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International audience; Exposure to titanium dioxide (TiO2) nanoparticles (NPs) is associated with lung remodeling, but the underlying mechanisms are unknown. Matrix metalloprotease (MMP)-1 is an important actor in matrix homeostasis and could therefore participate in TiO2 NP effects. Our aim was to evaluate the effects of TiO2 NPs on MMP-1 expression and activity in lung pulmonary fibroblasts and to understand the underlying mechanisms and assess the importance of the physicochemical characteristics of the particles in these effects. Human pulmonary fibroblasts (MRC-5 cell line and primary cells) were exposed to 10 or 100 μg/cm(2) TiO2 (two anatases, two anatase/rutile mix, one rutile NP, and one micrometric) and carbon black (CB) NPs for 6 to 48 hours. We examined cell viability, MMP-1 expression and activity, and the implication of oxidative stress, transforming growth factor (TGF)-β, extracellular MMP inducer, and IL-1β in MMP-1 expression. All TiO2 NPs induced MMP-1 (mRNA and protein expression), repression of procollagen-1, and α-actin expression, but only the two anatase/rutile mix induced MMP-1 activity. Micrometric TiO2 had smaller effects than TiO2 NPs, and CB NPs did not induce MMP-1. MMP-1 induction by TiO2 NPs was not related to TGF-β, oxidative stress, or EMPRIN expression but was related to IL-1β expression, which partly drives MMP-1 induction by two TiO2 NPs (one anatase/rutile mix and the rutile one). Taken together, our results show that TiO2 NPs are potent inducers and regulators of MMP-1 expression and activity, partly via an IL-1β-dependent mechanism. This may explain TiO2 lung remodeling effects.

International audience; In spite of the great promises that the development of nanotechnologies can offer, concerns regarding potential adverse health effects of occupational exposure to nanoparticle (NP) is raised. We recently identified metal oxide NP in lung tissue sections of welders, located inside macrophages infiltrated in fibrous regions. This suggests a role of these NP in the lung alterations observed in welders. We therefore designed a study aimed to investigate the pulmonary effects, in mice, of repeated exposure to NP administered at occupationally relevant doses. We therefore chose four metal oxide NPs representative of those found in the welder’s lungs: Fe2O3, Fe3O4, MnFe2O4 and CrOOH. These NPs were administered weekly for up to 3 months at two different doses: 5 μg, chosen as occupationally relevant to welding activity, and 50 μg, chosen as occupationally relevant to the context of an NP-manufacturing facility. Our results show that 3 month-repeated exposures to 5 μg NP induced limited pulmonary effects, characterized by the development of a mild peribronchiolar fibrosis observed for MnFe2O4 and CrOOH NP only. This fibrotic event was further extended in terms of intensity and localization after the repeated administration of 50 μg NP: all but Fe2O3 NP induced the development of peribronchiolar, perivascular and alveolar fibrosis, together with an interstitial inflammation. Our data demonstrate for the first time a potential risk for respiratory health posed by repeated exposure to NP at occupationally relevant doses. Given these results, the development of occupational exposure limits (OELs) specifically dedicated to NP exposure might therefore be an important issue to address.

International audience; Rationale: Sleep disorders may lead to stress-induced premature aging and telomere shortening.Objectives: To determine whether obstructive sleep apnea syndrome causing intermittent hypoxemic episodes was associated with telomere shortening independently from the comorbidities associated with this syndrome.Methods: We conducted a cross-sectional study in 161prospectivelly enrolled, untreated, middle-aged men free of known comorbidities related or unrelated to sleep apnea. Each participant underwent full standard overnight polysomnography. Patients with apnea sleep syndrome were naive to treatment.Measurements and main results: In univariate analysis, we found that telomere shortening was associated with older age, apnea-hypopnea index, oxygen desaturation index, waist circumference, and fat mass. After adjustment for age, only apnea-hypopnea index and oxygen desaturation index were significantly related to telomere shortening. The mean telomere length ratio was 0.70 ± 0.37 in the participants without sleep apnea, compared with 0.61 ± 0.22 and 0.53 ± 0.16 in those with mild to moderate and severe sleep apnea, respectively (P = 0.01). In multivariate analysis, we found that oxygen desaturation index was the only factor independently associated with telomere length. Arterial stiffness assessed by carotid-femoral pulse wave velocity correlated negatively with telomere length.Conclusions: Intermittent hypoxemia due to obstructive sleep apnea syndrome is a major contributor to telomere shortening in middle-aged men. Oxidative stress may explain this finding.

Several convergent destructive mechanisms such as oxidative stress, alveolar cell apoptosis, extracellular matrix proteolysis and chronic inflammation contribute to chronic obstructive pulmonary disease (COPD) development. Evidence suggests that oxidative stress contributes to the pathophysiology of COPD, particularly during exacerbations. Nuclear factor erythroid-2-related factor 2 (NRF2), a transcription factor expressed predominantly in epithelium and alveolar macrophages, has an essential protective role in the lungs through the activation of antioxidant response element-regulated antioxidant and cytoprotective genes. Animal models and human studies have identified NRF2 and several NRF2 target genes as a protective system against inflammation and oxidative stress from cigarette smoke, a major causative factor in COPD development. Hence, NRF2 targeting might provide clinical benefit by reducing both oxidative stress and inflammation in COPD.

Introduction La dysplasie bronchopulmonaire (DBP), caracterisee par un arret de l’alveolisation, se developpe chez les prematures et entraine des consequences pulmonaires durables. Le facteur de transcription p16Ink4 est implique dans le cycle cellulaire et son expression est augmentee dans les cellules progenitrices leucocytaires du sang de cordon des nouveau-nes prematures. Objectifs Determiner dans un modele murin de BPD, si la deletion de p16 favorise la regeneration pulmonaire chez les souris nouveau-nees, et protege contre les consequences pulmonaires a l’âge adulte. Methodes Des souris sauvages et p16−/− ont ete exposees a l’hyperoxie (85 % FiO2) ou a l’air ambiant du jour (j) 3 a 14. Les mesures suivantes ont ete realisees a j14, 16 et 60 et 120 : morphometrie, elastine (Weigert), collagene (rouge sirius), identification des lipofibroblastes (IHC : ADRP, lipidtox), etudes transcriptomique et lipidomique, voies du metabolisme lipidique LXR (par q PCR : SREBP, SCAP, ADRP). Puis des souris sauvages ont ete traitees quotidiennement a partir de j14 apres l’hyperoxie, par un activateur de la voie LXR, T0901317, ou la rosiglitazone activateur de PPARg. Resultats A j14, l’hyperoxie a provoque un arret de l’alveolisation et une augmentation des marqueurs de senescence et d’inflammation (avec polarisation des macrophages en M1). Les souris hyperoxiques p16−/− ne sont pas protegees contre cette hypoalveolarisation mais presentent moins d’inflammation (avec switch des macrophages de M1 a M2) par rapport aux souris sauvages. A l’âge adulte, la deletion de p16 permet une restauration de l’architecture pulmonaire, associee a une augmentation du nombre de lipofibroblastes (ADRP, lipidtox en IHC) et du metabolisme lipidique (expressions augmentees de SREBP, ADRP en qPCR, contenu augmente en lipides neutres). De la meme maniere, l’administration la rosiglitazone, et T0901317, a permis d’ameliorer significativement l’architecture pulmonaire. Conclusion La deletion de p16 limite les sequelles parenchymateuses de la DBP a l’âge adulte, via l’induction des lipofibroblastes, secondaire a l’activation de la voie LXR favorisant ainsi les processus de regeneration pulmonaire. L’activation de la voie LXR induit les memes benefices et pourrait ouvrir des perspectives therapeutiques dans la prise en charge de la DBP.

