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

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

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

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

CD151, a laminin receptor showing increased expression in asthmatic patients, contributes to airway hyperresponsiveness through calcium signaling.

BACKGROUND: Airway smooth muscle (ASM) contraction underpins airway constriction; however, underlying mechanisms for airway hyperresponsiveness (AHR) remain incompletely defined. CD151, a 4-transmembrane glycoprotein that associates with laminin-binding integrins, is highly expressed in the human lung. The role of CD151 in ASM function and its relationship to asthma have yet to be elucidated. OBJECTIVE: We sought to ascertain whether CD151 expression is clinically relevant to asthma and whether CD151 expression affects AHR. METHODS: Using immunohistochemical analysis, we determined the expression of CD151 in human bronchial biopsy specimens from patients with varying asthma severities and studied the mechanism of action of CD151 in the regulation of ASM contraction and bronchial caliber in vitro, ex vivo, and in vivo. RESULTS: The number of CD151+ ASM cells is significantly greater in patients with moderate asthma compared with those in healthy nonasthmatic subjects. From loss- and gain-of-function studies, we reveal that CD151 is required for and enhances G protein-coupled receptor (GPCR)-induced peak intracellular calcium release, the primary determinant of excitation-contraction coupling. We show that the localization of CD151 can also be perinuclear/cytoplasmic and offer an explanation for a novel functional role for CD151 in supporting protein kinase C (PKC) translocation to the cell membrane in GPCR-mediated ASM contraction at this site. Importantly, CD151-/- mice are refractory to airway hyperreactivity in response to allergen challenge. CONCLUSIONS: We identify a role for CD151 in human ASM contraction. We implicate CD151 as a determinant of AHR in vivo, likely through regulation of GPCR-induced calcium and PKC signaling. These observations have significant implications in understanding the mechanism for AHR and the efficacy of new and emerging therapeutics.

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.

Platinum (IV) coiled coil nanotubes selectively kill human glioblastoma cells.

Malignant glioma are often fatal and pose a significant therapeutic challenge. Here we have employed α-helical right handed coiled coils (RHCC) which self-assemble into tetrameric nanotubes that stably associate with platinum (Pt) (IV) compound. This Pt(IV)-RHCC complex showed superior in vitro and in vivo toxicity in human malignant glioma cells at up to 5 fold lower platinum concentrations when compared to free Pt(IV). Pt(IV)-RHCC nanotubes activated multiple cell death pathways in GB cells without affecting astrocytes in vitro or causing damage to normal mouse brain. This Pt(IV)-RHCC nanotubes may serve as a promising new therapeutic tool for low dose Pt(IV) prodrug application for highly efficient and selective treatment of human brain tumors. FROM THE CLINICAL EDITOR: The prognosis of malignant glioma remains poor despite medical advances. Platinum, one of the chemotherapeutic agents used, has significant systemic side effects. In this article, the authors employed α-helical right handed coiled coil (RHCC) protein nanotubes as a carrier for cisplatin. It was shown that the new compound achieved higher tumor kill rate but lower toxicity to normal cells and thus may hold promise to be a highly efficient treatment for the future.

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.

Suppression of influenza A virus replication in human lung epithelial cells by noncytotoxic concentrations bafilomycin A1.

Subcellular trafficking within host cells plays a critical role in viral life cycles, including influenza A virus (IAV). Thus targeting relevant subcellular compartments holds promise for effective intervention to control the impact of influenza infection. Bafilomycin A1 (Baf-A1), when used at relative high concentrations (≥10 nM), inhibits vacuolar ATPase (V-ATPase) and reduces endosome acidification and lysosome number, thus inhibiting IAV replication but promoting host cell cytotoxicity. We tested the hypothesis that much lower doses of Baf-A1 also have anti-IAV activity, but without toxic effects. Thus we assessed the antiviral activity of Baf-A1 at different concentrations (0.1-100 nM) in human alveolar epithelial cells (A549) infected with IAV strain A/PR/8/34 virus (H1N1). Infected and mock-infected cells pre- and cotreated with Baf-A1 were harvested 0-24 h postinfection and analyzed by immunoblotting, immunofluorescence, and confocal and electron microscopy. We found that Baf-A1 had disparate concentration-dependent effects on subcellular organelles and suppressed affected IAV replication. At concentrations ≥10 nM Baf-A1 inhibited acid lysosome formation, which resulted in greatly reduced IAV replication and release. Notably, at a very low concentration of 0.1 nM that is insufficient to reduce lysosome number, Baf-A1 retained the capacity to significantly impair IAV nuclear accumulation as well as IAV replication and release. In contrast to the effects of high concentrations of Baf-A1, very low concentrations did not exhibit cytotoxic effects or induce apoptotic cell death, based on morphological and FACS analyses. In conclusion, our results reveal that low-concentration Baf-A1 is an effective inhibitor of IAV replication, without impacting host cell viability.

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

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

Connexin 43 phosphorylation and degradation are required for adipogenesis.

Connexin-43 (Cx43) is a membrane phosphoprotein that mediates direct inter-cellular communication by forming gap junctions. In this way Cx43 can influence gene expression, differentiation and growth. Its role in adipogenesis, however, is poorly understood. In this study, we established that Cx43 becomes highly phosphorylated in early adipocyte differentiation and translocates to the plasma membrane from the endoplasmic reticulum. As preadipocytes differentiate, Cx43 phosphorylation declines, the protein is displaced from the plasma membrane, and total cellular levels are reduced via proteosomal degradation. Notably, we show that inhibiting Cx43 degradation or constitutively over-expressing Cx43 blocks adipocyte differentiation. These data reveal that transient activation of Cx43 via phosphorylation followed by its degradation is vital for preadipocyte differentiation and maturation of functional adipocytes.

