Publications by authors named "Roberto Motterlini"

135 Publications

Sensitive quantification of carbon monoxide in vivo reveals a protective role of circulating hemoglobin in CO intoxication.

Commun Biol 2021 Mar 29;4(1):425. Epub 2021 Mar 29.

Department of Molecular Chemistry and Biochemistry, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto, Japan.

Carbon monoxide (CO) is a gaseous molecule known as the silent killer. It is widely believed that an increase in blood carboxyhemoglobin (CO-Hb) is the best biomarker to define CO intoxication, while the fact that CO accumulation in tissues is the most likely direct cause of mortality is less investigated. There is no reliable method other than gas chromatography to accurately determine CO content in tissues. Here we report the properties and usage of hemoCD1, a synthetic supramolecular compound composed of an iron(II)porphyrin and a cyclodextrin dimer, as an accessible reagent for a simple colorimetric assay to quantify CO in biological samples. The assay was validated in various organ tissues collected from rats under normal conditions and after exposure to CO. The kinetic profile of CO in blood and tissues after CO treatment suggested that CO accumulation in tissues is prevented by circulating Hb, revealing a protective role of Hb in CO intoxication. Furthermore, hemoCD1 was used in vivo as a CO removal agent, showing that it acts as an effective adjuvant to O ventilation to eliminate residual CO accumulated in organs, including the brain. These findings open new therapeutic perspectives to counteract the toxicity associated with CO poisoning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-021-01880-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007703PMC
March 2021

Increased Sirt1 secreted from visceral white adipose tissue is associated with improved glucose tolerance in obese Nrf2-deficient mice.

Redox Biol 2021 01 24;38:101805. Epub 2020 Nov 24.

University Paris-Est Créteil, INSERM, IMRB, F-94010, Créteil, France. Electronic address:

Obesity is associated with metabolic dysregulation characterized by insulin resistance and glucose intolerance. Nuclear factor E2-related factor (Nrf2) is a critical regulator of the stress response and Nrf2-deficient mice (Nrf2) are protected against high fat diet (HFD)-induced metabolic derangement. We searched for factors that could underline this favorable phenotype and found that Nrf2 mice exhibit higher circulating levels of sirtuin 1 (Sirt1), a key player in cellular homeostasis and energy metabolism, compared to wild-type mice. Increased Sirt1 levels in Nrf2 mice were found not only in animals under standard diet but also following HFD. Interestingly, we report here that the visceral adipose tissue (eWAT) is the sole source of increased Sirt1 protein in plasma. eWAT and other fat depots displayed enhanced adipocytes lipolysis, increased fatty acid oxidation and glycolysis, suggesting autocrine and endocrine actions of Sirt1 in this model. We further demonstrate that removal of eWAT completely abolishes the increase in circulating Sirt1 and that this procedure suppresses the beneficial effect of Nrf2 deficiency on glucose tolerance, but not insulin sensitivity, following a HFD regime. Thus, in contrast to many other stressful conditions where Nrf2 deficiency exacerbates damage, our study indicates that up-regulation of Sirt1 levels specifically in the visceral adipose tissue of Nrf2 mice is a key adaptive mechanism that mitigates glucose intolerance induced by nutritional stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2020.101805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721645PMC
January 2021

LIPE-related lipodystrophic syndrome: clinical features and disease modeling using adipose stem cells.

Eur J Endocrinol 2021 Jan;184(1):155-168

Sorbonne Université, Inserm UMRS_938, Centre de Recherche Saint Antoine, Paris, France.

Objective: The term Multiple Symmetric Lipomatosis (MSL) describes a heterogeneous group of rare monogenic disorders and multifactorial conditions, characterized by upper-body adipose masses. Biallelic variants in LIPE encoding hormone-sensitive lipase (HSL), a key lipolytic enzyme, were implicated in three families worldwide. We aimed to further delineate LIPE-related clinical features and pathophysiological determinants.

