Permanent culture of macrophages at physiological oxygen attenuates the antioxidant and immunomodulatory properties of dimethyl fumarate.

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.

Design and synthesis of new hybrid molecules that activate the transcription factor Nrf2 and simultaneously release carbon monoxide.

The transcription factor Nrf2 and its downstream target heme oxygenase-1 (HO-1) are essential protective systems against oxidative stress and inflammation. The products of HO-1 enzymatic activity, biliverdin and carbon monoxide (CO), actively contribute to this protection, suggesting that exploitation of these cellular systems may offer new therapeutic avenues in a variety of diseases. Starting from a CO-releasing compound and a chemical scaffold exhibiting electrophilic characteristics (esters of fumaric acid), we report the synthesis of hybrid molecules that simultaneously activate Nrf2 and liberate CO. These hybrid compounds, which we termed "HYCOs", release CO to myoglobin and activate the CO-sensitive fluorescent probe COP-1, while also potently inducing nuclear accumulation of Nrf2 and HO-1 expression and activity in different cell types. Thus, we provide here the first example of a new class of pharmacologically active molecules that target the HO-1 pathway by combining an Nrf2 activator coordinated to a CO-releasing group.

The expression and relaxant effect of bitter taste receptors in human bronchi.

BACKGROUND: Bitter-taste receptors (TAS2Rs) have recently been involved in the relaxation of mouse and guinea pig airways, and increased expression of TAS2Rs was shown in blood leucocytes from asthmatic children. We sought to identify and characterize the TAS2Rs expressed in isolated human bronchi and the subtypes involved in relaxation. METHODS: Human bronchi were isolated from resected lungs and TAS2R transcripts were assessed with RT-qPCR. Relaxation to TAS2R agonists was tested in organ bath in the presence or absence of pharmacological modulators of the signalling pathways involved in bronchial relaxation. RESULTS: We detected the expression of TAS2R transcripts in human bronchi. The non-selective agonists chloroquine, quinine, caffeine, strychnine and diphenidol produced a bronchial relaxation as effective and potent as theophylline but much less potent than formoterol and isoproterenol. Denatonium, saccharin and colchicine did not produce relaxation. Receptor expression analysis together with the use of selective agonists suggest a predominant role for TAS2R5, 10 and 14 in bitter taste agonist-induced relaxation. The mechanism of relaxation was independent of the signalling pathways modulated by conventional bronchodilators and may be partly explained by the inhibition of phosphatidylinositol-3-kinases. CONCLUSIONS: The TAS2Rs may constitute a new therapeutic target in chronic obstructive lung diseases such as asthma.

Heme oxygenase-1 induction attenuates senescence in chronic obstructive pulmonary disease lung fibroblasts by protecting against mitochondria dysfunction.

Chronic obstructive pulmonary disease (COPD) is associated with lung fibroblast senescence, a process characterized by an irreversible proliferation arrest associated with secretion of inflammatory mediators. ROS production, known to induce senescence, is increased in COPD fibroblasts and mitochondria dysfunction participates in this process. Among the battery of cellular responses against oxidative stress damage, heme oxygenase (HO)-1 plays a critical role in defending the lung against oxidative stress and inflammation. Therefore, we investigated whether pharmacological induction of HO-1 by chronic hemin treatment attenuates senescence and improves dysfunctional mitochondria in COPD fibroblasts. Fibroblasts from smoker controls (S-C) and COPD patients were isolated from lung biopsies. Fibroblasts were long-term cultured in the presence or absence of hemin, and/or ZnPP or QC-15 (HO-1 inhibitors). Lung fibroblasts from smokers and COPD patients displayed in long-term culture a senescent phenotype, characterized by a reduced replicative capacity, an increased senescence and inflammatory profile. These parameters were significantly higher in senescent COPD fibroblasts which also exhibited decreased mitochondrial activity (respiration, glycolysis, and ATP levels) which led to an increased production of ROS, and mitochondria biogenesis and impaired mitophagy process. Exposure to hemin increased the gene and protein expression level of HO-1 in fibroblasts and diminished ROS levels, senescence, the inflammatory profile and simultaneously rescued mitochondria dysfunction by restoring mitophagy in COPD cells. The effects of hemin were abolished by a cotreatment with ZnPP or QC-15. We conclude that HO-1 attenuates senescence in COPD fibroblasts by protecting, at least in part, against mitochondria dysfunction and restoring mitophagy.

Vascular and angiogenic activities of CORM-401, an oxidant-sensitive CO-releasing molecule.

Carbon monoxide (CO) is generated by heme oxygenase-1 (HO-1) and displays important signaling, anti-apoptotic and anti-inflammatory activities, indicating that pharmacological agents mimicking its action may have therapeutic benefit. This study examined the biochemical and pharmacological properties of CORM-401, a recently described CO-releasing molecule containing manganese as a metal center. We used in vitro approaches, ex-vivo rat aortic rings and the EA.hy926 endothelial cell line in culture to address how CORM-401 releases CO and whether the compound modulates vascular tone and pro-angiogenic activities, respectively. We found that CORM-401 released up to three CO/mole of compound depending on the concentration of the acceptor myoglobin. Oxidants such as H2O2, tert-butyl hydroperoxide or hypochlorous acid increased the CO liberated by CORM-401. CORM-401 also relaxed pre-contracted aortic rings and vasorelaxation was enhanced in combination with H2O2. Consistent with the release of multiple CO molecules, CORM-401-induced vasodilation was three times higher than that elicited by CORM-A1, which exhibits a similar half-life to CORM-401 but liberates only one CO/mole of compound. Furthermore, endothelial cells exposed to CORM-401 accumulated CO intracellularly, accelerated migration in vitro and increased VEGF and IL-8 levels. Studies using pharmacological inhibitors revealed HO-1 and p38 MAP kinase as two independent and parallel mechanisms involved in stimulating migration. We conclude that the ability of CORM-401 to release multiple CO, its sensitivity to oxidants which increase CO release, and its vascular and pro-angiogenic properties highlight new advances in the design of CO-releasing molecules that can be tailored for the treatment of inflammatory and oxidative stress-mediated pathologies.