Les nanotechnologies produisent des matériaux à l’échelle du nanomètre appelés nanomatériaux. Les nanomatériaux ont des propriétés spécifiques, très intéressantes d’un point de vue industriel, ce qui explique leur production et leur utilisation croissantes dans des produits de consommation courante, et en particulier dans des produits d’hygiène personnelle. Cependant, des inquiétudes s’expriment concernant leur potentielle toxicité pour la santé humaine. Dans cet article, nous proposons une synthèse des connaissances actuelles concernant les effets des nanomatériaux sur la santé. Devant le champ quasi-vierge de ces connaissances, il apparaît urgent, d’une part, de développer les recherches en toxicologie sur ces nouveaux matériaux et, d’autre part, de disposer d’informations de la part des producteurs de nanomatériaux pour pouvoir mesurer les niveaux d’exposition des personnes qui les manipulent. Ceci est un préalable à la mise en place de mesures de précaution sanitaire appropriées, si cela s’avère nécessaire.

Here, we review several articles of original research published in 2008 that concerned the toxicity of metal and carbon-based nanoparticles. Articles were selected from the MEDLINE PubMed database, all published or pre-published during 2008 and relating to nanomaterials, -particles or -structures and toxicity or health. From the 746 articles, we concentrated on research into carbonaceous (carbon nanotubes [CNTs] and fullerenes) and metallic materials (pure metal, oxides), because these nanomaterials are produced and used worldwide and are the most relevant for public health. Unfortunately, due to the large variability in materials used and methods used conflicting data are generated hampering the risk assessment.

Tobacco may be involved in the decreased macrophage heme oxygenase-1 (HO-1) expression described in smoking-induced severe emphysema, via the nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)–BTB and CNC homology 1, basic leucine zipper transcription factor 1 (Bach1) pathway. We assessed in vitro effects of cigarette smoke condensate (CS) in the human monocyte/macrophage cell line (THP-1). CS exposure led to increased HO-1 and nuclear Nrf2 expression (6h) followed by decreased HO-1 expression concomitantly with nuclear Nrf2/Bach1 ratio decrease (72h). CS-induced mitogen-activated protein kinase (MAPK) phosphorylation. Extracellular-signal-regulated kinase1/2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK) inhibition completely abrogated CS effects on HO-1 expression and nuclear Nrf2/Bach1 translocation. These results suggest that ERK1/2 and JNK are involved in CS-induced biphasic HO-1 expression by a specific regulation of Nrf2/Keap1–Bach1.

Les nanotechnologies sont definies comme l’ensemble des techniques visant a concevoir, caracteriser et produire des materiaux a l’echelle du nanometre dans au moins une de leurs dimensions. Ces nanomateriaux sont eux-memes constitues de nano-objets (nanoparticules, nanotubes…). Leur dimension nanometrique leur confere, du fait des lois de la physique quantique, de nouvelles proprietes physicochimiques et des comportements inedits. Les applications des nanotechnologies sont multiples (cosmetologie, industrie, medecine…). La production et l’utilisation des nanomateriaux connaissent une croissance importante. Cependant, des inquietudes sont emises sur les effets potentiels des nanoparticules sur la sante, a court et a long terme. Ces questions sont motivees par la connaissance des effets toxiques sur la sante des particules micrometriques de la pollution atmospherique et par la crainte de voir ces effets s’amplifier du fait de la nanodimension de ces materiaux. Dans cet article, est proposee une synthese globale mais non exhaustive des connaissances actuelles concernant la penetration, la deposition, la translocation et l’elimination dans l’appareil respiratoire, ainsi que les effets respiratoires des nanoparticules metalliques (plus particulierement du dioxyde de titane) et des nanotubes de carbone. L’ensemble des etudes experimentales in vivo et in vitro actuellement disponibles met en evidence l’existence d’effets biologiques des nanoparticules sur l’appareil respiratoire avec, notamment, la generation de stress oxydant, un effet pro-inflammatoire, un effet prothrombotique, la possibilite de survenue de fibrose et d’emphyseme pulmonaire ou de dommages a l’ADN. Une meilleure connaissance des effets biologiques potentiels des nanoparticules est requise, afin de mettre en place des mesures de prevention appropriees en milieu de travail et/ou en population generale, si cela s’averait necessaire.

Telomere length is considered a marker for biological aging. Chronic obstructive pulmonary disease (COPD) may be associated with premature aging.To test the hypothesis that patients with COPD experience accelerated telomere shortening and that inflammation is linked to this process.We measured telomere length, using a quantitative polymerase chain reaction-based method, and plasma levels of various cytokines in 136 patients with COPD, 113 age- and sex-matched smokers, and 42 nonsmokers with normal lung function.Median (range) telomere length ratio was significantly lower in patients with COPD (0.57 [0.23-1.18]) than in control smokers (0.79 [0.34-1.58]) or nonsmokers (0.85 [0.38-1.55]) (P0.001). The difference remained highly significant when using logistic regression analysis adjusted for age, sex, and tobacco exposure. Both females and males with COPD had shorter telomere length than same-sex control subjects. Telomere length was related to age in patients and control subjects but was shorter in patients than in control subjects in all age groups. No relationship was found between telomere length and tobacco exposure in patients or control subjects, with no difference between control smokers and nonsmokers. In patients with COPD, telomere length was related to PaO2 (P0.001) and PaCO2 (P0.001) but not to lung function parameters or the BODE Index. Patients with COPD also had elevated plasma levels of various cytokines, interleukin-6 correlating negatively with telomere length (P0.001).Given that in vivo telomere length reflects cellular turnover and exposure to oxidative and inflammatory damage, our data support accelerated aging in COPD.

The search for new molecules to fight Pseudomonas aeruginosa is of paramount importance. Carbon monoxide (CO) is known to act as an effective inhibitor of the respiratory chain in P. aeruginosa, but the practical use of this gas as an antibacterial molecule is hampered by its toxicity and difficulty to manipulate. Here, we show that a water-soluble CO releaser (CORM-3) possesses bactericidal properties against laboratory and antibiotic-resistant P. aeruginosa. CORM-3 reduced the bacterial count by 4 logs 180 min after in vitro treatment. CORM-3-treated bacteria had a lower O(2) consumption than vehicle-treated bacteria, and the decrease in O(2) consumption temporally preceded the bactericidal action of CORM-3. These results support the hypothesis that the antimicrobial effect of CORM-3 is mediated by an interaction of CO liberated by the carrier with the bacterial respiratory chain. The antibacterial effect occurred at concentrations of CORM-3 that are 50-fold lower than toxic concentrations for eukaryotic cells. CORM-3 treatment compared to vehicle treatment decreased bacterial counts in the spleen and increased survival in immunocompetent and immunosuppressed mice following P. aeruginosa bacteremia. Our results suggest that CORMs could form the basis for developing a new therapeutic strategy against P. aeruginosa-induced infection.

