeling in asthma, and transforming growth factor β1 (TGFβ1) 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 TGFβ1-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 TGFβ1-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.

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.

Contractile airway smooth muscle (ASM) cells retain the ability for phenotype plasticity in response to multiple stimuli, which equips them with capacity to direct modeling and remodeling during development, and in disease states such as asthma. We have shown that endogenously expressed laminin is required for maturation of human ASM cells to a contractile phenotype, as occurs during ASM thickening in asthma. In this study, we profiled the expression of laminin-binding integrins alpha3beta1, alpha6beta1, and alpha7beta1, and tested whether they are required for laminin-induced myocyte maturation. Immunoblotting revealed that myocyte maturation induced by prolonged serum withdrawal, which was marked by the accumulation of contractile phenotype marker protein desmin, was also associated with the accumulation of alpha3A, alpha6A, and alpha7B. Flow cytometry revealed that alpha7B expression was a distinct feature of individual myocytes that acquired a contractile phenotype. siRNA knockdown of alpha7, but not alpha3 or alpha6, suppressed myocyte maturation. Thus, alpha7B is a novel marker of the contractile phenotype, and alpha7 expression is essential for human ASM cell maturation, which is a laminin-dependent process. These observations provide new insight into mechanisms that likely underpin normal development and remodeling associated with airways disease.

Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca2+homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca2+([Ca2+]i) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M3receptors (M3R) and Gαq/11cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with β-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-β-cyclodextrin (mβCD) reduced sensitivity but not maximum [Ca2+]iinduced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mβCD disrupted the colocalization of caveolae-1 and M3R, but [ N-methyl-3H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca2+]iflux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mβCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca2+]imobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca2+]imobilization leading to ASM contraction induced by submaximal concentrations of ACh.

© 2015, National Research Council of Canada. All rights reserved. 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.

Chronic airways diseases, including asthma, are associated with an increased airway smooth muscle (ASM) mass, which may contribute to chronic airway hyperresponsiveness. Increased muscle mass is due, in part, to increased ASM proliferation, although the precise molecular mechanisms for this response are not completely clear. Caveolae, which are abundant in smooth muscle cells, are membrane microdomains where receptors and signaling effectors can be sequestered. We hypothesized that caveolae and caveolin-1 play an important regulatory role in ASM proliferation. Therefore, we investigated their role in p42/p44 MAPK signaling and proliferation using human ASM cell lines. Disruption of caveolae using methyl-β-cyclodextrin and small interfering (si)RNA-knockdown of caveolin-1 caused spontaneous p42/p44 MAPK activation; additionally, caveolin-1 siRNA induced ASM proliferation in mitogen deficient conditions, suggesting a key role for caveolae and caveolin-1 in maintaining quiescence. Moreover, caveolin-1 accumulates twofold in myocytes induced to a contractile phenotype compared with proliferating ASM cells. Caveolin-1 siRNA failed to increase PDGF-induced p42/p44 MAPK activation and cell proliferation, however, indicating that PDGF stimulation actively reversed the antimitogenic control by caveolin-1. Notably, the PDGF induced loss of antimitogenic control by caveolin-1 coincided with a marked increase in caveolin-1 phosphorylation. Furthermore, the strong association of PDGF receptor-β with caveolin-1 that exists in quiescent cells was rapidly and markedly reduced with agonist addition. This suggests a dynamic relationship in which mitogen stimulation actively reverses caveolin-1 suppression of p42/p44 MAPK signal transduction. As such, caveolae and caveolin-1 coordinate PDGF receptor signaling, leading to myocyte proliferation, and inhibit constitutive activity of p42/p44 MAPK to sustain cell quiescence.