Methods: A gene panel was used to identify pathogenic variants. The disease features were reviewed at the French lipodystrophy reference center. The immunohistological, ultrastructural, and protein expression characteristics of lipomatous tissue were determined in surgical samples from one patient. The functional impact of variants was investigated by developing a model of adipose stem cells (ASCs) isolated from lipomatous tissue.

Results: We identified new biallelic LIPE null variants in three unrelated patients referred for MSL and/or partial lipodystrophy. The hallmarks of the disease, appearing in adulthood, included lower-limb lipoatrophy, upper-body and abdominal pseudo-lipomatous masses, diabetes and/or insulin resistance, hypertriglyceridemia, liver steatosis, high blood pressure, and neuromuscular manifestations. Ophthalmological investigations revealed numerous auto-fluorescent drusen-like retinal deposits in all patients. Lipomatous tissue and patient ASCs showed loss of HSL and decreased expression of adipogenic and mature adipocyte markers. LIPE-mutated ASCs displayed impaired adipocyte differentiation, decreased insulin response, defective lipolysis, and mitochondrial dysfunction.

Conslusions: Biallelic LIPE null variants result in a multisystemic disease requiring multidisciplinary care. Loss of HSL expression impairs adipocyte differentiation, consistent with the lipodystrophy/MSL phenotype and associated metabolic complications. Detailed ophthalmological examination could reveal retinal damage, further pointing to the nervous tissue as an important disease target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/EJE-20-1013DOI Listing
January 2021

Therapeutic effects of CO-releaser/Nrf2 activator hybrids (HYCOs) in the treatment of skin wound, psoriasis and multiple sclerosis.

Redox Biol 2020 07 1;34:101521. Epub 2020 Apr 1.

Inserm U955, Equipe 12, Créteil, 94010, France; University Paris Est, Faculty of Medicine, Créteil, 94010, France. Electronic address:

Carbon monoxide (CO) produced by heme oxygenase-1 (HO-1) or delivered by CO-releasing molecules (CO-RMs) exerts anti-inflammatory action, a feature also exhibited by the nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of the stress response. We have recently developed new hybrid molecules (HYCOs) consisting of CO-RMs conjugated to fumaric esters known to activate Nrf2/HO-1. Here we evaluated the biological activities of manganese (Mn) and ruthenium (Ru)-based HYCOs in human monocytes and keratinocytes in vitro as well as in vivo models of inflammation. The effects of HYCOs were compared to: a) dimethyl fumarate (DMF), a known fumaric ester used in the clinic; b) a CO-RM alone; or c) the combination of the two compounds. Mn-HYCOs donated CO and up-regulated Nrf2/HO-1 in vitro more efficiently than Ru-HYCOs. However, irrespective of the metal, a strong reduction in anti-inflammatory markers in monocytes stimulated by LPS was observed with specific HYCOs. This effect was not observed with DMF, CO-RM alone or the combination of the two, indicating the enhanced potency of HYCOs compared to the separate entities. Selected HYCOs given orally to mice accelerated skin wound closure, reduced psoriasis-mediated inflammation and disease symptoms equalling or surpassing the effect of DMF, and ameliorated motor dysfunction in a mouse model of multiple sclerosis. Thus, HYCOs have potent anti-inflammatory activities that are recapitulated in disease models in which inflammation is a prominent component. Prolonged daily administration of HYCOs (up to 40 days) is well tolerated in animals. Our results clearly confirm that HYCOs possess a dual mode of action highlighting the notion that simultaneous Nrf2 targeting and CO delivery could be a clinically relevant application to combat inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2020.101521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184182PMC
July 2020

The CO-releasing molecule CORM-3 protects adult cardiomyocytes against hypoxia-reoxygenation by modulating pH restoration.

Eur J Pharmacol 2019 Nov 3;862:172636. Epub 2019 Sep 3.