Introduction Le deficit en alpha-1 antitrypsine est associe a un risque accru d’emphyseme pulmonaire. L’objectif de cette etude est de decrire les caracteristiques des patients emphysemateux deficitaires. Methodes Etude de cohorte prospective multicentrique. Criteres d’inclusion : patients adultes, presentant un emphyseme identifie au scanner, deficitaires en alpha-1 antitrypsine et suivis en France. Les caracteristiques cliniques, fonctionnelles, la qualite de vie et les modalites therapeutiques sont recueillies tous les 6 mois pendant 5 ans. Resultats Deux cent un patients ont ete recenses par 56 centres entre 2005 et 2008. Les caracteristiques a l’inclusion des 110 premiers patients ont ete analysees. L’âge moyen etait de 50 ans (DS : 11,8), 62,7 % etaient des hommes, 90 % etaient fumeurs. Caracteristiques fonctionnelles (% theo.) : VEMS : 42,8 (19,6), CPT : 128,3 (21,7), CRF : 167,0 (46,0), test de marche de 6 minutes (metres) : 413 (130). 51 (46,4 %) patients recevaient un traitement substitutif ; le mode d’administration etait hebdomadaire (37,5 %), bimensuel (35,4 %) ou mensuel (25,5 %). Le seul facteur associe a la probabilite d’etre traite etait le centre de suivi du patient. Conclusion Cette etude revele les grandes disparites de presentation clinique et de prise en charge des patients emphysemateux deficitaires en alpha-1 antitrypsine et souligne la necessite d’une homogeneisation des pratiques.

Background: Oxidative stress, resulting from the increased oxidative burden and decreased level of antioxidant proteins, plays a role in the pathophysiology of smoking-related pulmonary emphysema. Expression of several antioxidant proteins, such as heme oxygenase-1 (HO-1), glutathione peroxidase 2 (GPX2) and NAD(P)H:quinone oxidoreductase 1 (NQO1), results from an equilibrium created by positive or negative regulation by the transcription factors Nrf2, Keap1 and Bach1, respectively. However, whether the expression of these transcription factors is altered in emphysema and could account for decreased expression of antioxidant proteins is not known. A study was undertaken to investigate the expression and subcellular localisation of Nrf2, Keap1 and Bach1 as potential regulators of HO-1, GPX2 and NQO1 in alveolar macrophages, a key cell in oxidative stress, in lung surgical specimens from non-smokers without emphysema and smokers with and without emphysema. Methods and results: Western blot, immunohistochemical and laser scanning confocal analysis revealed that the Nrf2 protein level decreased significantly in whole lung tissue and alveolar macrophages (cytosol and nucleus) in patients with emphysema compared with those without emphysema. Conversely, Bach1 and Keap1 levels were increased in patients with emphysema. These modifications were associated with a parallel decrease in the expression of HO-1, GPX2 and NQO1 at the cellular level, which was inversely correlated with airway obstruction and distension indexes, and increased macrophage expression of the lipid peroxidation product 4-hydroxy-2-nonenal. Silencing RNA experiments in vitro in THP-1 cells were performed to confirm the cause-effect relation between the loss of Nrf2 and the decrease in HO-1, NQO1 and GPX2 expression. Nrf2/Keap1-Bach1 equilibrium was altered in alveolar macrophages in pulmonary emphysema, which points to a decreased stress response phenotype. Conclusions: This finding opens a new view of the pathophysiology of emphysema and could provide the basis for new therapeutic approaches based on preservation and/or restoration of such equilibrium.

The use and commercial potential of engineered nanomaterials is increasing, but questions of occupational and public health safety remain. Here, we review research published in 2007 concerning toxicology of nanomaterials. Articles were selected from the Medline Pubmed database, published or pre-published during 2007, using keywords (nanomaterials or nanoparticles or nanostructures) and (toxicity or health). From the 238 articles, we chose to concentrate mainly on research into carbonaceous (carbon nanotubes [CNTs] and fullerenes) and metallic materials (pure metal, oxides), because of their relevance. The induction of oxidative stress was repeatedly reported, and new information on the movement of nanomaterials through membranes was publicized. Concerning CNTs, information was revealed on DNA damage in vitro and pulmonary and systemic in vivo effects. Several of the reports failed to follow recent expert recommendations concerning good practice for nanotoxicologic research, complicating the integration of...

Due to several commercial applications of manufactured nanoparticles (NPs), such as silver (Ag NPs), titanium dioxide (TiO2 NPs) and cerium dioxide (CeO2 NPs), knowledge of the toxicity of those NPs is of great importance. Many studies have linked exposure to fine and ultrafine particles (from air pollution) to an increase of morbidity or mortality due to various respiratory or cardiovascular diseases. Several works have focused on the effects of pulmonary exposure to manufactured NPs. It has been shown that exposure to NPs may lead to an inflammatory response, pulmonary fibrosis and emphysema. However, less is known on the effect of exposure to NPs on the offspring. It has been reported that exposure during pregnancy to TiO2 NPs and SiO2 NPs is associated with fetal hypertrophy, neurotoxicity, and exposure to carbon black NPs induced renal disease in the offspring. NPs may interfere with normal fetal lung development. In addition, a recent study reported that pulmonary exposure of newborn mice to TiO2 NPs was associated with pulmonary inflammation and impaired lung development. Therefore, the aim of this study is to assess the impact of exposure by the respiratory route to various NP during pregnancy on lung development of the offspring in a mouse model, and to determine the key parameters involved in lung alterations. For this study, we used three metal NPs: TiO2, Ag, and CeO2 with the same size and shape, to assess the impact of NPs physico-chemical properties in the potential effects on lung development. C57Bl6/J pregnant mice were exposed weekly to 100 μg NPs by nonsurgical intratracheal instillation. Analysis (biological, histological and functional) of the lungs of the offspring were made at different time of lung development: one day before delivery (18GD for gestational day), 14 days after birth (pulmonary alveolization) and 21 days after birth (lung maturity). Preliminary results show that the exposure to TiO2 NPs during pregnancy did not affect the number of fetuses per litter, or the weight of uterus and placenta. However, the weight of fetuses whose mothers were exposed to TiO2 NPs is significantly decreased. A decrease of VEGF-A gene expression, involved in lung development, was also observed at 18GD. These preliminary results suggest that exposure to TiO2 NPs during pregnancy could affect the development of the offspring. For the remainder of this work, pregnant mice will be exposed to other NPs (Ag and CeO2) in order to see if this effect depends on the type of NP. Moreover additional analyzes will be performed to confirm whether lung alterations are observed at later times after birth, and to understand the key parameters and mechanism associated with these changes.

The toxic effect of high concentrations of CO gas in living organisms is coherently typified at biochemical levels by the high affinity of CO for hemoglobin and cytochromes, heme-dependent proteins that are indispensable for oxygen transport and mitochondrial respiration. However, the basal production of CO during heme degradation and the ability of heme oxygenase-1 (HO-1) to increase CO availability pose the question of how this gaseous molecule interacts with metal centers within the intracellular milieu to serve as one of the most unconventional signaling mediators. Emerging evidence indicates that the diverse and multifaceted beneficial effects exerted by "low concentrations" of CO cannot be explained solely by the activation of classic prototypic targets (i.e., guanylate cyclase/potassium channels) but entails the dynamic and concerted activation/inhibition of a group of CO-responsive proteins. As the complexity of the temporal and spatial action of CO is progressively being appreciated, this review aims to (a) highlight the current knowledge on certain metal-containing proteins that interact directly with CO; (b) analyze the latest notions on their functional role in response to CO; and finally (c) propose a rational view on the mode these CO targets may interrelate with and be regulated by the HO/CO pathway.

Oxidative stress is thought to play a major role in the pathogenesis of chronic obstructive pulmonary disease, characterized by impaired lung function. A large number (> or =33) of GT repeats (L-allele) in the gene of the powerful antioxidant enzyme heme oxygenase-1 has been associated with susceptibility to accelerated lung function decline. In contrast, beta-carotene may help to protect against accelerated decline. To determine whether high serum levels of beta-carotene might counterbalance the greater susceptibility of L-allele carriers, the authors analyzed the annual decline in forced expiratory volume in 1 second (FEV1) in a general population sample of 523 French subjects (20-44 years, 50% men) examined in 1992 and 2000 as part of the European Community Respiratory Health Survey. Analysis of covariance, adjusted for sex as well as baseline age, body mass index, smoking, and FEV1, showed that, among subjects with low beta-carotene levels, L-allele carriers experienced a steeper mean FEV1 decline than did noncarriers (mean = -58.8, 95% confidence interval: -73.2, -44.5 vs. mean = -34.7, 95% confidence interval: -38.9, -29.8 ml/year) (p = 0.009), whereas among subjects with high beta-carotene levels, the FEV1 decline was not different in L-allele carriers and noncarriers (two-sided p = 0.9). The results suggest that high levels of beta-carotene might counterbalance the effects on FEV1 decline of a genetically determined deficiency in antioxidant response.

