Role of the cystic fibrosis transmembrane conductance channel in human airway smooth muscle.

Patients with cystic fibrosis (CF) suffer from asthma-like symptoms and gastrointestinal cramps, attributed to a mutation in the CF transmembrane conductance regulator (CFTR) gene present in a variety of cells. Pulmonary manifestations of the disease include the production of thickened mucus and symptoms of asthma, such as cough and wheezing. A possible alteration in airway smooth muscle (ASM) cell function of patients with CF has not been investigated. The aim of this study was to determine whether the (CFTR) channel is present and affects function of human ASM cells. Cell cultures were obtained from the main or lobar bronchi of patients with and without CF, and the presence of the CFTR channel detected by immunofluorescence. Cytosolic Ca(2+) was measured using Fura-2 and dual-wavelength microfluorimetry. The results show that CFTR is expressed in airway bronchial tissue and in cultured ASM cells. Peak Ca(2+) release in response to histamine was significantly decreased in CF cells compared with non-CF ASM cells (357 +/- 53 nM versus 558 +/- 20 nM; P < 0.001). The CFTR pharmacological blockers, glibenclamide and N-phenyl anthranilic acid, significantly reduced histamine-induced Ca(2+) release in non-CF cells, and similar results were obtained when CFTR expression was varied using antisense oligonucleotides. In conclusion, these data show that the CFTR channel is present in ASM cells, and that it modulates the release of Ca(2+) in response to contractile agents. In patients with CF, a dysfunctional CFTR channel could contribute to the asthma diathesis and gastrointestinal problems experienced by these patients.

The effects of interleukin-8 on airway smooth muscle contraction in cystic fibrosis.

BACKGROUND: Many cystic fibrosis (CF) patients display airway hyperresponsiveness and have symptoms of asthma such as cough, wheezing and reversible airway obstruction. Chronic airway bacterial colonization, associated with neutrophilic inflammation and high levels of interleukin-8 (IL-8) is also a common occurrence in these patients. The aim of this work was to determine the responsiveness of airway smooth muscle to IL-8 in CF patients compared to non-CF individuals. METHODS: Experiments were conducted on cultured ASM cells harvested from subjects with and without CF (control subjects). Cells from the 2nd to 5th passage were studied. Expression of the IL-8 receptors CXCR1 and CXCR2 was assessed by flow cytometry. The cell response to IL-8 was determined by measuring intracellular calcium concentration ([Ca2+](i)), cell contraction, migration and proliferation. RESULTS: The IL-8 receptors CXCR1 and CXCR2 were expressed in both non-CF and CF ASM cells to a comparable extent. IL-8 (100 nM) induced a peak Ca2+ release that was higher in control than in CF cells: 228 +/- 7 versus 198 +/- 10 nM (p < 0.05). IL-8 induced contraction was greater in CF cells compared to control. Furthermore, IL-8 exposure resulted in greater phosphorylation of myosin light chain (MLC20) in CF than in control cells. In addition, MLC20 expression was also increased in CF cells. Exposure to IL-8 induced migration and proliferation of both groups of ASM cells but was not different between CF and non-CF cells. CONCLUSION: ASM cells of CF patients are more contractile to IL-8 than non-CF ASM cells. This enhanced contractility may be due to an increase in the amount of contractile protein MLC20. Higher expression of MLC20 by CF cells could contribute to airway hyperresponsiveness to IL-8 in CF patients.

IFN-gamma, IL-4 and IL-13 modulate responsiveness of human airway smooth muscle cells to IL-13.