U955-IMRB, Equipe 03, Inserm, UPEC, Ecole Nationale Vétérinaire d'Alfort, Créteil, France.

Several studies have reported that CORM-3, a water-soluble carbon monoxide releasing molecule, elicits cardioprotection against myocardial infarction but the mechanism remains to be investigated. Numerous reports indicate that inhibition of pH regulators, the Na/H exchanger (NHE) and Na/HCO symporter (NBC), protect cardiomyocytes from hypoxia/reoxygenation injury by delaying the intracellular pH (pHi) recovery at reperfusion. Our goal was to explore whether CORM-3-mediated cytoprotection involves the modulation of pH regulation. When added at reoxygenation, CORM-3 (50 μM) reduced the mortality of cardiomyocytes exposed to 3 h of hypoxia and 2 h of reoxygenation in HCO-buffered solution. This effect was lost when using inactive iCORM-3, which is depleted of CO and used as control, thus implicating CO as the mediator of this cardioprotection. Interestingly, the cardioprotective effect of CORM-3 was abolished by switching to a bicarbonate-free medium. This effect of CORM-3 was also inhibited by 5-hydroxydecanoate, a mitochondrial ATP-dependent K (mK) channel inhibitor (500 μM) or PD098059, a MEK1/2 inhibitor (10 μM). In additional experiments and in the absence of hypoxia-reoxygenation, intracellular pH was monitored in cardiomyocytes exposed to cariporide to block NHE activity. CORM-3 inhibited alkalinisation and this effect was blocked by PD098059 and 5-HD. In conclusion, CORM-3 protects the cardiomyocyte against hypoxia-reoxygenation injury by inhibiting a bicarbonate transporter at reoxygenation, probably the Na/HCO symporter. This cardioprotective effect of CORM-3 requires the activation of mK channels and the activation of MEK1/2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejphar.2019.172636DOI Listing
November 2019

Design and Biological Evaluation of Manganese- and Ruthenium-Based Hybrid CO-RMs (HYCOs).

ChemMedChem 2019 09 23;14(18):1684-1691. Epub 2019 Aug 23.

Université Paris Est, ICMPE (UMR 7182), CNRS, UPEC, 94320, Thiais, France.

Interest in the therapeutic effects of carbon monoxide (CO), a product of heme degradation catalyzed by the enzyme heme oxygenase-1 (HO-1), has led to the development of CO-releasing molecules (CO-RMs) for the controlled delivery of this gas in vivo. We recently proposed conjugating a cobalt-based CO-RM with various activators of nuclear factor erythroid 2-related factor 2 (Nrf2), the transcription factor that regulates HO-1 expression, in order to exploit the beneficial effects of exogenous and endogenous CO. In this study, we describe the preparation of hybrid molecules (termed HYCOs) conjugating a fumaric acid derivative as an Nrf2 activator to a Mn- or a Ru-based CO-RM known to be pharmacologically active. With the exception of an acyl-manganese complex, these hybrids were obtained by associating the two bioactive entities by means of a linker of variable structure. X-ray diffraction analyses and preliminary biological investigations are also presented.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.201900426DOI Listing
September 2019

Heme oxygenase-1-Dependent anti-inflammatory effects of atorvastatin in zymosan-injected subcutaneous air pouch in mice.

PLoS One 2019 9;14(5):e0216405. Epub 2019 May 9.

Department of Biochemistry and Molecular Genetics, American University of Beirut, Beirut, Lebanon.

