Combined Beta-agonists and corticosteroids do not inhibit extracellular matrix protein production in vitro.

Background. Persistent asthma is characterized by airway remodeling. Whereas we have previously shown that neither ß(2)-agonists nor corticosteroids inhibit extracellular matrix (ECM) protein release from airway smooth muscle (ASM) cells, the effect of their combination is unknown and this forms the rationale for the present study. Methods. ASM cells from people with and without asthma were stimulated with TGFß1 (1 ng/ml) with or without budesonide (10(-8) M) and formoterol (10(-10) and 10(-8) M), and fibronectin expression and IL-6 release were measured by ELISA. Bronchial rings from nonasthmatic individuals were incubated with TGFß1 (1 ng/ml) with or without the drugs, and fibronectin expression was measured using immunohistochemistry. Results. Budesonide stimulated fibronectin deposition, in the presence or absence of TGFß1, and this was partially reversed by formoterol (10(-8) M) in both asthmatic and nonasthmatic cells. Budesonide and formoterol in combination failed to inhibit TGFß-induced fibronectin in either cell type. A similar pattern of expression of fibronectin was seen in bronchial rings. TGFß1-induced IL-6 release was inhibited by the combination of drugs. Conclusion. Current combination asthma therapies are unable to prevent or reverse remodeling events regulated by ASM cells.

Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement.

CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1ß, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1ß, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1ß and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.

The phosphoinositide 3'-kinase p110δ modulates contractile protein production and IL-6 release in human airway smooth muscle.

Transforming growth factor (TGF) ß1 increases pro-inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3' kinase (PI3K) is one of the signaling pathways implicated in TGFß1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFß1 induced pro-inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFß1 increased 110δ mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 µM), the p110ß inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10 µM) had any effect on basal IL-6, calponin or smooth muscle α-actin (α-SMA) expression. However, TGFß1 increased calponin and α-SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFß1 induced IL-6 release in a dose related manner in all groups of ASM cells. PI3K p110δ is important for TGFß1 induced production of the contractile proteins calponin and α-SMA and the proinflammatory cytokine IL-6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling.

Dual ERK and phosphatidylinositol 3-kinase pathways control airway smooth muscle proliferation: differences in asthma.

Hyperplasia of airway smooth muscle (ASM) within the bronchial wall of asthmatic patients has been well documented and is likely due to increased muscle proliferation. We have shown that ASM cells obtained from asthmatic patients proliferate faster than those obtained from non-asthmatic patients. In ASM from non-asthmatics, mitogens act via dual signaling pathways (both ERK- and PI 3-kinase-dependent) to control growth. In this study we are the first to examine whether dual pathways control the enhanced proliferation of ASM from asthmatics. When cells were incubated with 0.1% or 1% FBS, ERK activation was significantly greater in cells from asthmatic subjects (P < 0.05). In contrast, when cells were stimulated with 10% FBS, ERK activity was significantly greater in the non-asthmatic cells. However, cell proliferation in asthmatic cells was still significantly higher in cells stimulated by both 1% and 10% FBS. Pharmacological inhibition revealed that although dual proliferative pathways control ASM growth in cells from non-asthmatics stimulated with 10% FBS to an equal extent ([(3)H]-thymidine incorporation reduced to 57.2 +/- 6.9% by the PI 3-kinase inhibitor LY294002 and 57.8 +/- 1.1% by the ERK-pathway inhibitor U0126); in asthmatics, the presence of a strong proliferative stimulus (10% FBS) reduces ERK activation resulting in a shift to the PI 3-kinase pathway. The underlying mechanism appears to be upregulation of an endogenous MAPK inhibitor--MKP-1--that constrains ERK signaling in asthmatic cells under strong mitogenic stimulation. This study suggests that the PI 3-kinase pathway may be an attractive target for reversing hyperplasia in asthma.

A phosphodiesterase 4 inhibitor inhibits matrix protein deposition in airways in vitro.