Airway neutrophilia has been recognized as a predominant feature of acute lung disorders. While it has been shown that IL-17 induces expression of the CXC chemokines in the airways leading to neutrophil recruitment, the IL-17R expression in human ASM cells and the molecular mechanism by which IL-17 mediates neutrophilic chemo-attractant CXCL-8 (IL-8) production have not been determined. Our study showed that ASM cells express steady state IL-17R protein, mRNA and surface-bound receptor. Interestingly, airway sections from COPD patients revealed IL-17R-positive immunostaining within ASM bundles. IL-17 was capable of stimulating CXCL-8 protein release from ASM cells which was significantly decreased by neutralizing anti-IL-17 mAb. Furthermore, IL-17 induction of CXCL-8 mRNA and protein release from ASM cells was abrogated by transcriptional inhibitor actinomycin D. CXCL-8 promoter reporter analysis using wild type and site specific mutant constructs demonstrated a key role for AP1 and NF-kappaB binding sites in IL-17-induced CXCL-8 expression. These data demonstrate that IL-17 mediates CXCL-8 expression in ASM cells via a transcriptional mechanism depending on NF-kappaB and AP-1 pathways. Together, our findings suggest that ASM cells play an important role in airway neutrophilia.

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.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.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.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.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.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.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 7–11). 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.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.

In anaphylactic shock (AS), the relative effects of the autacoids including histamine, prostaglandins, and leukotrienes on causing cardiovascular collapse and the extent to which receptor blocking agents and pathway inhibitors may prevent this collapse are not clear. In a ragweed model of anaphylaxis, we examined whether pretreatment with H1, H2, H3 receptor blockers, and cyclooxygenase and leukotriene pathway inhibitors was useful in preventing the depression in left ventricular (LV) contractility known to occur in this model. The dose of allergen was varied to produce similar degrees of shock between treatments. The animals were studied under pentobarbital anesthesia in which the treatment studies were approximately 3 wk apart. LV volumes were measured by sonomicrometric techniques. During challenge, mean arterial blood pressure (Pa), cardiac output (Q), and LV end-diastolic pressure (LVEDP) decreased approximately 50% compared with preshock values in all treatments. Histamine H3 receptor blockade was associated with higher heart rates (HR) and higher stroke work (SW) (p0.05) as compared with the other treatment studies. We conclude that histamine H3 activation by inhibiting adrenergic neural norepinephrine release contributes to cardiovascular collapse in AS.

We examined the effect of anaphylactic shock on the longitudinal distribution of pulmonary vascular resistance (PVR) in ragweed-sensitized dogs in which PVR was partitioned into an upstream arterial component (Rus) and a downstream venous and capillary component (Rds). We also assessed whether Rus and Rds would be reduced by pretreatment with histamine H1- and H2-receptor blocking agents and with cyclooxygenase and lipoxygenase pathway inhibitors. Anesthetized animals were examined on separate occasions 3 wk apart in which one of the treatments was randomly given. The pulmonary arterial occlusion technique was used to determine segmental pressure drops. During ragweed challenge, PVR increased ≈4 times compared with the preshock value (3.04 vs. 12.07 mmHg ⋅ l−1⋅ min; P< 0.05). Although both Rus and Rds increased postshock, the greatest relative increase occurred in Rds. None of the treatments reduced partitioned resistances compared with no treatment. Our results show that, under conditions of anaphylactic shock, increases in Rus and Rds could not be ascribed to release of histamine or products of the cyclooxygenase and lipoxygenase pathways.

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 α7β1 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 α7β1 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.—Tran, T., Teoh, C. M., Tam, J. K. C., Qiao, Y., Chin, C. Y., Chong, O. K., Stewart, A. G., Harris, T., Wong, W. S. F., Guan, S. P., Leung, B. P., Gerthoffer, W. T., Unruh, H., and Halayko, A. J. Laminin drives survival signals to promote a contractile smooth muscle phenotype and airway hyperreactivity.