Statins exert pleiotropic and beneficial anti-inflammatory and antioxidant effects. We have previously reported that macrophages treated with statins increased the expression of heme oxygenase-1 (HO-1), an inducible anti-inflammatory and cytoprotective stress protein, responsible for the degradation of heme. In the present study, we investigated the effects of atorvastatin on inflammation in mice and analyzed its mechanism of action in vivo. Air pouches were established in 8 week-old female C57BL/6J mice. Atorvastatin (5 mg/kg, i.p.) and/or tin protoporphyrin IX (SnPPIX), a heme oxygenase inhibitor (12 mg/kg, i.p.), were administered for 10 days. Zymosan, a cell wall component of Saccharomyces cerevisiae, was injected in the air pouch to trigger inflammation. Cell number and levels of inflammatory markers were determined in exudates collected from the pouch 24 hours post zymosan injection by flow cytometry, ELISA and quantitative PCR. Analysis of the mice treated with atorvastatin alone displayed increased expression of HO-1, arginase-1, C-type lectin domain containing 7A, and mannose receptor C-type 1 in the cells of the exudate of the air pouch. Flow cytometry analysis revealed an increase in monocyte/macrophage cells expressing HO-1 and in leukocytes expressing MRC-1 in response to atorvastatin. Mice treated with atorvastatin showed a significant reduction in cell influx in response to zymosan, and in the expression of proinflammatory cytokines and chemokines such as interleukin-1α, monocyte chemoattractant protein-1 and prostaglandin E2. Co-treatment of mice with atorvastatin and tin protoporphyrin IX (SnPPIX), an inhibitor of heme oxygenase, reversed the inhibitory effect of statin on cell influx and proinflammatory markers, suggesting a protective role of HO-1. Flow cytometry analysis of air pouch cell contents revealed prevalence of neutrophils and to a lesser extent of monocytes/macrophages with no significant effect of atorvastatin treatment on the modification of their relative proportion. These findings identify HO-1 as a target for the therapeutic actions of atorvastatin and highlight its potential role as an in vivo anti-inflammatory agent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216405PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508873PMC
January 2020

TLR4 activation alters labile heme levels to regulate BACH1 and heme oxygenase-1 expression in macrophages.

Free Radic Biol Med 2019 06 24;137:131-142. Epub 2019 Apr 24.

Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany. Electronic address:

Heme oxygenase (HO)-1, a stress-inducible enzyme that converts heme into carbon monoxide (CO), iron and biliverdin, exerts important anti-inflammatory effects in activated macrophages. HO-1 expression is mainly governed by a mutual interplay between the transcriptional factor NRF2 and the nuclear repressor BTB and CNC homology 1 (BACH1), a heme sensor protein. In the current study we hypothesized that alterations in the levels of intracellular labile heme in macrophages stimulated by lipopolysaccharide (LPS), a prototypical pro-inflammatory Toll-like receptor (TLR)4 agonist, are responsible for BACH1-dependent HO-1 expression. To this end, labile heme was determined in both mouse bone marrow-derived macrophages (mBMDMs) and human monocyte-derived macrophages (hMDMs) using an apo-horseradish peroxidase-based assay. We found that LPS raised the levels of labile heme, depressed BACH1 protein and up-regulated HO-1 in mBMDMs. In contrast, in hMDMs LPS decreased labile heme levels while increasing BACH1 expression and down-regulating HO-1. These effects were abolished by the TLR4 antagonist TAK-242, suggesting that TLR4 activation triggers the signaling cascade leading to changes in the labile heme pool. Studies using mBMDMs from BACH1-/- and NRF2-/- mice revealed that regulation of HO-1 and levels of labile heme after LPS stimulation are strictly dependent on BACH1, but not NRF2. A strong interplay between BACH1-mediated HO-1 expression and intracellular levels of labile heme was also confirmed in hMDMs with siRNA knockdown studies and following inhibition of de novo heme synthesis with succinylacetone. Finally, CORM-401, a compound that liberates CO, counteracted LPS-dependent down-regulation of HO-1 and restored levels of labile heme in hMDMs. In conclusion, alterations of labile heme levels in macrophages following TLR4 stimulation play a crucial role in BACH1-mediated regulation of HO-1 expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2019.04.024DOI Listing
June 2019

Human and murine macrophages exhibit differential metabolic responses to lipopolysaccharide - A divergent role for glycolysis.

