Macrophage metalloelastase (MMP-12) deficiency does not alter bleomycin-induced pulmonary fibrosis in mice

BACKGROUND: Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in the interstitium resulting in respiratory failure. The role of remodeling mediators such as metalloproteinases (MMPs) and their inhibitors (TIMPs) in the fibrogenic process remains misunderstood. In particular, macrophage metalloelastase, also identified as MMP-12, is known to be involved in remodeling processes under pathological conditions. However, MMP-12 involvement in pulmonary fibrosis is unknown. Here we investigated fibrotic response to bleomycin in MMP-12 deficient mice. MATERIALS AND METHODS: C57BL/6 mice, Balb/c mice and MMP-12 -/- mice with a C57BL/6 background received 0.3 mg bleomycin by intranasal administration. 14 days after, mice were anesthetized and underwent either bronchoalveolear lavage (BAL) or lung removal. Collagen deposition in lung tissue was determined by Sircol™ collagen assay, MMP activity in BAL fluid was analyzed by zymography, and other mediators were quantified in BAL fluid by ELISA. Real time PCR was performed to assess gene expression in lung removed one or 14 days after bleomycin administration. Student t test or Mann & Whitney tests were used when appropriate for statistical analysis. RESULTS: The development of pulmonary fibrosis in "fibrosis prone" (C57BL/6) mice was associated with prominent MMP-12 expression in lung, whereas MMP-12 expression was weak in lung tissue of "fibrosis resistant" (Balb/c) mice. MMP-12 mRNA was not detected in MMP-12 -/- mice, in conformity with their genotype. Bleomycin elicited macrophage accumulation in BAL of MMP-12 -/- and wild type (WT) mice, and MMP-12 deficiency had no significant effect on BAL cells composition. Collagen content of lung was increased similarly in MMP-12 -/- and WT mice 14 days after bleomycin administration. Bleomycin elicit a raise of TGF-β protein, MMP-2 and TIMP-1 protein and mRNA in BAL fluids and lung respectively, and no significant difference was observed between MMP-12 -/- and WT mice considering those parameters. CONCLUSION: The present study shows that MMP-12 deficiency has no significant effect on bleomycin-induced fibrosis

The absence of reactive oxygen species production protects mice against bleomycin-induced pulmonary fibrosis

BACKGROUND: Reactive oxygen species and tissue remodeling regulators, such as metalloproteinases (MMPs) and their inhibitors (TIMPs), are thought to be involved in the development of pulmonary fibrosis. We investigated these factors in the fibrotic response to bleomycin of p47(phox )-/- (KO) mice, deficient for ROS production through the NADPH-oxidase pathway. METHODS: Mice are administered by intranasal instillation of 0.1 mg bleomycin. Either 24 h or 14 days after, mice were anesthetized and underwent either bronchoalveolar lavage (BAL) or lung removal. RESULTS: BAL cells from bleomycin treated WT mice showed enhanced ROS production after PMA stimulation, whereas no change was observed with BAL cells from p47(phox )-/- mice. At day 1, the bleomycin-induced acute inflammatory response (increased neutrophil count and MMP-9 activity in the BAL fluid) was strikingly greater in KO than wild-type (WT) mice, while IL-6 levels increased significantly more in the latter. Hydroxyproline assays in the lung tissue 14 days after bleomycin administration revealed the absence of collagen deposition in the lungs of the KO mice, which had significantly lower hydroxyproline levels than the WT mice. The MMP-9/TIMP-1 ratio did not change at day 1 after bleomycin administration in WT mice, but increased significantly in the KO mice. By day 14, the ratio fell significantly from baseline in both strains, but more in the WT than KO strains. CONCLUSIONS: These results suggest that NADPH-oxidase-derived ROS are essential to the development of pulmonary fibrosis. The absence of collagen deposition in KO mice seems to be associated with an elevated MMP-9/TIMP-1 ratio in the lungs. This finding highlights the importance of metalloproteinases and protease/anti-protease imbalances in pulmonary fibrosis

Analyse cellulaire et moléculaire des mécanismes physiopathologiques conduisant au remodelage tissulaire et au développement de la fibrose pulmonaire chez la souris (étude de l'implication des métalloprotéinases et des radicaux libres oxygènes)

RENNES1-BU Santé (352382103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Nitric oxide synthase in post-ischaemic remodelling: new pathways and mechanisms