Epidural thoracic fentanyl infusions provide effective preoperative analgesia after thoracotomy; however, side effects can limit the effectiveness of this technique. This study evaluates epinephrine as an adjunct to continuous thoracic epidural fentanyl infusions after thoracotomy. Thirty-eight patients were studied in a prospective, randomized, double-blind trial comparing fentanyl alone to fentanyl with epinephrine 1:300,000. Epidural infusion rates were titrated to equivalent pain relief using a visual analog scale. With the addition of epinephrine, there was a significant reduction in fentanyl requirements (0.82 +/- 0.07 vs 1.19 +/- 0.11 micrograms.kg-1.h-1, P = 0.005, repeated-measures analysis of variance) and in plasma fentanyl concentrations (steady state: 0.91 +/- 0.13 vs 1.65 +/- 0.23 ng/mL, P = 0.007, repeated-measures analysis of variance). There were no differences in pain scores, side effects, spirometry, patient satisfaction scores, or hemodynamic variables. This study demonstrates that adding epinephrine 1:300,000 to continuous thoracic epidural infusions decreases fentanyl requirements titrated for effective analgesia. The reduction in fentanyl requirements was associated with reduced fentanyl plasma concentrations.

This study was designed to determine if leaving a stylet in the left Bronch-Cath endobronchial tube (DLT) for the entire intubating procedure improves the accuracy of placement on the initial attempt, without introducing complications.Sixty ASA 1-3 patients were randomized to one of two groups. In Group 1 (n = 30), the stylet was retained for the entire intubation procedure and in Group 2 (n = 30), the stylet was removed once the bronchial cuff had passed the vocal cords. In both groups, the DLT was turned 110 degrees counterclockwise and advanced until resistance was encountered. Placement was assessed by auscultation and fibreoptic bronchoscopy (FOB). After surgery, the DLT was replaced by a single-lumen endotracheal tube. The thoracic surgeon (blinded to the method of intubation, and using a FOB) assessed the appearance of the tracheobronchial mucosa.The two groups were similar with respect to sex, height, weight, DLT size, surgeon and expertise of the laryngoscopist. When the stylet was retained, the DLT was correctly placed 60% of the time compared with 17%, if the stylet was removed, (P = 0.001). Seven out of 30 DLTs in Group 2 were initially placed into the right mainstem bronchus, (P = 0.005). The average time to confirmation of correct tube placement by FOB was increased in Group 2, (P = 0.01). Although the observed incidence of left bronchial, mucosal petechiae and erythema was greater in Group 2, this was not statistically significant, (P = 0.063).Retaining the stylet for the entire intubation procedure allows for a more rapid, accurate placement of the DLT without increasing the incidence of tracheobronchial mucosa injury.

Many victims of accidental hypothermia have been successfully resuscitated with cardiopulmonary bypass, but questions remain regarding treatment indications and efficacy. To assess the role of cardiopulmonary bypass in resuscitation from hypothermia, a collective literature review was performed. Data on 68 hypothermic patients resuscitated with cardiopulmonary bypass were analyzed. Impairment from alcohol, drug abuse, or mental illness was the most common predisposing factor for accidental hypothermia. Mean initial core temperature was 21 degrees C. Sixty-one patients (90%) were in cardiac arrest. Femoral-femoral bypass was used in 72% of patients. Overall survival was 60%. Eighty percent of survivors returned to their previous level of function. Sixty-seven percent of nonsurvivors died because of inability to establish a cardiac rhythm or wean from bypass. Patient age, type of cardiopulmonary bypass (femoral-femoral or atrial-aortic), and initial core temperature were not significant prognostic indicators. There were no survivors among the 6 patients with a core temperature less than 15 degrees C. Patients in cardiac arrest had a higher mortality than patients who were not (p = 0.02). Climbing and avalanche victims had a higher mortality than other hypothermic patients (p = 0.003). The possibility of publication bias must be considered before firm conclusions can be drawn from this collective literature review. Controlled studies comparing the efficacy of cardiopulmonary bypass and alternative warming techniques have not been done. Nevertheless, cardiopulmonary bypass has several advantages over other warming methods for profoundly hypothermic patients. Tissue perfusion and oxygenation are maintained while rapid warming occurs. Cardiopulmonary bypass resuscitation is recommended for hypothermic patients in arrest and for patients with core temperatures lower than 25 degrees C, irrespective of rhythm. Patients in stable condition with temperatures between 25 degrees and 28 degrees C can be treated with cardiopulmonary bypass or conventional warming methods.