BACKGROUND: Airway smooth muscle (ASM) cells may contribute to airway remodeling through the release of growth factors, cytokines, and extracellular matrix (ECM) proteins. The effect of current asthma therapies on this release is not known. OBJECTIVE: We examined the effect of corticosteroids, long-acting beta(2)-agonists, and a phosphodiesterase 4 (PDE4) inhibitor on ASM-released connective tissue growth factor (CTGF), collagen I, fibronectin, versican, and IL-6. METHODS: Airway smooth muscle cells from individuals with and without asthma were stimulated with TGF-beta with or without the drugs and CTGF and ECM protein expression measured by real-time PCR, cell surface, or matrix-associated ELISA. IL-6 release was measured by ELISA. Bronchial rings from individuals without asthma were incubated with TGF-beta with or without the drugs. RESULTS: Neither corticosteroids nor long-acting beta(2)-agonists reduced TGF-beta-induced CTGF, collagen I, or fibronectin in either cell type, whereas corticosteroids alone induced the expression of CTGF, collagen I, and fibronectin. These drugs did not prevent the accumulation of TGF-beta-induced proteins in bronchial rings, whereas the PDE4 inhibitor roflumilast inhibited TGF-beta-induced CTGF, collagen I, and fibronectin. CONCLUSION: In our model, current asthma therapies are not able to inhibit matrix protein deposition from ASM cells. The results of this study suggest that the PDE4 inhibitor roflumilast may have a role in regulating the ECM and therefore aspects of airway remodeling in asthma. CLINICAL IMPLICATIONS: Although current asthma therapies are effective in reducing inflammation and symptoms, reversal or prevention of structural changes contributing to remodeling may require additional therapy, which could include PDE4 inhibitors.

Connective tissue growth factor and vascular endothelial growth factor from airway smooth muscle interact with the extracellular matrix.

Airway remodeling describes the structural changes that occur in the asthmatic airway that include airway smooth muscle hyperplasia, increases in vascularity due to angiogenesis, and thickening of the basement membrane. Our aim in this study was to examine the effect of transforming growth factor-beta on the release of connective tissue growth factor and vascular endothelial growth factor from human airway smooth muscle cells derived from asthmatic and nonasthmatic patients. In addition we studied the immunohistochemical localization of these cytokines in the extracellular matrix after stimulating bronchial rings with transforming growth factor-beta. Connective tissue growth factor and vascular endothelial growth factor were released from both cell types and colocalized in the surrounding extracellular matrix. Prostaglandin E2 inhibited the increase in connective tissue growth factor mRNA but augmented the release of vascular endothelial growth factor. Matrix metalloproteinase-2 decreased the amount of connective tissue growth factor and vascular endothelial growth factor, but not fibronectin deposited in the extracellular matrix. This report provides the first evidence that connective tissue growth factor may anchor vascular endothelial growth factor to the extracellular matrix and that this deposition is decreased by matrix metalloproteinase-2 and prostaglandin E2. This relationship has the potential to contribute to the changes that constitute airway remodeling, therefore providing a novel focus for therapeutic intervention in asthma.

Connective tissue growth factor induces extracellular matrix in asthmatic airway smooth muscle.

Transforming growth factor (TGF)-beta and connective tissue growth factor may be implicated in extracellular matrix protein deposition in asthma. We have recently reported that TGF-beta increased connective tissue growth factor expression in airway smooth muscle cells isolated from patients with asthma. In this study, we examined fibronectin and collagen production and signal transduction pathways after stimulation with TGF-beta and connective tissue growth factor. In both asthmatic and nonasthmatic airway smooth muscle cells, TGF-beta and connective tissue growth factor led to the production of fibronectin and collagen I. Fibronectin and collagen expression was extracellular regulated kinase-dependent in both cell types but phosphoinositide-3 kinase-dependent only in asthmatic airway smooth muscle cells. p38 was implicated in fibronectin but not collagen expression in both cell types. TGF-beta induction of fibronectin and collagen was in part mediated by an autocrine action of connective tissue growth factor. Phosphorylation of SMAD-2 may represent an additional pathway because this was increased in asthmatic cells. Our results suggest that these two cytokines may be important in the deposition of extracellular matrix proteins and that the signal transduction pathways may be different in asthmatic and nonasthmatic cells.

pDMAEMA is internalised by endocytosis but does not physically disrupt endosomes.

Earlier workers proposed that poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) facilitates cell transfection by being endocytosed, complexed with DNA, and subsequently acting as a "proton sponge" to burst endosomes/lysosomes and release DNA to the cytosol. It also seemed feasible that the cytotoxicity of pDMAEMA might result from lysosomal bursting, which can induce cell death. Experiments were performed to determine the extent of cytotoxicity of uncomplexed pDMAEMA, the mode of cell death it induces (i.e. apoptosis or necrosis), its mechanism of entry into cells, and its ability to disrupt endosomes/lysosomes and release molecules into the cell cytosol. The results indicate that (i). pDMAEMA is highly cytotoxic and induces rapid, primarily necrotic cell death, (ii). it is internalised into cells via fluid-phase endocytosis, and (iii). although pDMAEMA affected the morphology of late endosomes/lysosomes, it did not physically disrupt them to release their contents to the cytosol. The lack of endosomal disruptive activity suggests that this is not involved in the cytotoxicity of pDMAEMA or in its ability to transfect cells. Further work will be required to establish the molecular mechanism(s) by which pDMAEMA facilitates transfection.