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.

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.

Effects of extensively oxidized low-density lipoprotein on mitochondrial function and reactive oxygen species in porcine aortic endothelial cells.

Atherosclerotic cardiovascular disease is the leading cause of mortality in the Western world. Dysfunction of the mitochondrial respiratory chain and overproduction of reactive oxygen species (ROS) are associated with atherosclerosis and cardiovascular disease. Oxidation increases the atherogenecity of LDL. Oxidized LDL may be apoptotic or nonapoptotic for vascular endothelial cells (EC), depending on the intensity of oxidation. A previous study demonstrated that nonapoptotic oxidized LDL increased activity of mitochondrial complex I in human umbilical vein EC. The present study examined the impact of extensively oxidized LDL (eoLDL) on oxygen consumption and the activities of key enzymes in the mitochondrial respiratory chain of cultured porcine aortic EC. Oxygraphy detected that eoLDL significantly reduced oxygen consumption in various mitochondrial complexes. Treatment with eoLDL significantly decreased NADH-ubiquinone dehydrogenase (complex I), succinate cytochrome c reductase (complex II/III), ubiquinone cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV) activities and the NAD+-to-NADH ratio in EC compared with mildly oxidized LDL, LDL, or vehicle. Butylated hydroxytoluene, a potent antioxidant, normalized eoLDL-induced reductions in complex I and III enzyme activity in EC. Mitochondria-associated intracellular ROS and release of ROS from EC were significantly increased after eoLDL treatment. These findings suggest that eoLDL impairs enzyme activity in mitochondrial respiratory chain complexes and increases ROS generation from mitochondria of arterial EC. Collectively, these effects could contribute to vascular injury and atherogenesis under conditions of hypercholesterolemia and oxidative stress.

Impairment of mitochondrial respiratory chain activity in aortic endothelial cells induced by glycated low-density lipoprotein.

Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated LDL (glyLDL) were detected in patients with diabetes. Our previous studies demonstrated that glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). This study examined the effects of glyLDL on oxygen consumption in mitochondria and the activities of key enzymes in the mitochondrial electron transport chain (ETC) in cultured porcine aortic EC. The results demonstrated that glyLDL treatment significantly impaired oxygen consumption in Complexes I, II/III, and IV of the mitochondrial ETC in EC compared to LDL or vehicle control detected using oxygraphy. Incubation with glyLDL significantly reduced the mitochondrial membrane potential, the NAD(+)/NADH ratio, and the activities of mitochondrial ETC enzymes (NADH-ubiquinone dehydrogenase, succinate cytochrome c reductase, ubiquinone cytochrome c reductase, and cytochrome c oxidase) in EC compared to LDL or control. The abundance of mitochondria-associated ROS and the release of ROS from EC were significantly increased after glyLDL treatment. The findings suggest that glyLDL attenuates the activities of key enzymes in the mitochondrial ETC, decreases mitochondrial oxygen consumption, reduces mitochondrial membrane potential, and increases ROS generation in EC, which potentially contribute to mitochondrial dysfunction in diabetic patients.

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.

Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle.

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 Ca(2+) 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 Ca(2+) ([Ca(2+)](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 M(3) receptors (M(3)R) and Galpha(q/11) cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with beta-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-beta-cyclodextrin (mbetaCD) reduced sensitivity but not maximum [Ca(2+)](i) induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mbetaCD disrupted the colocalization of caveolae-1 and M(3)R, but [N-methyl-(3)H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca(2+)](i) flux, 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 mbetaCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca(2+)](i) mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca(2+)](i) mobilization leading to ASM contraction induced by submaximal concentrations of ACh.

Laminin-binding integrin alpha7 is required for contractile phenotype expression by human airway myocytes.

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.

Insulin increases the expression of contractile phenotypic markers in airway smooth muscle.

We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 microM) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated p70(S6K) (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 microM) or the PI 3-kinase inhibitor LY-294002 (10 microM), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus.

Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle.

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-beta-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-beta 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.

Effects of oxidized and glycated low-density lipoproteins on transcription and secretion of plasminogen activator inhibitor-1 in vascular endothelial cells.

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1 were frequently detected in patients with coronary artery disease (CAD) or diabetes. Low-density lipoprotein (LDL) is a classical risk factor of CAD. Oxidation and glycation increase the atherogenecity of LDL. Previous studies demonstrated that oxidized LDL (oxLDL) or glycated LDL (gly-LDL) increased the release of PAI-1 from endothelial cells (ECs). The present study examined the effects of oxLDL and gly-LDL on the transcription, expression, secretion, and subcellular distribution of PAI-1 in cultured human ECs. Treatment with LDL significantly increased the promoter activity, mRNA level, and the release of PAI-1 from ECs by two- to threefold compared to controls. Oxidation or glycation significantly enhanced the effects of LDL on PAI-1 production in ECs compared to LDL (four- to fivefold vs. controls). No significant differences were detected between the effects of oxLDL and gly-LDL. Abundant PAI-1 antigens were detected in the perinuclear region of ECs and overlapped with giantin, a marker of Golgi apparatus. Treatment with brefeldin A disturbed the stack structure of Golgi apparatus and almost completely inhibited the release of PAI-1 from ECs induced by the lipoproteins and at basal conditions. The results suggest that oxidation and glycation enhanced the effects of LDL on the production of PAI-1 in ECs through increasing the transcription of PAI-1. Intact Golgi apparatus is required for PAI-1 generation from ECs induced by LDL or its modified forms.