The effect of chronic right ventricular (RV) pressure overload on left ventricular (LV) systolic function in chronic obstructive lung disease is unclear. To examine LV systolic performance in pulmonary emphysema, a chronic canine model was developed in which pulmonary artery pressure could be elevated to a level found in human disease. Severe emphysema was produced by the repeated instillations of the enzyme papain into the lung. Sonomicrometry was used to assess LV dimensions along the septal-lateral, apex-base, and anterior-posterior orthogonal axes of the LV. With the animal conscious, measurements of LV systolic function were obtained over a wide range of LV circumferential end-ejection stresses at baseline and after 1 yr of emphysema (post-1-yr study). In the emphysema group (n = 5), the results showed that at the post-1-yr study, measurements of LV ejection fraction, mean velocity of circumferential shortening, and rate of anterior-posterior dimensional shortening were reduced compared with those obtained at the baseline study. In the emphysema group, end-systolic volume was increased for a given end-systolic pressure or stress at the post-1-yr study compared with baseline values, while fractional shortening measured along the three axes was decreased. There were no similar changes in systolic parameters in control groups. We conclude that chronic RV pressure overload may cause an impairment in LV systolic performance in chronic emphysema.

Background Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 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 TGFβ1-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 TGFβ1-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 TGFβ1 (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 TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin. Conclusions We conclude that TGFβ1-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.

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.

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)-1β, 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-1β, 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-1β, 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.

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 Atg 12-5 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 proapoptotic 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-alpha 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. Original Publication:Saeid Ghavami, Mark M Mutawe, Pawan Sharma, Behzad Yeganeh, Karol D McNeill, Thomas Klonisch, Helmut Unruh, Hessam H Kashani, Dedmer Schaafsma, Marek Jan Los and Andrew J Halayko, Mevalonate Cascade Regulation of Airway Mesenchymal Cell Autophagy and Apoptosis: A Dual Role for p53, 2011, PLOS ONE, (6), 1, 0016523.http://dx.doi.org/10.1371/journal.pone.0016523Licensee: Public Library of Science (PLoS)http://www.plos.org/

Thirty patients were prospectively randomized to receive either thoracic or lumbar epidural fentanyl infusion for postthoracotomy pain. Epidural catheters were inserted, and placement was confirmed with local anesthetic testing before operation. General anesthesia consisted of nitrous oxide, oxygen, isoflurane, intravenous fentanyl citrate (5 μg/kg), and vecuronium bromide. Pain was measured by a visual analogue scale (0 = no pain to 10=worst pain ever). Postoperatively, patients received epidural fentanyl in titrated doses every 15 minutes until the visual analogue scale score was less than 4 or until a maximum fentanyl dose of 150 μg by bolus and an infusion rate of 150 μg/h was reached. The visual analogue scale score of patients who received thoracic infusion decreased from 8.8 ± 0.5 to 5.5 ± 0.7 ( p ≤ 0.05) by 15 minutes and to 3.5 ± 0.4 ( p ≤ 0.05) by 45 minutes. The corresponding values in the lumbar group were 8.8 ± 0.6 to 7.8 ± 0.7 at 15 minutes and 5.3 ± 0.9 at 45 minutes ( p ≤ 0.05). The infusion rate needed to maintain a visual analogue scale score of less than 4 was lower in the thoracic group (1.55 ± 0.13 μg · kg −1 · h −1 ) than in the lumbar group (2.06 ± 0.19 μg · kg −1 · h −1 ) during the first 4 hours after operation ( p ≤ 0.05). The epidural fentanyl infusion rates could be reduced at 4, 24, and 48 hours after operation without compromising pain relief. Four patients in the lumbar group required naloxone hydrochloridc intravenously. Three of these patients had respiratory rates of less than 6/min and were difficult to arouse. The fourth patient was difficult to arouse and had an arterial carbon dioxide tension of 83 mm Hg. We conclude that thoracic epidural fentanyl infusion is better than lumbar infusion for postthoracotomy pain control because of more rapid onset, smaller dose requirements, and less respiratory depression.