Carbon nanotubes (CNTs) are cylinders of one or several coaxial graphite layer(s) (single-walled [SW]CNTs or multiwalled [MW]CNTs, respectively) with a catalytic material (iron, nickel or cobalt) often present inside and/or at their extremity, as well as variable amounts of inert synthesis support, depending on the synthesis method [1]. Their diameter is in the order of nanometers (depending on the number of walls) and they can reach several micrometers in length. Owing to their unique electrical properties, unusual strength and particular effectiveness in heat conduction [2], CNTs are particularly promising nanomaterials for industrial use (see [101] for inventory) and, therefore, one can easily imagine that their production will continue to increase in the future. However, the same novel properties that make CNTs interesting raise concerns about their potential adverse effects on biological systems, which could lead to health issues, particularly when thinking of their potential use in the medical field.

Chronic exposure to particulate air pollution is associated with lung function impairment. To determine the molecular mechanism(s) of this phenomenon, we investigated, in an alveolar human epithelial cell line (A549), whether diesel exhaust particles (DEPs), a main component of particulate air pollution, modulates the expression and activity of the matrix metalloprotease (MMP)-1, a collagenase involved in alveolar wall degradation. Interaction of DEPs with cigarette smoke, which also produces structural and functional lung alterations, was also investigated. A noncytotoxic concentration of DEPs induced an increase in MMP-1 mRNA and protein expression and activity in A549 cells without modifying the expression of the MMP inhibitors TIMP-1 and -2. This effect was not potentiated when cells were coexposed to noncytotoxic concentrations of cigarette smoke condensate. DEP-induced MMP-1 was associated with increased ERK 1/2 phosphorylation and upregulation of expression and activity of the NADPH oxidase analog NOX4. Cell transfection with a NOX4 small interfering RNA prevented these phenomena, showing the critical role of a NOX4 ERK 1/2 pathway in DEP-induced MMP-1 expression and activity. Similar results to those observed in A549 cells were obtained in another human lung epithelial cell line, NCI-H292. Furthermore, experiments in mice intratracheally instilled with DEPs confirmed the in vitro findings, showing the induction of NOX4 and MMP-1 protein expression in alveolar epithelial cells. We conclude that alveolar alterations secondary to MMP-1 induction could explain lung function impairment associated with exposure to particulate pollution.

Objective To determine the consequences of pharmacologic up-regulation of heme oxygenase 1 (HO-1), and inhibition of HO-1 by injection of an anti–HO-1 small interfering RNA (siRNA), in vivo in the acute phase of a mouse model of nonautoimmune arthritis. Methods In the K/BxN mouse serum transfer model, which mimics human inflammatory arthritis without lymphocyte influence, HO-1 was up-regulated by intraperitoneal injection of cobalt protoporphyrin IX (CoPP), a potent pharmacologic inducer, and was inhibited using a specific siRNA. The clinical progress of arthritis was monitored by measurement of paw thickness. Interleukin-1β (IL-1β), IL-6, tumor necrosis factor α (TNFα), serum antioxidant, and nitric oxide (NO) levels, prostaglandin E2 (PGE2) production, and matrix metalloproteinase 9 (MMP-9) activity were measured in serum. At the end of the experiments, joints were examined for immunohistopathologic changes. Results Intraperitoneal injection of CoPP alleviated disease symptoms, such as joint swelling, cartilage degradation, and proliferation of inflammatory tissue in joints, in the acute phase of inflammatory arthritis. The CoPP-induced expression of HO-1 in the joints and liver was associated with marked decreases in IL-1β, IL-6, and TNFα levels, PGE2 secretion, and MMP-9 activity in serum, and with a marked increase in systemic antioxidant activity. In contrast, NO production in serum and inducible NO synthase expression in chondrocytes were not affected by HO-1 induction. Specific inhibition of HO-1 by in vivo delivery of anti–HO-1 siRNA repressed the protective effects. Conclusion Our data provide the first evidence that pharmacologically induced up-regulation of HO-1 triggers a robust protective antiinflammatory response in a model of nonautoimmune arthritis in mice. This suggests that exogenously induced HO-1 may have potential as therapy in the acute phase of inflammatory arthritis in humans.

Cellular senescence--defined as a stable cell-cycle arrest combined with stereotyped phenotypic changes--might play a causal role in various lung diseases, including chronic obstructive pulmonary disease (COPD), which is predicted to become the third leading cause of death worldwide by 2020. COPD is characterized by slowly progressive airflow obstruction and emphysema due to destruction of the lung parenchyma and is often complicated by pulmonary hypertension (PH). No curative treatment is available. Senescent cells, which accumulate with age, are increased in lungs from patients with COPD and express a robust senescence-associated secretory phenotype (SASP), which is proinflammatory. The aim of this review is to show how senescent cells can drive the lung alterations seen in COPD, which mechanisms may be involved, and whether therapeutic interventions may slow or delay the in vitro cell-senescence process and in vivo lung alterations.

Surface tunability and their ability to translocate plasma membranes make chemically functionalized carbon nanotubes (f-CNTs) promising intracellular delivery systems for therapeutic or diagnostic purposes in the central nervous system (CNS). The present study aimed to determine the biological impact of different types of multiwalled CNTs (MWNTs) on primary neuronal and glial cell populations isolated from fetal rat frontal cortex (FCO) and striatum (ST). Neurons from both brain regions were generally not affected by exposure to MWNTs as determined by a modified LDH assay. In contrast, the viability of mixed glia was reduced in ST-derived mixed glial cultures, but not in FCO-derived ones. Cytotoxicity was independent of MWNT type or dose, suggesting an inherent sensitivity to CNTs. Characterization of the cell populations in mixed glial cultures prior to nanotube exposure showed higher number of CD11b/c positive cells in the ST-derived mixed glial cultures. After exposure to MWNTs, CNT were uptaken more effectively by CD11b/c positive cells (microglia), compared to GFAP positive cells (astrocytes). When exposed to conditioned media from microglia enriched cultures exposed to MWNTs, ST-derived glial cultures secreted more NO than FCO-derived cells. These results suggested that the more significant cytotoxic response obtained from ST-derived mixed glia cultures was related to the higher number of microglial cells in this brain region. Our findings emphasize the role that resident macrophages of the CNS play in response to nanomaterials and the need to thoroughly investigate the brain region-specific effects toward designing implantable devices or delivery systems to the CNS. journal article research support, non-u.s. gov't 2015 Aug 25 2015 06 26 imported

Nanotechnologies, defined as techniques aimed to conceive, characterize and produce material at the nanometer scale, represent a fully expanding domain, and one can predict without risk that production and utilization of nanomaterials will increase exponentially in the coming years. Applications of nanotechnologies are numerous, in constant development, and their potential use in the medical field as diagnosis and therapeutics tools is very attractive. The size particularity of these nanomaterials gives them novel properties, allowing them to adopt new comportments because of the laws of quantum physics that exist at this scale. However, worries are expressed regarding the exact properties that make these nanomaterials attractive, and questions are raised regarding their potential toxicity, their long-term secondary effects or their biodegradability, particularly when thinking of their use in the (nano)medical field. These questions are justified by the knowledge of the toxic effects of atmospheric pollution micrometric particles on health, and the fear to get an amplification of these effects because of the size of the materials blamed. In this paper, we first expose the sensed medical applications of nanomaterials, and the physicochemical and molecular determinants potentially responsible for nanomaterials biological effects. Finally, we present a synthesis of the actual knowledge regarding toxicological effects of nanomaterials. It is clear that, in regard to the almost empty field of what is known on the subject, there's an urge to better understand biological effects of nanomaterials, which will allow their safe use, in particular in the nanomedicine field.