BACKGROUND: IL-13 is a critical mediator of allergic asthma and associated airway hyperresponsiveness. IL-13 acts through a receptor complex comprised of IL-13Ralpha1 and IL-4Ralpha subunits with subsequent activation of signal transducer and activator of transcription 6 (STAT6). The IL-13Ralpha2 receptor may act as a decoy receptor. In human airway smooth muscle (HASM) cells, IL-13 enhances cellular proliferation, calcium responses to agonists and induces eotaxin production. We investigated the effects of pre-treatment with IL-4, IL-13 and IFN-gamma on the responses of HASM cells to IL-13. METHODS: Cultured HASM were examined for expression of IL-13 receptor subunits using polymerase chain reaction, immunofluorescence microscopy and flow cytometry. Effects of cytokine pre-treatment on IL-13-induced cell responses were assessed by looking at STAT6 phosphorylation using Western blot, eotaxin secretion and calcium responses to histamine. RESULTS: IL-13Ralpha1, IL-4Ralpha and IL-13Ralpha2 subunits were expressed on HASM cells. IL-13 induced phosphorylation of STAT6 which reached a maximum by 30 minutes. Pre-treatment with IL-4, IL-13 and, to a lesser degree, IFN-gamma reduced peak STAT6 phosphorylation in response to IL-13. IL-13, but not IFN-gamma, pre-treatment abrogated IL-13-induced eotaxin secretion. Pre-treatment with IL-4 or IL-13 abrogated IL-13-induced augmentation of the calcium transient evoked by histamine. Cytokine pre-treatment did not affect expression of IL-13Ralpha1 and IL-4Ralpha but increased expression of IL-13Ralpha2. An anti-IL-13Ralpha2 neutralizing antibody did not prevent the cytokine pre-treatment effects on STAT6 phosphorylation. Cytokine pre-treatment increased SOCS-1, but not SOCS-3, mRNA expression which was not associated with significant increases in protein expression. CONCLUSION: Pre-treatment with IL-4 and IL-13, but not IFN-gamma, induced desensitization of the HASM cells to IL-13 as measured by eotaxin secretion and calcium transients to histamine. The mechanism of IL-4 and IL-13 induced desensitization does not appear to involve either downregulation of receptor expression or induction of the IL-13Ralpha2 or the SOCS proteins.

Covered Stent and Coils Embolization of a Pulmonary Artery Pseudoaneurysm After Gunshot Wound.

We report the first case of endovascular covered stent implantation for the treatment of a large pulmonary artery pseudoaneurysm (PAPA) following a right thoracic gunshot wound. After resuscitation and hemodynamic stabilization, a CT angiography was performed to analyze the neck size of the PAPA and its position relative to the branches of the parent artery. Covered stent implantation with additional coil embolization was successfully performed. At the 4-year follow-up, the stents remained patent and there was neither pseudoaneurysm recurrence nor treatment-related complication.

[Acute visceral ischaemia, early complication of the aortic syndrome: how to detect and manage it?]. / Les malperfusions viscérales aiguës, complications précoces du syndrome aortique: comment les détecter et les prendre en charge?

Visceral malperfusion as a complication of aortic dissection represent 20 % of all type A or B aortic dissections. Visceral ischaemia is a factor of poor prognosis resulting in a higher mortality for these patients, yet, its clinical diagnosis is often underestimated. In all cases, the cross-sectional imaging, especially computed tomography, is needed to precise the mechanism of ischaemia. It allows differentiating dynamic from static ischaemia and thus to adapt its management. Endovascular treatment of ischaemia has been developed because it allows to a rapid treatment with a high success and low complication rates. Early results of aortic stentgrafting and aortic fenestration are up to 96 %, but late changes on the morphology or function reperfused gut was not studied.

MAP kinases mediate interleukin-13 effects on calcium signaling in human airway smooth muscle cells.

Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of allergic asthma through animal models that have shown that IL-13 is both necessary and sufficient to cause airway hyperresponsiveness (AHR). Airway smooth muscle (ASM) is a primary effector of AHR, and IL-13 increases the responsiveness of ASM, by increasing Ca(2+) release intracellularly, to bronchoconstrictors such as histamine. The mechanisms and signaling pathways mediating this effect are incompletely understood. We have investigated the pathways through which IL-13 regulates the Ca(2+) response to histamine in primary human ASM cell cultures. Functional IL-13 receptors were demonstrated by IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mitogen-activated protein kinases (MAPKs). IL-13 increased Ca(2+) responses to histamine. The augmentation of Ca(2+) signaling was not affected by inhibition of STAT6 or p38 MAPK signaling but was prevented by concurrent inhibition of c-jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) MAPKs. This inhibition did not affect the IL-13-induced increase in histamine receptors. We conclude that IL-13 induces potentiation of Ca(2+) responses to contractile agonists by affecting mechanisms downstream of receptors. JNK and ERK MAPKs modulate these mechanisms.