BACKGROUND In chronic obstructive lung disease, a right to left ventricular septal shift that occurs as a consequence of right ventricular pressure overload is the usual mechanism given to explain a decrease in left ventricular (LV) diastolic performance. The purpose of the present study was to examine the extent to which this mechanism could account for a decrease in LV diastolic function in a canine model in which pulmonary artery pressure was elevated to a level found in human disease. METHODS AND RESULTS Severe emphysema was produced in dogs by repeated instillations of the enzyme papain into the lung. To assess LV diastolic function, we used sonomicrometry, in which three pairs of subendocardial crystal transducers were implanted along the three orthogonal axes of the LV. LV end-diastolic dimensions and pressure-strain relations along the three axes, as well as the time constant of LV isovolumic relaxation (T), were measured before (baseline) and after 1 year of emphysema (post-1-year study). The results showed that after 1 year of pulmonary hypertension, LV pressure-strain relations were decreased along the septal-lateral and anterior-posterior axes, but a right to left ventricular septal shift was not detected. The relation of average midwall circumferential stress to midwall circumferential strain was used to describe the intrinsic compliance of the LV. The results showed that myocardial stiffness increased in emphysema but that chamber volume was not reduced. At the post-1-year study, T was abnormally increased in the emphysema group in response to augmented preload and afterload compared with preemphysema measurements. CONCLUSIONS We conclude that mechanisms other than ventricular interdependence may be operative in leading to altered LV diastolic filling in chronic emphysema.

Transforming growth factor-beta (TGF-beta) can regulate cell growth and differentiation as well as production of extracellular matrix proteins. Elevated production of TGF-beta has been associated with human and rodent chronic inflammatory and fibrotic diseases. Using immunohistochemical staining, we have examined lung sections of patients with advanced idiopathic pulmonary fibrosis (IPF), a disease characterized by chronic inflammation and fibrosis and demonstrated a marked and consistent increase in TGF-beta production in epithelial cells and macrophages when compared to patients with nonspecific inflammation and those with no inflammation or fibrosis. In patients with advanced IPF, intracellular staining with anti-LC (1-30) TGF-beta antibody was seen prominently in bronchiolar epithelial cells. In addition, epithelial cells of honeycomb cysts and hyperplastic type II pneumocytes stained intensely. Anti-CC (1-30) TGF-beta antibody, which reacts with extracellular TGF-beta, was localized in the lamina propria of bronchioles and in subepithelial regions of honeycomb cysts in areas of dense fibroconnective tissue deposition. The close association of subepithelial TGF-beta to the intracellular form in advanced IPF suggests that TGF-beta was produced and secreted primarily by epithelial cells. Because of the well-known effects of TGF-beta on extracellular matrix formation and on epithelial cell differentiation, the increased production of TGF-beta in advanced IPF may be pathogenic to the pulmonary fibrotic and regenerative responses seen in this disease.