RESUME Les oxydants pourraient etre impliques dans la Physiopathologie de la BPCO (Broncho-Pneumopathie Chronique Obstructive), definie par une diminution des debits aeriens progressive et incompletement reversible. L’HO-1 (heme oxygenase-1 ) est un antioxydant puissant. Il a ete montre qu’un polymorphisme microsatellite du promoteur du gene de l’HO-1 (repetition de paires de bases (GT)) etait capable de moduler la transcription du gene en reponse au stress oxydant. Un promoteur long serait associe a une expression de la proteine HO-1 moindre. Nous avons emis l’hypothese que ce polymorphisme pourrait etre associe au declin de la fonction pulmonaire chez des sujets exposes a une agression oxydante importante comme les fumeurs. Pour tester cette hypothese, nous avons genotype 749 sujets (âges de 20 a 44 ans, 50 % d’hommes et 40 % de non-fumeurs) examines en 1992 et en 2000 dans le cadre du recueil des donnees francaises de l’etude ECRHS. Nous avons compare les porteurs d’allele long (L) ((GT)n ≥ 33 repetitions sur un ou deux allele (s)) aux autres. En longitudinal, nous avons observe que le declin de la fonction pulmonaire etait accelere chez les porteurs de l’allele L par rapport aux autres. En outre, il y avait une interaction entre l’allele L et le tabagisme : le declin de la fonction pulmonaire etait accelere dans le groupe des gros fumeurs porteurs de l’allele L par rapport aux gros fumeurs non-porteurs de l’allele L et aux porteurs de l’allele L non-gros fumeurs. Nos resultats suggerent donc qu’un promoteur long du gene de l’HO-1 serait associe, chez les gros fumeurs, au developpement d’un trouble ventilatoire obstructif.

In the United States sepsis annually affects 700,000 people and accounts for about 210,000 deaths. Respiratory failure has long been known to be a frequent occurrence of this pathological condition and to represent a major contributor to the high associated mortality [1]. This contribution discusses of the effects of sepsis and septic shock on respiratory muscle function and focuses on some of the possible mechanisms involved in the genesis of these effects.

Heme oxygenase (HO), the heme-degrading enzyme, has shown anti-inflammatory effects in several models of pulmonary diseases. HO is induced in airways during asthma; however, its functional role is unclear. Therefore, we evaluated the role of HO on airway inflammation [evaluated by bronchoalveolar lavage (BAL) cellularity and BAL levels of eotaxin, PGE2, and proteins], mucus secretion (evaluated by analysis of MUC5AC gene expression and periodic acid-Schiff staining), oxidative stress (evaluated by quantification of 4-hydroxynonenal adducts and carbonylated protein levels in lung homogenates), and airway responsiveness to histamine in ovalbumin (OVA)-sensitized and multiple aerosol OVA or saline-challenged guinea pigs (6 challenges, once daily, OVA group and control group, respectively). Airway inflammation, mucus secretion, oxidative stress, and responsiveness were significantly increased in the OVA group compared with the control group. HO upregulation by repeated administrations of hemin (50 mg/kg ip) significantly decreased airway responsiveness in control animals and airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These effects were reversed by the concomitant administration of the HO inhibitor tin protoporphyrin-IX (50 μmol/kg ip). Repeated administrations of tin protoporphyrin-IX alone significantly increased airway responsiveness in control animals but did not modify airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These results suggest that upregulation of the HO pathway has a significant protective effect against airway inflammation, mucus hypersecretion, oxidative stress, and hyperresponsiveness in a model of allergic asthma in guinea pigs.

Contexte La biometrologie des particules minerales non fibreuses (PMNF) est un ensemble de techniques permettant d’evaluer le niveau de retention en PMNF dans differents milieux biologiques. Ces analyses aident a l’identification d’expositions professionnelles ou environnementales a rapprocher de diverses maladies, notamment respiratoires. Leur interpretation necessite de connaitre le profil de retention pulmonaire dans des series de patients indemnes d’exposition significative (temoins). Objectif L’objectif de cette etude est d’evaluer l’evolution du niveau de retention pulmonaire en PMNF et de valider l’hypothese d’une augmentation sur une periode de 20 ans, du niveau de retention en particules de titane dans 2 series de patients non exposes professionnellement a ces PMNF. Patients et methodes Deux series de patients atteints de cancer broncho-pulmonaire ont ete recrutees a partir de services de pneumologie : serie 1 (entre 1994 et 1999, n = 33, âge moyen : 58,7 ans, tous fumeurs) et serie 2 (entre 2009 et 2014, n = 36, âge moyen : 63,2 ans, 31 fumeurs). L’analyse detaillee de l’interrogatoire professionnel par des experts en pathologie professionnelle ne retrouvait pas d’exposition a des PMNF. Les echantillons de parenchyme pulmonaire preleves lors de l’intervention d’exerese pulmonaire ont ete analyses en microscopie electronique a transmission couplee a un dispositif d’analyse chimique elementaire, afin d’identifier et de quantifier les PMNF mesurant au moins 0,1 μm. Resultats Il a ete observe une augmentation de la retention pulmonaire pour la totalite des PMNF entre les series 1 et 2. L’element le plus notable etait l’augmentation du niveau de retention en particules de titane (mediane : 5 × 107 et 8,8 × 107 p/g, pour les series 1 et 2, respectivement ; p = 0,005). Au sein de la serie 2, il n’y avait pas d’influence du sexe, de l’âge, du tabagisme ou du mode de recueil des echantillons (paraffine/formaldehyde) sur le resultat de la mesure de concentration des particules de titane. Discussion-Conclusion Ces resultats, obtenus sur des series importantes comparativement aux donnees de la litterature, confirment l’hypothese d’une augmentation du niveau de retention pulmonaire en particules de titane entre 1994–1999 et 2009–2014 chez des personnes sans exposition professionnelle specifique identifiee a des PMNF. Il apparait important de documenter les sources d’expositions potentielles responsables de cette augmentation. La prise en compte du resultat de populations « temoins » est essentielle a l’interpretation des resultats biometrologiques chez les patients atteints d’une pathologie respiratoire pour laquelle le role d’une exposition professionnelle a des particules minerales est suspecte.

Nitric oxide (NO) has been implicated in the development of heart failure, although the source, significance, and functional role of the different NO synthase (NOS) isoforms in this pathology are controversial. The presence of a neuronal-type NOS isoform (NOS1) in the cardiac sarcoplasmic reticulum has been recently discovered, leading to the hypothesis that NOS1-derived NO may notably alter myocardial inotropy. However, the regulation and role(s) of NOS1 in cardiac diseases remain to be determined. Using an experimental model of myocardial infarction (MI) in senescent rats, we demonstrated a significant increase in cardiac NOS1 expression and activity in MI, coupled with the translocation of this enzyme to the sarcolemma through interactions with caveolin-3. The enhanced NOS1 activity counteracts the decrease in cardiac NOS3 expression and activity observed in heart failure. We demonstrated an increased interaction between NOS1 and its regulatory protein HSP90 in post-MI hearts, a potential mechanism for the higher NOS1 activity in this setting. Finally, preferential in vivo inhibition of NOS1 activity enhanced basal post-MI left ventricular dysfunction in senescent rats. These results provide the first evidence that increased NOS1-derived NO production may play a significant role in the autocrine regulation of myocardial contractility after MI in aging rats.

