Roger Marthan

Fraktalkine Produced by Airway Smooth Muscle Cells Contributes to Mast Cell Recruitment in Asthma

Human airway smooth muscle cells (HASMC) secrete fractalkine (FKN), a chemokine the concentration of which is increased in asthmatic patients. HASMC also induce mast cell chemotaxis, as a component of asthma inflammation. We therefore evaluated the role of smooth muscle-derived FKN in mast cell migration. We assessed the capacity of recombinant FKN to induce human mast cell chemotaxis. This effect implicates a calcium-independent pathway involving actin reorganization and protein kinase C-δ. We found that HASMC constitutively produce FKN, the synthesis of which is reinforced upon proinflammatory stimulation. Under basal experimental conditions, FKN production by HASMC is not sufficient to induce mast cell chemotaxis. However, pretreatment of mast cells with the neuropeptide vasoactive intestinal peptide (VIP) increases FKN potency to attract mast cells. Since we observed, in asthmatic patients, an increase in both FKN and VIP expression by airway smooth muscle and a positive correlation between VIP staining and mast cell infiltration of the smooth muscle layer, we conclude that HASMC-derived FKN may contribute to mast cell recruitment in asthma.

Inflammation of bronchial smooth muscle in allergic asthma

Background: Recent observations in asthma suggest that bronchial smooth muscle is infiltrated by inflammatory cells including mast cells. Such an infiltration may contribute to airway remodelling that is partly due to an increase in smooth muscle mass. Whether muscle increase is the result of smooth muscle cell hypertrophy remains controversial and has not been studied by ultrastructural analysis. A morphometric analysis of airway smooth muscle (ASM) was undertaken in asthmatic patients using electron microscopy to examine the interactions between ASM cells and inflammatory cells. Methods: ASM specimens were obtained from 14 asthmatic subjects and nine non-asthmatic controls undergoing fibreoptic endoscopy. Inflammatory cell counts were assessed by immunohistochemistry, and ultrastructural parameters were measured using electron microscopy in a blinded fashion on smooth muscle cells and inflammatory cells. Results: ASM from asthmatic patients was infiltrated by an increased number of mast cells and lymphocytes. Smooth muscle cells and their basal lamina were thicker in asthmatic patients (9.5 (0.8) and 1.4 (0.2) μm) than in controls (6.7 (0.4) and 0.7 (0.1) μm). In asthmatics the extracellular matrix was frequently organised in large amounts between ASM cells. Myofibroblasts within smooth muscle bundles were only observed in asthmatics, some of them displaying a close contact with ASM cells. Conclusion: In asthma, airway myositis is characterised by a direct interaction between ASM cells and mast cells and lymphocytes. Smooth muscle remodelling was present, including cell hypertrophy and abnormal extracellular matrix deposition moulding ASM cells.

Role of reactive oxygen species and gp91phox in endothelial dysfunction of pulmonary arteries induced by chronic hypoxia.

1 This study investigates the role of nitric oxide (NO) and reactive oxygen species (ROS) on endothelial function of pulmonary arteries in a mice model of hypoxia‐induced pulmonary hypertension. 2 In pulmonary arteries from control mice, the NO‐synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME) potentiated contraction to prostaglandin F2α (PGF2α) and completely abolished relaxation to acetylcholine. In extrapulmonary but not intrapulmonary arteries, acetylcholine‐induced relaxation was slightly inhibited by polyethyleneglycol‐superoxide dismutase (PEG‐SOD) or catalase. 3 In pulmonary arteries from hypoxic mice, ROS levels (evaluated using dihydroethidium staining) were higher than in controls. In these arteries, relaxation to acetylcholine (but not to sodium nitroprusside) was markedly diminished. L‐NAME abolished relaxation to acetylcholine, but failed to potentiate PGF2α‐induced contraction. PEG‐SOD or catalase blunted residual relaxation to acetylcholine in extrapulmonary arteries, but did not modify it in intrapulmonary arteries. Hydrogen peroxide elicited comparable (L‐NAME‐insensitive) relaxations in extra‐ and intrapulmonary arteries from hypoxic mice. 4 Exposure of gp91phox–/– mice to chronic hypoxia also decreased the relaxant effect of acetylcholine in extrapulmonary arteries. However, in intrapulmonary arteries from hypoxic gp91phox–/– mice, the effect of acetylcholine was similar to that obtained in mice not exposed to hypoxia. 5 Chronic hypoxia increases ROS levels and impairs endothelial NO‐dependent relaxation in mice pulmonary arteries. Mechanisms underlying hypoxia‐induced endothelial dysfunction differ along pulmonary arterial bed. In extrapulmonary arteries from hypoxic mice, endothelium‐dependent relaxation appears to be mediated by ROS, in a gp91phox‐independent manner. In intrapulmonary arteries, endothelial dysfunction depends on gp91phox, the latter being rather the trigger than the mediator of impaired endothelial NO‐dependent relaxation.