To determine whether myocardial dysfunction contributes to vascular collapse in anaphylactic shock, we examined left ventricular (LV) contractility, coronary blood flow, and myocardial lactate metabolism during antigen challenge in eight dogs that were sensitized to ragweed pollen extract (anaphylaxis group). Findings in the anaphylaxis group were contrasted to those in another group of dogs in which mean blood pressure was decreased to the same extent by arteriolar vasodilation with nitroprusside. The animals were examined under nonhypoxic conditions while anesthetized and ventilated. LV mechanics were examined with subendocardial crystals placed primarily along the anterior-posterior minor axis of the LV. During antigen challenge, a depression in LV contractility was observed in the anaphylaxis group as assessed by fractional dimensional shortening, stroke volume, and the slope of the end-systolic pressure-dimension relationship. During anaphylaxis, moreover, coronary vasodilation rather than coronary vasoconstriction was observed, and evidence of myocardial ischemia as assessed by altered myocardial lactate metabolism was not found. Our results indicate that depressed LV contractility occurs in anaphylactic shock. The results further suggest that the mechanism may be due to a direct effect of mediators of anaphylaxis on the myocardium to produce systolic dysfunction.

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 FEV1in 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. FEV1was 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 FEV1decline from six months before azithromycin initiation, and rates of FEV1increase 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, FEV1tended 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.

Although Eurocollins solution (ECS) is commonly used for lung preservation, its vascular effects and their time course and response to pharmocological interventions are not well understood. The effect of 4 °C ECS on the pulmonary circulation was assessed in excised canine left lower lobes. The roles of static oxygen inflation and prostacyclin infusion during ECS perfusion were also examined. The lobes were divided into five groups: time control (A), ECS with oxygen (B), ECS without oxygen (C), ECS with glycine buffer (D), and ECS with prostacyclin (E); group D was the control for E. Eurocollins solution had no effect on gas exchange but had a marked effect on the pulmonary circulation. Vascular conductance decreased from 22.6 to 18.9 mL/min/cm H 2 O and from 21.3 to 14.1 mL/min/cm H 2 O with average vascular closure increasing by 1.2 and 2.1 cm H 2 O in groups B and C, respectively. The decreased vascular conductance and increased vascular closure was associated with a reduction in vascular compliance from 1.63 to 1.25 mL/cm H 2 O. When prostacyclin was added to ECS, the reduction in vascular closure was much less and was associated with a decrease in vascular closure and no loss of vascular compliance. Eurocollins solution increases pressure cost for perfusion by causing both vascular obstruction and increased tone, especially when oxygen is not provided. This is significantly overcome by addition of prostacyclin infusion during ECS perfusion.

Muscarinic receptors and platelet-derived growth factor (PDGF) receptors synergistically induce proliferation of airway smooth muscle (ASM), but the pathways that regulate these effects are not yet completely identified. We hypothesized that glycogen synthase kinase-3 (GSK-3), a kinase that represses several promitogenic signaling pathways in its unphosphorylated form, is cooperatively inhibited by PDGF and muscarinic receptors in immortalized human ASM cell lines. PDGF or methacholine alone induced rapid GSK-3 phosphorylation. This phosphorylation was sustained only for PDGF; however, methacholine potentiated PDGF-induced sustained GSK-3 phosphorylation. Synergistic effects of methacholine also were observed on PDGF-induced retinoblastoma protein (Rb) phosphorylation and cell proliferation. Suppression of GSK-3 inhibitory function using SB 216763 also augmented PDGF-induced Rb phosphorylation and cell cycle progression; this synergy was similar in magnitude to that seen for methacholine with PDGF. GSK-3 phosphorylation induced by methacholine required PKC, since it was abolished by GF 109203X and Gö 6976; however, inhibition of PKC had no effect on cell responses to PDGF. PKC inhibition also specifically abolished the synergistic effect of methacholine on PDGF-induced GSK-3 phosphorylation and cell proliferation. Collectively, these results show that GSK-3 plays a key repressive role in ASM cell proliferation. Moreover, muscarinic receptors mediate PKC-dependent GSK-3 inhibition, and this appears to be a primary mechanism underpinning augmentation of PDGF-induced cell growth.