Effects of decorin and biglycan on human airway smooth muscle cell proliferation and apoptosis.

Proteoglycans (PG) are altered in the asthmatic airway wall. Because PGs are known to affect cell proliferation and apoptosis, we hypothesized that alterations in PG might influence the airway smooth muscle (ASM) hyperplasia observed in the asthmatic airway. Human ASM cells were seeded on plastic or plates coated with decorin (Dcn), biglycan (Bgn), or collagen type I (Col I) (1, 3, and 10 microg/ml). Cells were stimulated with platelet-derived growth factor (PDGF), and cell number was assessed at 0, 48, and 96 h. Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation and apoptosis by annexin V and propidium iodide staining at 48 h post-PDGF stimulation. A significant decrease in cell number was observed with cells seeded on Dcn (10 microg/ml) at 0, 48, and 96 h (P < 0.01). Dcn induced both decreases in BrdU incorporation and increases in annexin V staining (P < 0.05). Bgn decreased cell number at time 0 only (P < 0.05) and affected neither proliferation nor apoptosis. Col I (10 mug/ml) caused a significant increase in cell number at 48 and 96 h (P < 0.01). Adding exogenous Dcn (1-30 microg/ml) to the medium had no effect on cell number. Exposing Dcn-coated matrices to chondroitinase ABC, an enzyme that degrades glycosaminoglycan side chains, reversed the Dcn-induced decrease in cell number. These studies demonstrate that different PGs have variable effects on ASM cell proliferation and apoptosis. Recently described decreases in Dcn in the asthmatic airway wall could potentially permit more exuberant ASM growth.

Immunotherapy as a disease modifier.

Immunotherapy was formerly the treatment of allergic disease by repeated exposure to allergen. Other approaches to immune modulation have emerged using new knowledge of T cell function. The so-called hygiene hypothesis argues that alterations in our environment have resulted in a failure of the immune system to develop normally, such that excessive Th2 type responses to antigen exposures occur. These observations have prompted therapies designed to promote a shift from Th-2 to Th-1 responses. These therapies include bacterial vaccines and stimulation of the immune system with Toll like receptor ligands or bacterial nucleotide immunostimulatory sequences (CpG motifs). Traditional desensitization immunotherapy is being re-examined and anti-IgE therapy is also enjoying a measure of success.

Interleukin-8: novel roles in human airway smooth muscle cell contraction and migration.

In patients with cystic fibrosis (CF) and asthma, elevated levels of interleukin-8 (IL-8) are found in the airways. IL-8 is a CXC chemokine that is a chemoattractant for neutrophils through CXCR1 and CXCR2 G protein-coupled receptors. We hypothesized that IL-8 acts directly on airway smooth muscle cells (ASMC) in a way that may contribute to the enhanced airway responsiveness and airway remodeling observed in CF and asthma. The aim of this study was to determine whether human ASMC (HASMC) express functional IL-8 receptors (CXCR1 and CXCR2) linked to cell contraction and migration. Experiments were conducted on cells harvested from human lung specimens. Real-time PCR and fluorescence-activated cell sorting analysis showed that HASMC expressed mRNA and protein for both CXCR1 and CXCR2. Intracellular Ca(2+) concentration ([Ca(2+)](i)) increased from 115 to 170 nM in response to IL-8 (100 nM) and decreased after inhibition of phospholipase C (PLC) with U-73122. On blocking the receptors with specific neutralizing antibodies, changes in [Ca(2+)](i) were abrogated. IL-8 also contracted the HASMC, decreasing the length of cells by 15%, and induced a 2.5-fold increase in migration. These results indicate that HASMC constitutively express functional CXCR1 and CXCR2 that mediate IL-8-triggered Ca(2+) release, contraction, and migration. These data suggest a potential role for IL-8 in causing abnormal airway structure and function in asthma and CF.

The effects of extracellular purines and pyrimidines on human airway smooth muscle cells.