Airway remodeling in asthma: New mechanisms and potential for pharmacological intervention

The chronic inflammatory response within the airways of asthmatics is associated with structural changes termed airway remodeling. This remodeling process is a key feature of severe asthma. The 5-10% of patients with a severe form of the disease account for the higher morbidity and health costs related to asthma. Among the histopathological characteristics of airway remodeling, recent reports indicate that the increased mass of airway smooth muscle (ASM) plays a critical role. ASM cell proliferation in severe asthma implicates a gallopamil-sensitive calcium influx and the activation of calcium-calmodulin kinase IV leading to enhanced mitochondrial biogenesis through the activation of various transcription factors (PGC-1α, NRF-1 and mt-TFA). The altered expression and function of sarco/endoplasmic reticulum Ca(2+) pump could play a role in ASM remodeling in moderate to severe asthma. Additionally, aberrant communication between an injured airway epithelium and ASM could also contribute to disease severity. Airway remodeling is insensitive to corticosteroids and anti-leukotrienes whereas the effect of monoclonal antibodies (the anti-IgE omalizumab, the anti-interleukin-5 mepolizumab or anti-tumor necrosis factor-alpha) remains to be investigated. This review focuses on potential new therapeutic strategies targeting ASM cells, especially Ca(2+) and mitochondria-dependent pathways.

Functional significance of air trapping detected in moderate asthma.

Abstract. The aim of this study was to evaluate bronchial and lung abnormalities in patients suffering from moderate asthma as defined by international guidelines, with special attention to air trapping on CT in comparison with that detected in smoking and non-smoking normal subjects. Twenty-two patients classified as moderate asthma and control subjects including healthy volunteers, smokers (n = 10) or non-smokers (n = 12) were prospectively explored by high-resolution CT (HRCT) performed at suspended full inspiration and expiration. The same expiratory protocol was performed 15 min after inhalation of 200 μg of salbutamol. Patients underwent pulmonary function tests within the same week and bronchodilator response was assessed following inhalation of salbutamol. Abnormalities of bronchi and lung parenchyma on inspiratory CT and air trapping on expiratory CT, in dependent and non-dependent areas, were assessed and scored semi-quantitatively by two independent observers. Comparison of score mean values between the different groups was performed using Mann-Whitney test and Spearman correlation between CT findings and pulmonary function tests were calculated. Mosaic perfusion was observed in 23 % of asthmatics. Air-trapping scores were significantly higher in asthmatic patients than in non-smoking control subjects (p = 0.003), but not than in smokers. This difference was ascribed to non-dependent zones of the lung for which air-trapping scores were also higher in asthmatic patients (p = 0.003) and in smoking subjects (p = 0.004) than in normal controls. In the asthmatic group, a significant positive correlation was found between airways resistance and bronchial dilatation score (p = 0.01), and between small airways obstruction index and mosaic perfusion score (p = 0.05). In addition, both FEV1 and reversibility of small airways obstruction values correlated with air-trapping score (p = 0.03 and p = 0.007, respectively). No change could be detected in air-trapping score following salbutamol inhalation. Patients suffering from moderate asthma present mosaic perfusion and larger areas of air trapping than normal subjects, particularly in non-dependent areas of the lung. These lung abnormalities are related to small airways obstruction.

Involvement of TRPV1 and TRPV4 channels in migration of rat pulmonary arterial smooth muscle cells.