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.

Background Cells exhibiting dysregulated growth may express telomerase reverse transcriptase (TERT), the dual function of which consists of maintaining telomere length, in association with the RNA template molecule TERC, and controlling cell growth. Here, we investigated lung TERT in human and experimental pulmonary hypertension (PH) and its role in controlling pulmonary artery smooth muscle cell (PA-SMC) proliferation. Methods and Results Marked TERT expression or activity was found in lungs from patients with idiopathic PH and from mice with PH induced by hypoxia or serotonin-transporter overexpression (SM22-5HTT+ mice), chiefly within PA-SMCs. In cultured mouse PA-SMCs, TERT was expressed on growth stimulation by serum. The TERT inhibitor imetelstat and the TERT activator TA65 abrogated and stimulated PA-SMC growth, respectively. PA-SMCs from PH mice showed a heightened proliferative phenotype associated with increased TERT expression, which was suppressed by imetelstat treatment. TERC−/− mice at generation 2 and TERT−/− mice at generations 2, 3, and 4 developed less severe PH than did wild-type mice exposed to chronic hypoxia, with less distal pulmonary artery muscularization and fewer Ki67-stained proliferating PA-SMCs. Telomere length differed between TERC−/− and TERT−/− mice, whereas PH severity was similar in the 2 strains and across generations. Chronic imetelstat treatment reduced hypoxia-induced PH in wild-type mice or partially reversed established PH in SM22-5HTT+ mice while simultaneously decreasing TERT expression. Opposite effects occurred in mice treated with TA65. Conclusions Telomerase exerts telomere-independent effects on PA-SMC growth in PH and may constitute a treatment target for PH.

Rationale Chronic obstructive pulmonary disease (COPD) is often associated with age-related systemic abnormalities that adversely affect the prognosis. Whether these manifestations are linked to the lung alterations or are independent complications of smoking remains unclear. Objectives To look for aging-related systemic manifestations and telomere shortening in COPD patients and smokers with minor lung destruction responsible for a decline in the diffusing capacity for carbon monoxide (DLCO) corrected for alveolar volume (KCO). Methods Cross-sectional study in 301 individuals (100 with COPD, 100 smokers without COPD, and 101 nonsmokers without COPD). Measurements and Main Results Compared to control smokers, patients with COPD had higher aortic pulse-wave velocity (PWV), lower bone mineral density (BMD) and appendicular skeletal muscle mass index (ASMMI), and shorter telomere length (TL). Insulin resistance (HOMA-IR) and glomerular filtration rate (GFR) were similar between control smokers and COPD patients. Smokers did not differ from nonsmokers for any of these parameters. However, smokers with normal spirometry but low KCO had lower ASMMI values compared to those with normal KCO. Moreover, female smokers with low KCO, had lower BMD and shorter TL compared to those with normal KCO. Conclusions Aging-related abnormalities in patients with COPD are also found in smokers with minor lung dysfunction manifesting as a KCO decrease. Decreased KCO might be useful, particularly among women, for identifying smokers at high risk for aging-related systemic manifestations and telomere shortening.

International audience; The development and the increasing production of manufactured nanoparticles (NP) are raising safety concerns because of the potential effects of NP on human health, particularly at the respiratory level. Several studies have shown that exposure to manufactured NP can induce pathogenic biological effects, with lung remodelling (fibrosis, emphysema…), depending on the physicochemical characteristics of the NP. Currently, oxidative stress and inflammation are the most widely accepted paradigms of NP toxicity; however, the exact underlying mechanisms in the biological effects of NP still remain unknown. Autophagy is a physiological process that allows the autodigestion of the subcellar components and which is also involved in the elimination of intracellular pathogens. It has been shown that this process can negatively regulate inflammation and oxidative stress. Thus, the hypothesis of this study is that a defective autophagy could be a new mechanism explaining, at least in part, NP effects. We choose to focus on TiO2 NP since it is one of the most abundantly produced and widely used NP. We used seven TiO2 NPs presenting different physicochemicals properties (size, crystal phase, surface embedding) and compared their effects to those of micron-size TiO2 and carbon black (CB - for chemical composition effects). NPs were characterized by electron microscopy, dynamic light scattering and X-ray diffraction. Murine macrophages (RAW cell line) were exposed to 50 μg/mL TiO2 and CB NP for 6 hours. Effects of NP on the autophagy process were analysed by looking at the expression of autophagy markers (LC3-II and p62) and lysosomal proteins (LAMPs and cathepsins). We also analysed the cytoskeleton network by fluorescence microscopy. Moreover, expression of inflammatory cytokines was determined in macrophages exposed to NP. All particles, except the micrometric one, induced an increase of LC3-II protein expression, indicating an accumulation of autophagosomes. These particles also increased p62 protein level, suggesting an autophagy blockade. This perturbation of the autophagy process by NP does not seem to result from a disruption of the cytoskeleton but from a defect in the lysosome function as suggested by a decrease of mature cathepsins expression. Moreover, preliminary results showed that exposure to TiO2 NP induce inflammation, at different levels depending on their physicochemicals characteristics. These results suggest that some TiO2 NP, depending on their physicochemical properties, can block the autophagy process. The future work will be to understand the mechanisms explaining this autophagy dysfunction and to determine its consequence on the toxicity induced by these NP.

Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3′,5′-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.

The rapid development of nanotechnologies is raising safety concerns because of the potential effects of engineered nanomaterials on human health, particularly at the respiratory level. Since the last decades, many in vivo studies have been interested in the pulmonary effects of different classes of nanomaterials. It has been shown that some of them can induce toxic effects, essentially depending on their physico-chemical characteristics, but other studies did not identify such effects. Inflammation and oxidative stress are currently the two main mechanisms described to explain the observed toxicity. However, the exact underlying mechanism(s) still remain(s) unknown and autophagy could represent an interesting candidate. Autophagy is a physiological process in which cytoplasmic components are digested via a lysosomal pathway. It has been shown that autophagy is involved in the pathogenesis and the progression of human diseases, and is able to modulate the oxidative stress and pro-inflammatory responses. A growing amount of literature suggests that a link between nanomaterial toxicity and autophagy impairment could exist. In this review, we will first summarize what is known about the respiratory effects of nanomaterials and we will then discuss the possible involvement of autophagy in this toxicity. This review should help understand why autophagy impairment could be taken as a promising candidate to fully understand nanomaterials toxicity.

We hypothesized that O2 tension influences the redox state and the immunomodulatory responses of inflammatory cells to dimethyl fumarate (DMF), an activator of the nuclear factor Nrf2 that controls antioxidant genes expression. This concept was investigated in macrophages permanently cultured at either physiological (5% O2) or atmospheric (20% O2) oxygen levels and then treated with DMF or challenged with lipopolysaccharide (LPS) to induce inflammation. RAW 264.7 macrophages cultured at 20% O2 exhibited a pro-oxidant phenotype, reflected by a lower content of reduced glutathione, higher oxidized glutathione and increased production of reactive oxygen species when compared to macrophages continuously grown at 5% O2. At 20% O2, DMF induced a stronger antioxidant response compared to 5% O2 as evidenced by a higher expression of heme oxygenase-1, NAD(P)H:quinone oxydoreductase-1 and superoxide dismutase-2. After challenge of macrophages with LPS, several pro-inflammatory (iNOS, TNF-α, MMP-2, MMP-9), anti-inflammatory (arginase-1, IL-10) and pro-angiogenic (VEGF-A) mediators were evaluated in the presence or absence of DMF. All markers, with few interesting exceptions, were significantly reduced at 5% O2. This study brings new insights on the effects of O2 in the cellular adaptation to oxidative and inflammatory stimuli and highlights the importance of characterizing the effects of chemicals and drugs at physiologically relevant O2 tension. Our results demonstrate that the common practice of culturing cells at atmospheric O2 drives the endogenous cellular environment towards an oxidative stress phenotype, affecting inflammation and the expression of antioxidant pathways by exogenous modulators.