Redox Biol 2019 04 20;22:101147. Epub 2019 Feb 20.

Institute of Transfusion Medicine, Hannover Medical School, Hannover, 30625, Germany. Electronic address:

Macrophages adopt different phenotypes in response to microenvironmental changes, which can be principally classified into inflammatory and anti-inflammatory states. Inflammatory activation of macrophages has been linked with metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis. In contrast to mouse macrophages, little information is available on the link between metabolism and inflammation in human macrophages. In the current report it is demonstrated that lipopolysaccharide (LPS)-activated human peripheral blood monocyte-derived macrophages (hMDMs) fail to undergo metabolic reprogramming towards glycolysis, but rely on oxidative phosphorylation for the generation of ATP. By contrast, activation by LPS led to an increased extracellular acidification rate (glycolysis) and decreased oxygen consumption rate (oxidative phosphorylation) in mouse bone marrow-derived macrophages (mBMDMs). Mitochondrial bioenergetics after LPS stimulation in human macrophages was unchanged, but was markedly impaired in mouse macrophages. Furthermore, treatment with 2-deoxyglucose, an inhibitor of glycolysis, led to cell death in mouse, but not in human macrophages. Finally, glycolysis appeared to be critical for LPS-mediated induction of the anti-inflammatory cytokine interleukin-10 in both human and mouse macrophages. In summary, these findings indicate that LPS-induced immunometabolism in human macrophages is different to that observed in mouse macrophages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2019.101147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396203PMC
April 2019

MR (Mineralocorticoid Receptor) Induces Adipose Tissue Senescence and Mitochondrial Dysfunction Leading to Vascular Dysfunction in Obesity.

Hypertension 2019 02;73(2):458-468

From the Department of Physiology, INSERM UMRS 1138 Team 1, Centre de Recherche des Cordeliers, Sorbonne University, Paris, France (C.L., R.P.-R., N.B., F.J., A.N.D.C.).

Adipose tissue (AT) senescence and mitochondrial dysfunction are associated with obesity. Studies in obese patients and animals demonstrate that the MR (mineralocorticoid receptor) contributes to obesity-associated cardiovascular complications through its specific role in AT. However, underlying mechanisms remain unclear. This study aims to elucidate whether MR regulates mitochondrial function in obesity, resulting in AT premature aging and vascular dysfunction. Obese (db/db) and lean (db/+) mice were treated with an MR antagonist or a specific mitochondria-targeted antioxidant. Mitochondrial and vascular functions were determined by respirometry and myography, respectively. Molecular mechanisms were probed by Western immunoblotting and real-time polymerase chain reaction in visceral AT and arteries and focused on senescence markers and redox-sensitive pathways. db/db mice displayed AT senescence with activation of the p53-p21 pathway and decreased SIRT (sirtuin) levels, as well as mitochondrial dysfunction. Furthermore, the beneficial anticontractile effects of perivascular AT were lost in db/db via ROCK (Rho kinase) activation. MR blockade prevented these effects. Thus, MR activation in obesity induces mitochondrial dysfunction and AT senescence and dysfunction, which consequently increases vascular contractility. In conclusion, our study identifies novel mechanistic insights involving MR, adipose mitochondria, and vascular function that may be of importance to develop new therapeutic strategies to limit obesity-associated cardiovascular complications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/HYPERTENSIONAHA.118.11873DOI Listing
February 2019

Carbon monoxide-induced metabolic switch in adipocytes improves insulin resistance in obese mice.

JCI Insight 2018 11 15;3(22). Epub 2018 Nov 15.

Inserm U955, Team 12, Créteil, France.