Pulmonary hypertension, the main disease of the pulmonary circulation, is characterized by an increase in pulmonary vascular resistance, involving proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). However, cellular and molecular mechanisms underlying these phenomena remain to be identified. In the present study, we thus investigated in rat intrapulmonary arteries (1) the expression and the functional activity of TRPV1 and TRPV4, (2) the PASMC migration triggered by these TRPV channels, and (3) the associated reorganization of the cytoskeleton. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis demonstrated expression of TRPV1 and TRPV4 mRNA in rat intrapulmonary arteries. These results were confirmed at the protein level by western blot. Using microspectrofluorimetry (indo-1), we show that capsaicin and 4α-phorbol-12,13-didecanoate (4α-PDD), selective agonists of TRPV1 and TRPV4, respectively, increased the intracellular calcium concentration of PASMC. Furthermore, stimulation of TRPV1 and TRPV4 induced PASMC migratory responses, as assessed by two different methods (a modified Boyden chamber assay and a wound-healing migration assay). This response cannot seem to be attributed to a proliferative effect as assessed by BrdU and Wst-1 colorimetric methods. Capsaicin- and 4α-PDD-induced calcium and migratory responses were inhibited by the selective TRPV1 and TRPV4 blockers, capsazepine and HC067047, respectively. Finally, as assessed by immunostaining, these TRPV-induced migratory responses were associated with reorganization of the F-actin cytoskeleton and the tubulin and intermediate filament networks. In conclusion, these data point out, for the first time, the implication of TRPV1 and TRPV4 in rat PASMC migration, suggesting the implication of these TRPV channels in the physiopathology of pulmonary hypertension.

Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores.

Intracellular Ca2+ release channels such as ryanodine receptors play crucial roles in the Ca2+-mediated signaling that triggers excitation-contraction coupling in muscles. Although the existence and the role of these channels are well characterized in skeletal and cardiac muscles, their existence in smooth muscles, and more particularly in the myometrium, is very controversial. We have now clearly demonstrated the expression of ryanodine receptor Ca2+ release channels in rat myometrial smooth muscle, and for the first time, intracellular Ca2+ concentration experiments with indo 1 on single myometrial cells have revealed the existence of a functional ryanodine- and caffeine-sensitive Ca2+ release mechanism in 30% of rat myometrial cells. RT-PCR and RNase protection assay on whole myometrial smooth muscle demonstrate the existence of all three ryr mRNAs in the myometrium: ryr3 mRNA is the predominant subtype, with much lower levels of expression for ryr1 and ryr2 mRNAs, suggesting that the ryanodine Ca2+ release mechanism in rat myometrium is largely encoded by ryr3. Moreover, using intracellular Ca2+ concentration measurements and RNase protection assays, we have demonstrated that the expression, the percentage of cells responding to ryanodine, and the function of these channels are not modified during pregnancy.Intracellular Ca(2+) release channels such as ryanodine receptors play crucial roles in the Ca(2+)-mediated signaling that triggers excitation-contraction coupling in muscles. Although the existence and the role of these channels are well characterized in skeletal and cardiac muscles, their existence in smooth muscles, and more particularly in the myometrium, is very controversial. We have now clearly demonstrated the expression of ryanodine receptor Ca(2+) release channels in rat myometrial smooth muscle, and for the first time, intracellular Ca(2+) concentration experiments with indo 1 on single myometrial cells have revealed the existence of a functional ryanodine- and caffeine-sensitive Ca(2+) release mechanism in 30% of rat myometrial cells. RT-PCR and RNase protection assay on whole myometrial smooth muscle demonstrate the existence of all three ryr mRNAs in the myometrium: ryr3 mRNA is the predominant subtype, with much lower levels of expression for ryr1 and ryr2 mRNAs, suggesting that the ryanodine Ca(2+) release mechanism in rat myometrium is largely encoded by ryr3. Moreover, using intracellular Ca(2+) concentration measurements and RNase protection assays, we have demonstrated that the expression, the percentage of cells responding to ryanodine, and the function of these channels are not modified during pregnancy.