International audience

Objective: To study the expression (mRNA and protein) and activity of the constitutive isoforms of nitric oxide synthase (NOS1 and NOS3) in a skeletal muscle of septic patients. Design: Prospective study. Setting: An adult trauma/surgical intensive care unit in an urban teaching hospital. Patients: Sixteen septic patients and 21 controls. Interventions: None. Measurements and Main Results: Samples of the rectus abdominis muscle were obtained during surgical procedure. NOS mRNA, protein, and activity were detected by reverse-transcriptase polymerase chain reaction, Western blot, and the conversion of [ 3H ]L-arginine to [ 3H ]L-citrulline, respectively. The main results of this study are as follows: a) Levels of NOS1 mRNA and protein were significantly higher than those of NOS3 in the rectus abdominis muscle of control patients; b) NOS1 expression was down-regulated in septic patients, whereas NOS3 was up-regulated; c) these modulations were associated with a reduction in constitutive NOS activity; and d) modifications of NOS1 and NOS3 protein expression were correlated significantly with the severity of sepsis, assessed by the Simplified Acute Physiology Score II. Conclusions: Sepsis induces reciprocal expressional modifications of NOS1 and NOS3 in human skeletal muscle, which decreases muscular constitutive NOS activity. These modifications may have implications for muscle impairment in septic patients.

Since increased tyrosine phosphorylation has been observed in response to brain ischemia, we investigated whether riluzole (an inhibitor of glutamate neurotransmission with neuroprotective properties) affects tyrosine phosphorylation stimulated by N -methyl- d -aspartate (NMDA) in rat hippocampal slices. Riluzole produced an extremely potent concentration-related inhibition of NMDA (1 mM)-stimulated protein tyrosine phosphorylation (IC 50 =0.5±0.03 μM, mean±S.D.), but failed to affect that evoked by phorbol 12-myristate 13-acetate (PMA, an activator of protein kinase C, 0.1 and 1 μM). These results suggest that inhibition of tyrosine phosphorylation may contribute to the neuroprotective effects of riluzole against excitotoxic injury.

Peroxynitrite anion (ONOO(-)) is a potent biological oxidant produced by the near diffusion-limited reaction of superoxide and nitric oxide. Peroxynitrite has been implicated in diverse forms of free radical-induced tissue injury. Experimental evidence showed that exogenous and endogenous peroxynitrite causes alterations of the structure and function of mitochondrial proteins, leading to mitochondrial dysfunction and cellular or organ injury. These data are discussed along with its physiopathological implications.

Skeletal muscle failure is a frequent manifestation of sepsis that affects prognosis and rehabilitation by impairing respiration and ambulation. Animal studies have shown that the inducible NO synthase (NOS2) is expressed in skeletal muscles during sepsis, likely affecting muscular function, by promoting the formation of the strong oxidant peroxynitrite. In contrast, whether human skeletal muscle expresses a functional NOS2 in similar conditions is unknown. We studied NOS2 expression (mRNA and protein) and activity and its role in contractile function in samples from rectus abdominis muscle obtained during surgical procedure in 16 septic patients and in 21 controls. Peroxynitrite formation was detected by immunohistochemical detection of nitrotyrosine residues. The main results of this study are as follows: (1) A significant increase in NOS2 mRNA, protein, and activity was found in muscles from septic patients, the expression of NOS2 protein positively correlating with sepsis severity. (2) Contractile force was significantly lower in septic than in control muscles. This phenomenon was not reverted by muscle incubation ex vivo with the NOS inhibitor L-NMMA, indicating that NO was not involved in force reduction at the time of biopsy. (3) NOS2 expression in skeletal myocytes was strongly co-localized with nitrotyrosine, revealing muscular peroxynitrite generation during the septic process, before the muscle was biopsied. Exposure of control muscles to an amount of peroxynitrite similar to that generated in septic muscles during the septic process resulted in a nonreversible reduction in force generation. These results suggest that NOS2 could be involved in the decreased muscular force of septic patients via the local generation of peroxynitrite.

We have assessed, by intravital microscopy in rats, the effects of different anaesthetics on diaphragmatic arteriolar diameter. Rats were anaesthetized with etomidate, propofol or thiopental (groups E, P and T, respectively) and the diameters of the arterioles were measured sequentially at baseline and after topical application of either mefenamic acid (MA, 20 mumol litre-1) or N omega-nitro-L-arginine (NNA, 300 mumol litre-1), inhibitors of prostaglandins and nitric oxide, respectively. In group E, baseline arteriolar diameters were significantly higher than those in the two other groups (P0.01). MA and NNA induced significant constriction in the three groups (P0.001). However, whereas constriction induced by NNA was similar in the three groups, constriction induced by MA was significantly higher in group E compared with groups P and T (P0.05). We conclude that diaphragmatic arteriolar diameters in rats were greater during etomidate than during thiopental or propofol anaesthesia. This phenomenon may be mediated by prostaglandins.

1 We investigated by intravital microscopy in rats, the in vivo direct effects of theophylline on the diameters of second and third order diaphragm arterioles. 2 Theophylline (1–100 μm) dilated second and third order diaphragm arterioles significantly, and with an amplitude which was not statistically different from the one obtained with adenosine (1–100 μm). Enprofylline (1–100 μm), a theophylline analogue with poor adenosine-receptor antagonism but with similar or higher phosphodiesterases inhibition properties than theophylline, also dilated diaphragm arterioles, causing however, a significantly smaller dilatation than theophylline. 3 Neither the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 50 nm), nor the A2 adenosine receptor antagonist 3,7-dimethyl-1-proparglyxanthine (DMPX, 10 μm) reduced significantly theophylline-induced arteriolar dilatation. 4 Theophylline (100 nm) abolished adenosine-induced arteriolar dilatation. 5 The dilatation induced by theophylline was unchanged by the nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine (NNA, 300 μm). 6 Theophylline-induced arteriolar dilatation was abolished by the prostaglandin synthesis inhibitors mefenamic acid or indomethacin (20 μm). 7 These findings show that theophylline induced a significant dilatation of diaphragm arterioles via the release of prostaglandins. British Journal of Pharmacology (1998) 124, 1355–1362; doi:10.1038/sj.bjp.0701962

To evaluate the effect of systemic hypoxia on leukocyte-endothelium interactions in peripheral tissues, we studied by intravital microscopy leukocyte rolling velocity and adherence in venules of rat cremaster muscle. We examined the possible roles of changes in blood oxygenation, peripheral tissue oxygenation, changes in local shear rate, and the involvement of integrins. Six groups of rats submitted to either control normoxic conditions, or systemic hypoxia (PO2 = 51 mm Hg) associated with either low O2 tension of Krebs superfusing the muscle, high O2 tension of the Krebs superfusing the muscle, anti-lymphocyte function-associated antigen (LFA)-1beta antibody, pentoxifylline, or normoxic conditions associated with partial occlusion of the artery perfusing the muscle. We found that: (1) systemic moderate hypoxia resulting from purely respiratory disturbance even in the absence of local stop-flow phenomenon or circulatory shock can induce an increase in leukocyte adhesion and a decrease in leukocyte rolling velocity in the microcirculation of peripheral tissues; (2) to be present, this increase in leukocyte adhesion does not require tissue hypoxia of the peripheral tissue but the effect of systemic hypoxia on rolling velocity is prevented by tissue oxygenation; (3) this increase in leukocyte adhesion is mediated by CD11/CD18 integrins but is not due to changes in local shear rate.