The three isoforms of nitric oxide synthase (NOS), spatially confined in specific intracellular compartments in cardiac cells, have distinct roles in the regulation of contractility in pathophysiological situations. Recently, evidence has emerged that implicates NOS in modulating myocardial remodelling during cardiac stress, including after ischaemic insults. As long as they remain in a coupled state the NOS mostly attenuate hypertrophic remodelling through both cGMP-dependent and independent mechanisms. We review the evidence provided from the phenotype of genetic mouse models as well as from in vitro cell experiments dissecting the signalling effectors involved in the NOS-mediated regulation that justify new therapeutic interventions on the NOS-cGMP axis to attenuate the development of heart failure

Ion channels as effectors of cyclic nucleotide pathways: Functional relevance for arterial tone regulation

International audienceNumerous mediators and drugs regulate blood flow or arterial pressure by acting on vascular tone, involving cyclic nucleotide intracellular pathways. These signals lead to regulation of several cellular effectors, including ion channels that tune cell membrane potential, Ca2+ influx and vascular tone. The characterization of these vasocontrictive or vasodilating mechanisms has grown in complexity due to i) the variety of ion channels that are expressed in both vascular endothelial and smooth muscle cells, ii) the heterogeneity of responses among the various vascular beds, and iii) the number of molecular mechanisms involved in cyclic nucleotide signalling in health and disease. This review synthesizes key data from literature that highlight ion channels as physiologically relevant effectors of cyclic nucleotide pathways in the vasculature, including the characterization of the molecular mechanisms involved. In smooth muscle cells, cation influx or chloride efflux through ion channels are associated with vasoconstriction, whereas K+ efflux repolarizes the cell membrane potential and mediates vasodilatation. Both categories of ion currents are under the influence of cAMP and cGMP pathways. Evidence that some ion channels are influenced by CN signalling in endothelial cells will also be presented. Emphasis will also be put on recent data touching a variety of determinants such as phosphodiesterases, EPAC and kinase anchoring, that complicate or even challenge former paradigms

Beta-3 adrenoceptors as new therapeutic targets for cardiovascular pathologies.

Catecholamines play a key role in the regulation of cardiovascular function, classically through ß(1/2)-adrenoreceptors (AR) activation. After ß(3)-AR cloning in the late 1980s, convincing evidence for ß(3)-AR expression and function in cardiovascular tissues recently initiated a reexamination of their involvement in the pathophysiology of cardiovascular diseases. Their upregulation in diseased cardiovascular tissues and resistance to desensitization suggest they may be attractive therapeutic targets. They may substitute for inoperant ß(1/2)-AR to mediate vasodilation in diabetic or atherosclerotic vessels. In cardiac ventricle, their contractile effects are functionally antipathetic to those of ß(1/2)-AR; in normal heart, ß(3)-ARs may mediate a moderate negative inotropic effect, but in heart failure, it may protect against adverse effects of excessive catecholamine stimulation by action on excitation-contraction coupling, electrophysiology, or remodelling. Thus, prospective studies in animals and patients at different stages of heart failure should lead to identify the best therapeutic window to use ß(3)-AR agonists and/or antagonists

Organ culture mimics the effects of hypoxia on membrane potential, K + channels and vessel tone in pulmonary artery

BACKGROUND AND PURPOSE: Blood vessel culture is gaining interest for use with transfection-based techniques, but alters the contractile properties of the vessels. The present study tested the effects of culture on the intrinsic tone of rat pulmonary arteries (PAs) and examined the function and expression of K(+) channels regulating the resting membrane potential (E(m)) and tone of pulmonary artery smooth muscle cells (PASMCs). EXPERIMENTAL APPROACH: Rat intrapulmonary arteries were isolated and cultured under standard and modified conditions. Contractile responses of fresh and cultured PA were compared using vessel myograph. Electrophysiology experiments on isolated PASMCs used the patch-clamp technique. K(+) channel expression was quantified using reverse transcription and real-time PCR. KEY RESULTS: After 4 days in culture vessels contracted to phenylephrine, but relaxation to carbachol was significantly impaired. Contractile responses to 10 mM KCl, 4-aminopyridine and tetraethylammonium increased, and vessels developed an uncharacteristic relaxation response to Ca(2+)-free solution, nifedipine and levcromakalim. PASMCs from cultured vessels were depolarized and K(+) currents reduced, in association with down-regulation of K(v)1.5, K(v)2.1 and TWIK-related acid-sensitive K(+) channel-1 mRNA. These changes were partially reversed by increased oxygenation of the culture medium or removing the endothelium before culture. CONCLUSIONS AND IMPLICATIONS: Culture of PA for 3–4 days induced loss of functional K(+) channels, depolarization of PASMCs, Ca(2+) influx, intrinsic tone and spontaneous constrictions, similar to the effects of chronic hypoxia. This limits the use of cultured vessels for studying excitation–contraction coupling, although oxygenating the culture medium and removing the endothelium can help to retain normal smooth muscle function