Role of DHEA in cardiovascular diseases

Dehydroepiandrosterone (DHEA) is a steroid hormone derived from cholesterol synthesized by the adrenal glands. DHEA and its 3β-sulphate ester (DHEA-S) are the most abundant circulating steroid hormones. In human, there is a clear age-related decline in serum DHEA and DHEA-S and this has suggested that a relative deficiency in these steroids may be causally related to the development of a series of diseases associated with aging including cardiovascular diseases (CVD). This commentary aims to highlight the action of DHEA in CVD and its beneficial effect in therapy. We thus discuss the possible impact of serum DHEA decline and DHEA supplementation in diseases such as hypertension, coronary artery disease and atherosclerosis. More specifically, we provide evidence for a beneficial action of DHEA in the main disease of the pulmonary circulation: pulmonary hypertension. We also examine the potential cellular mechanism of action of DHEA in terms of receptors (membrane/nuclear) and associated signaling pathways (ion channels, calcium signaling, PI3K/AKT/eNos pathway, cGMP, RhoA/RhoK pathway). We show that DHEA acts as an anti-remodeling and vasorelaxant drug. Since it is a well-tolerated and inexpensive drug, DHEA may prove to be a valuable molecule in CVD but it deserves further studies both at the molecular level and in large clinical trials.

IgE-induced passive sensitization of human isolated bronchi and lung mast cells

Passive sensitization of human isolated lung with serum from atopic asthmatic patients provides an opportunity to study the link between airway hyper-responsiveness and the allergic process. To directly demonstrate the role of immunoglobulin E (IgE) in the effect of the atopic serum, we have compared the effect of passively sensitizing both human bronchi and isolated lung mast cells with either serum from atopic asthmatic patients or human monoclonal IgE. Peripheral bronchi ( < 5 mm in internal diameter) were dissected out from human lung obtained at thoractomy and isometric contraction was studied in response to a variety of immunological stimuli according to the sensitization protocol. Mast cells were also isolated from human lung and histamine release was measured under similar experimental conditions. A contractile response was elicited by either the specific antigen or anti-IgE (0.6-600 ng.mL-1) but not anti-immunoglobulin G (IgG) 0.2-20 micrograms.mL-1) in airways sensitized with atopic serum (total IgE concentration of approximately 1,000 international units (IU).mL-1). The maximal contractile response to anti-IgE was 75 +/- 22% of the response to 1 mM acetylcholine. Similarly, anti-IgE released histamine from isolated lung mast cells sensitized with atopic serum up to 22.4 +/- 2% of total histamine measured within mast cells. When isolated airways or mast cells were sensitized with human monoclonal IgE (1,000 IU.mL-1), response to anti-IgE in terms of contractile response or histamine release, respectively, were not significantly different from those obtained following passive sensitization with atopic serum. Finally, the bronchial contractile response to anti-IgE depended not only on the concentration of anti-IgE but also on that of IgE (300-2,000 IU.mL-1) used to sensitize the airways. These results indicate that the effect of antigen or anti-IgE in peripheral bronchi passively sensitized with atopic serum is mimicked when sensitization is carried out directly with human monoclonal IgE.

Assessment of bronchial inflammation using an automated cell recognition system based on colour analysis

The aim of this study was to develop an automated system of cell recognition based upon colour analysis suitable for microscopic examination of bronchial inflammation. Human bronchi obtained from 17 patients undergoing thoracotomy were embedded in glycolmethacrylate to perform immunohistochemistry with antibodies against: neutrophil elastase, tryptase, chymase, eosinophil cationic protein, CD68, CD3 and immunoglobulin E. The image analysis system calculates three independent criteria (optic density, hue density, hue) combined with morphological parameters to specifically recognize a positive staining. This automated analysis was applied to the study of bronchial inflammation in smokers and nonsmokers in terms of the absolute number of cells and the expression of different markers by a single cell. The use of these criteria enabled the characterization of a positive stain on single (intraclass correlation coefficient (ICC) = 0.88 or serial (ICC = 0.84) sections. Cell counts obtained by the automated system were highly reproducible. Regarding bronchial inflammation, it was found that the number of inflammatory cells was significantly higher in smokers than in nonsmokers, the majority of these cells bearing immunoglobulin E. These results demonstrate that such computerized analysis of colours is a valuable method for quantifying inflammatory cells in bronchial tissue and for analysing the expression of different markers by a single cell.