Background Systemic hypotension may complicate the early postoperative period after lung transplantation. A release of proinflammatory cytokines secondary to lung ischemia/reperfusion injury could be involved in the pathogenesis of this early hemodynamic failure (EHF). Study objective To assess prospectively whether the occurrence of EHF is associated with a release of cytokines in the systemic circulation. Design Blood samples were taken daily during the first postoperative week in 26 patients who underwent a double or a single-lung transplantation. These patients were divided into three groups: 7 patients who experienced EHF and subsequently died (EHF group); 15 patients without EHF (control group); and 4 patients without EHF but with an identified sepsis (sepsis group). The serum levels of interleukin (IL)-1p, tumor necrosis factor-α (TNF-α), IL-6, and IL-8 were compared among the three groups. Results In the EHF group, the levels of each cytokine peaked at day 1 postoperatively. Cytokine levels at day 1 were significantly higher in the EHF group than in the control group (p Conclusion We conclude that EHF is associated with a massive release of proinflammatory cytokines that could play a determinant role in the pathogenesis of this complication.

Manufactured nanomaterials (MNMs) have the potential to improve everyday life as they can be utilised in numerous medical applications and day-to-day consumer products. However, this increased use has led to concerns about the potential environmental and human health impacts. The protein p53 is a key transcription factor implicated in cellular defence and reparative responses to various stress factors. Additionally, p53 has been implicated in cellular responses following exposure to some MNMs. Here, the role of the MNM mediated p53 induction and activation and its downstream effects following exposure to five well-characterised materials [namely two types of TiO2, two carbon black (CB), and one single-walled carbon nanotube (SWCNT)] were investigated. MNM internalisation, cellular viability, p53 protein induction and activation, oxidative stress, inflammation and apoptosis were measured in murine cell line and primary pulmonary macrophage models. It was observed that p53 was implicated in the biological responses to MNMs, with oxidative stress associated with p53 activation (only following exposure to the SWCNT). We demonstrate that p53 acted as an antioxidant and anti-inflammatory in macrophage responses to SWCNT and CB NMs. However, p53 was neither involved in MNM-induced cellular toxicity, nor in the apoptosis induced by these MNMs. Moreover, the physicochemical characteristics of MNMs seemed to influence their biological effects-SWCNT the materials with the largest surface area and a fibrous shape were the most cytotoxic in this study and were capable of the induction and activation of p53.

1. We investigated, by intravital microscopy in rats, the role of the subtypes of adenosine receptors A1 (A1/AR) and A2 (A2AR) in mediating adenosine-induced vasodilatation of second and third order arterioles of the diaphragm. 2. Adenosine, and the A1AR selective agonists R(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA) and N6-cyclo-pentyl-adenosine (CPA) induced a similar concentration-dependent dilatation of diaphragmatic arterioles. The non selective A2AR subtype agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl) ethyl]adenosine (DPMA) also dilated diaphragmatic arterioles but induced a significantly smaller dilatation than adenosine. By contrast the selective A(2a)AR subtype agonist 2-[p-(2-carboxyethyl)phenyl amino]-5'-N-ethyl carboxamido adenosine (CGS 21680) did not modify diaphragmatic arteriolar diameter. 3. The non selective adenosine receptor antagonist 1,3-dipropyl-8-p-sulphophenylxanthine (SPX, 100 microM) and the selective A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 50 nM) significantly attenuated adenosine-induced dilatation of diaphragmatic arterioles. By contrast, adenosine significantly dilated diaphragmatic arterioles in the presence of A2AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 10 microM). 4. The dilatation induced by adenosine was unchanged by the mast cell stabilizing agent sodium cromoglycate (cromolyn, 10 microM). 5. The nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine (L-NOARG, 300 microM) attenuated the dilatation induced by adenosine, and by the A1AR and A2AR agonists. 6. The ATP-dependent K+ channel blocker glibenclamide (3 microM) significantly attenuated diaphragmatic arteriolar dilatation induced by adenosine and by the A1AR agonists R-PIA and CPA. By contrast, glibenclamide did not significantly modify arteriolar dilatation induced by the A2AR agonist DPMA. 7. These findings suggest that adenosine-induced dilatation of diaphragmatic arterioles in the rat is predominantly mediated by the A1AR, via the release of NO and activation of the ATP-dependent K+ channels.

Engineered nanoparticles are increasingly used in medical applications and day-to-day consumer products, leading to concerns about the potential environmental and human health impacts. Silver nanoparticles are particularly prevalent because of their use as anti-bacterial agents in many commonly available products. Nanoparticles (NPs) are believed to accumulate, often preferentially, in the liver. This study therefore investigates the effect of a silver NP (20 nm) on the liver, and in particular, the role of Kupffer cells (KCs; resident liver macrophages) in the overall inflammatory response in the organ. Cytokine expression in the normal liver was measured in terms of IL2, IL4, TNF-α, IFN-γ and IL10 released from the organ with significant up-regulation of TNF-α and IL10 being observed. For livers in which the KC population was specifically targeted and destroyed this cytokine increase was significantly decreased in comparison to the normal tissue. IL10 was secreted at approximately three times the concentration of TNF-α in all the test cases. The high levels of IL10 released from the normal tissue in comparison to the KC depleted livers suggest that the cytokine may help to protect against a pro-inflammatory response to these Ag NPs. This may indicate a potentially important role for KCs in the anti-inflammatory response and suggests that tolerance to the Ag NPs is favoured over a fully activated immune response. In addition, albumin production was measured as an indicator of hepatic function. It was noted that the liver function was unaffected by the Ag NPs.

Objective— Carbon monoxide–releasing molecules (CORMs) represent a pharmacological alternative to CO gas inhalation. Here, we questioned whether CORM-3, a well-characterized water-soluble CORM, could prevent and reverse pulmonary hypertension (PH) in chronically hypoxic mice and in smooth muscle promoter 22 serotonin transporter mice overexpressing the serotonin transporter in smooth muscle cells (SMCs). Approach and Results— Treatment with CORM-3 (50 mg/kg per day once daily) for 3 weeks prevented PH, right ventricular hypertrophy, and distal pulmonary artery muscularization in mice exposed to chronic hypoxia and partially reversed PH in smooth muscle promoter 22 serotonin transporter mice by reducing Ki67 dividing pulmonary artery SMCs (PA-SMCs). In these models, CORM-3 markedly increased lung p21 mRNA and protein levels and p21-stained PA-SMCs. These effects contrasted with the transient pulmonary vasodilatation and rise in lung cGMP levels induced by a single injection of CORM-3 in mice exposed to acute hypoxia. Studies in cultured rat PA-SMCs revealed that the inhibitory effects of CORM-3 on cell growth were independent of cGMP formation but associated with increased p21 mRNA and protein levels. Protection against PH by CORM-3 required increased lung expression of p21, as indicated by the inability of CORM-3 to prevent chronic hypoxia-induced PH in p21-deficient mice and to alter the growth of PA-SMCs derived from p21-deficient mice. CORM-3–induced p21 overexpression was linked to p53 activation as assessed by the inability of CORM-3 to prevent PH and induce p21 expression in p53-deficient mice and in PA-SMCs derived from p53-deficient mice. Conclusions— CORM-3 inhibits pulmonary vascular remodeling via p21, which may represent a useful approach for treating PH.