Extracellular matrix proteins modulate asthmatic airway smooth muscle cell proliferation via an autocrine mechanism.

BACKGROUND: Airway remodeling is a key feature of persistent asthma and includes alterations in the extracellular matrix protein profile around the airway smooth muscle (ASM) and hyperplasia of the ASM. We have previously shown that nonasthmatic ASM cells in culture produce a range of extracellular matrix protein proteins and that asthmatic ASM cells proliferate faster than cells from nonasthmatic patients. OBJECTIVE: In this study, we compared the profile of extracellular matrix proteins produced by nonasthmatic and asthmatic ASM cells. We also examined the influence of these extracellular matrix protein proteins and conditioned medium derived from nonasthmatic or asthmatic ASM cells on the proliferation of nonasthmatic and asthmatic ASM cells. METHODS: Extracellular matrix proteins were measured by ELISA; proliferation of ASM cells was measured by tritiated thymidine incorporation. RESULTS: Production of perlecan and collagen I by the cells from asthmatic patients were significantly increased. In contrast, laminin alpha1 and collagen IV were decreased. Chondroitin sulfate was detectable only in the cells from nonasthmatic patients. Compared with nonasthmatic extracellular matrix proteins, proteins from asthmatic cells enhanced ASM cell proliferation. Conditioned medium from asthmatic ASM cells did not induce greater proliferation compared with conditioned medium from nonasthmatic cells. CONCLUSIONS: The data show that the profile of extracellular matrix protein components is altered in asthmatic cells and that this altered profile and not soluble mediators secreted from the ASM cells has the potential to influence the proliferation of these cells. These changes are likely to contribute to the airway wall remodeling that occurs in asthma.

PAR-2 activation, PGE2, and COX-2 in human asthmatic and nonasthmatic airway smooth muscle cells.

The protease-activated receptor-2 (PAR-2) is present on human airway smooth muscle (ASM) cells and can be activated by mast cell tryptase, trypsin, or an activating peptide (AP). Trypsin induced significant increases in PGE2 release from human ASM cells after 6 and 24 h and also induced cyclooxygenase (COX)-2 mRNA expression and COX-2 protein. Tryptase and the PAR-2 AP did not alter PGE2 release or COX-2 protein levels, suggesting a lack of PAR-2 involvement. When we compared results in asthmatic and nonasthmatic muscle cells, both trypsin and bradykinin induced less PGE2 from asthmatic ASM cells, and bradykinin induced significantly less COX-2 mRNA in asthmatic cells. Significantly less PGE2 was released from proliferating ASM cells from asthmatic patients. In conclusion, trypsin induces PGE2 release and COX-2 in human ASM cells, which is unlikely to be via PAR-2 activation. In addition, ASM cells from asthmatic patients produce significantly less PGE2 and COX-2 compared with nonasthmatic cells. These findings may contribute to the increase in muscle mass evident in asthmatic airways.

Expression of connective tissue growth factor in asthmatic airway smooth muscle cells.

There is strong evidence to implicate transforming growth factor-beta in the remodeling that occurs in asthma, as levels are increased in bronchial lavage fluid and gene expression is increased in bronchial tissue. Transforming growth factor-beta is also known to increase the release of collagen from airway smooth muscle. Here we identify for the first time a possible mechanism for the effects of transforming growth factor-beta. Transforming growth factor-beta specifically induces mRNA and protein for connective tissue growth factor in airway smooth muscle, and moreover, we report that the connective tissue growth factor response is greater in airway smooth muscle cultured from patients with asthma compared with patients without asthma. This occurs at both the level of mRNA (37.53 +/- 11.62- and 13.59 +/- 3.12-fold increase at 24 hours compared with time 0, respectively, p < 0.02) and protein production (67.57 +/- 27.80- and 3.58 +/- 0.6-fold increase at 24 hours compared with time 0, respectively, p < 0.03). The differential connective tissue growth factor response to transforming growth factor-beta in asthmatic airway smooth muscle identifies a potential role for connective tissue growth factor in the remodeling that is characteristic of severe persistent asthma.