Extracellular ATP and UTP modulate the function of many cell types through the stimulation of specific P2 receptors, and the inhalation of UTP has been proposed as a therapeutic means of increasing mucociliary clearance in cystic fibrosis patients. The aim of this study was to determine whether P2 receptors are present and functional in human airway smooth muscle (HASM) cells. Experiments were conducted on primary cultures of HASM cells. Reverse transcription-polymerase chain reaction and Western blot analysis showed that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are expressed. Exposure to extracellular ATP, UTP, ADP, and UDP at concentrations ranging from 10(-6) to 10(-4) M, produced significant increases in intracellular Ca(2+) that peaked to 491 +/- 51 nM (p < 0.001) with ATP 10(-5) M and to 321 +/- 30 nM with UTP 10(-4) M. ATP and UTP also induced HASM cell contraction, decreasing cell length by 9.9 +/- 4.3 and 5.6 +/- 2.0%, respectively. Pretreatment of the cells with UTP for short periods of time (10 and 30 min) enhanced the peak Ca(2+) release to UTP, whereas repeated and prolonged pretreatment with UTP decreased it. These results indicate that several subtypes of P2Y receptors are present and functional in HASM cells. They also show that the response of the receptors is increased after short periods of exposure to UTP and decreased after prolonged and repeated exposure. Considering that ATP and UTP are endogenous mediators and that analogs of UTP could be used as a therapeutic modality, the role of extracellular triphosphate nucleotides in physiological and pathophysiological processes in the airways warrants further investigation.

Effects of extracellular triphosphate nucleotides and nucleosides on airway smooth muscle cell proliferation.

Extracellular ATP and uridine triphosphate (UTP) have a range of effects on a wide variety of cells through the activation of P(2) receptors. The aim of this work was to establish if stimulation with ATP and UTP enhances airway smooth muscle (ASM) cell proliferation and to determine the type of receptor mediating this effect. Proliferation of rat ASM cells was assessed through bromodeoxyuridine (BrdU) uptake and by cell counting. At concentrations of 10(-6) and 10(-5) M, ATP and UTP induced significant increases in BrdU incorporation. ATP analogs specific for the P(2X) and P(2Y1) receptor subtypes had no effect. UDP (a P(2Y6) receptor agonist) produced significant decreases in BrdU incorporation and cell counts. Adenosine, the metabolite of ATP, produced an increase in cell proliferation through stimulation of the A(1) receptor. A(2) and A(3) receptor stimulation had no effect. Reverse transcription and polymerase chain reaction analysis showed that mRNA transcripts for the P(2Y2), P(2Y4), P(2Y6), A(1), A(2), and A(3) receptor subtypes were present in cultured ASM cells. These data show that extracellular UTP, ATP, and their metabolites may affect airway remodeling by increasing or by reducing (P(2Y6) receptor) ASM cell proliferation.

Airway smooth muscle cells express functional neurokinin-1 receptors and the nerve-derived preprotachykinin-a gene: regulation by passive sensitization.

Preprotachykinin-A (PPT-A) gene-derived neuropeptides, namely substance P (SP) and neurokinin (NK)A, and their receptors participate in allergen-induced airway responses. Whether airway smooth muscle cells (ASMC) may react directly to SP through expression of the NK-1 receptor or express the gene for the synthesis of SP, the PPT-A gene, is unknown. We demonstrated using reverse transcription-polymerase chain reaction that tracheal SMC (TSMC) from atopic Brown Norway rats contained mRNA transcripts for the full-length isoform of the NK-1 receptor. Flow cytometric analysis indicated that the NK-1 receptor was expressed on the surface of TSMC. This receptor was functional as demonstrated by calcium mobilization in response to SP stimulation. The expression of the NK-1 receptor was not altered in passively sensitized TSMC in response to antigenic stimulation, although this stimulation increased the expression of the chemokine RANTES (regulated on activation, normal T cells expressed and secreted). Using different sets of PCR primers, we showed that TSMC also express the beta, alpha, and its alternative splicing product delta, and possibly the gamma mRNA transcript isoforms of the PPT-A gene. Gene sequencing of the PCR-amplified beta isoform confirmed that it is a transcript product of the rat PPT-A gene, and the production of SP by TSMC was confirmed by enzyme immunoassay. We also showed the beta isoform increased after cell stimulation with rat sera, whether sensitized or not. In conclusion, both the PPT-A gene and NK-1 receptors are expressed by TSMC, which suggests the possibility of autocrine neuropeptidergic mechanisms in these cells. However, these mechanisms are not upregulated by passive sensitization.