Obesity is characterized by accumulation of adipose tissue and is one the most important risk factors in the development of insulin resistance. Carbon monoxide-releasing (CO-releasing) molecules (CO-RMs) have been reported to improve the metabolic profile of obese mice, but the underlying mechanism remains poorly defined. Here, we show that oral administration of CORM-401 to obese mice fed a high-fat diet (HFD) resulted in a significant reduction in body weight gain, accompanied by a marked improvement in glucose homeostasis. We further unmasked an action we believe to be novel, by which CO accumulates in visceral adipose tissue and uncouples mitochondrial respiration in adipocytes, ultimately leading to a concomitant switch toward glycolysis. This was accompanied by enhanced systemic and adipose tissue insulin sensitivity, as indicated by a lower blood glucose and increased Akt phosphorylation. Our findings indicate that the transient uncoupling activity of CO elicited by repetitive administration of CORM-401 is associated with lower weight gain and increased insulin sensitivity during HFD. Thus, prototypic compounds that release CO could be investigated for developing promising insulin-sensitizing agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.123485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302946PMC
November 2018

HYCO-3, a dual CO-releaser/Nrf2 activator, reduces tissue inflammation in mice challenged with lipopolysaccharide.

Redox Biol 2019 01 26;20:334-348. Epub 2018 Oct 26.

Inserm U955, Equipe 12, Créteil 94000, France; University Paris-Est, Faculty of Medicine, Créteil 94000, France. Electronic address:

Oxidative stress and inflammation are predominant features of several chronic diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a major arbiter in counteracting these insults via up-regulation of several defensive proteins, including heme oxygenase-1 (HO-1). HO-1-derived carbon monoxide (CO) exhibits anti-inflammatory actions and can be delivered to tissues by CO-releasing agents. In this study we assessed the pharmacological and anti-inflammatory properties of HYCO-3, a dual activity compound obtained by conjugating analogues of the CO-releasing molecule CORM-401 and dimethyl fumarate (DMF), an immunomodulatory drug known to activate Nrf2. HYCO-3 induced Nrf2-dependent genes and delivered CO to cells in vitro and tissues in vivo, confirming that the two expected pharmacological properties of this agent are achieved. In mice challenged with lipopolysaccharide, orally administered HYCO-3 reduced the mRNA levels of pro-inflammatory markers (TNF-α, IL-1β and IL-6) while increasing the expression of the anti-inflammatory genes ARG1 and IL-10 in brain, liver, lung and heart. In contrast, DMF or CORM-401 alone or their combination decreased the expression of pro-inflammatory genes but had limited influence on anti-inflammatory markers. Furthermore, HYCO-3 diminished TNF-α and IL-1β in brain and liver but not in lung and heart of Nrf2 mice, indicating that the CO-releasing part of this hybrid contributes to reduction of pro-inflammation and that this effect is organ-specific. These data demonstrate that the dual activity of HYCO-3 results in enhanced efficacy compared to the parent compounds indicating the potential exploitation of hybrid compounds in the development of effective anti-inflammatory therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2018.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223233PMC
January 2019

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

Aging Cell 2018 Dec 19;17(6):e12837. Epub 2018 Oct 19.

Inserm U955, Equipe 04, Créteil, France.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/acel.12837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260925PMC
December 2018

Modulation of cellular bioenergetics by CO-releasing molecules and NO-donors inhibits the interaction of cancer cells with human lung microvascular endothelial cells.

Pharmacol Res 2018 10 6;136:160-171. Epub 2018 Sep 6.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland; Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland. Electronic address:

Interactions between cancer cells and the endothelium play a crucial role during metastasis. Here we examined the effects of a carbon monoxide-releasing molecule (CORM-401) and a nitric oxide donor (PAPA NONOate) given alone or in combination on breast cancer cell adhesion and transmigration across the lung microvascular endothelium. We further explored whether the effects of CO and NO on cancer-endothelial cells interactions are linked with changes in cellular bioenergetics in breast cancer or endothelial cells. We found that CORM-401 and PAPA NONOate alone or in combination markedly decreased transmigration of breast cancer cells across human lung microvascular endothelial cells (hLMVEC), while cancer cell adhesion to the endothelium was diminished only by a combination of the two compounds. In hLMVECs, CORM-401 decreased glycolysis and stimulated mitochondrial respiration, while in breast cancer cells CORM-401 decreased both glycolysis and mitochondrial respiration. In contrast, PAPA NONOate decreased mitochondrial respiration and slightly stimulated glycolysis in both cell lines. When both donors were given together, mitochondrial respiration and glycolysis were both profoundly inhibited, and cancer-endothelial cells interactions were additively suppressed. Intercellular adhesion molecule-1 (ICAM-1), involved in breast cancer cell adhesion to hLMVECs, was downregulated by CORM-401 and PAPA NONOate, when applied alone, while a combination of both compounds did not cause any enhancement of ICAM-1 downregulation. In conclusion, our findings demonstrate that CO and NO differently affect cellular bioenergetics of cancer and endothelial cells and suggest that this phenomenon may contribute to additive anti-adhesive and anti-transmigratory effects of CO and NO. Pharmacological attenuation of metabolism represents a novel, effective way to prevent cancer cell interactions with the endothelium, that is an energy-demanding process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phrs.2018.09.005DOI Listing
October 2018

CORM-401 induces calcium signalling, NO increase and activation of pentose phosphate pathway in endothelial cells.

FEBS J 2018 04 8;285(7):1346-1358. Epub 2018 Mar 8.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland.

Carbon monoxide-releasing molecules (CO-RMs) induce nitric oxide (NO) release (which requires NADPH), and Ca -dependent signalling; however, their contribution in mediating endothelial responses to CO-RMs is not clear. Here, we studied the effects of CO liberated from CORM-401 on NO production, calcium signalling and pentose phosphate pathway (PPP) activity in human endothelial cell line (EA.hy926). CORM-401 induced NO production and two types of calcium signalling: a peak-like calcium signal and a gradual increase in cytosolic calcium. CORM-401-induced peak-like calcium signal, originating from endoplasmic reticulum, was reduced by thapsigargin, a SERCA inhibitor, and by dantrolene, a ryanodine receptors (RyR) inhibitor. In contrast, the phospholipase C inhibitor U73122 did not significantly affect peak-like calcium signalling, but a slow and progressive CORM-401-induced increase in cytosolic calcium was dependent on store-operated calcium entrance. CORM-401 augmented coupling of endoplasmic reticulum and plasmalemmal store-operated calcium channels. Interestingly, in the presence of NO synthase inhibitor (l-NAME) CORM-401-induced increases in NO and cytosolic calcium were both abrogated. CORM-401-induced calcium signalling was also inhibited by superoxide dismutase (poly(ethylene glycol)-SOD). Furthermore, CORM-401 accelerated PPP, increased NADPH concentration and decreased the ratio of reduced to oxidized glutathione (GSH/GSSG). Importantly, CORM-401-induced NO increase was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN), but neither by dantrolene nor by an inhibitor of large-conductance calcium-regulated potassium ion channel (paxilline). The results identify the primary role of CO-induced NO increase in the regulation of endothelial calcium signalling, that may have important consequences in controlling endothelial function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.14411DOI Listing
April 2018

Mesenchymal stem cells sense mitochondria released from damaged cells as danger signals to activate their rescue properties.

Cell Death Differ 2017 07 19;24(7):1224-1238. Epub 2017 May 19.

Université Paris-Est, UMR-S955, UPEC, Créteil, Paris, France.

Mesenchymal stem cells (MSCs) protect tissues against cell death induced by ischemia/reperfusion insults. This therapeutic effect seems to be controlled by physiological cues released by the local microenvironment following injury. Recent lines of evidence indicate that MSC can communicate with their microenvironment through bidirectional exchanges of mitochondria. In particular, in vitro and in vivo studies report that MSCs rescue injured cells through delivery of their own mitochondria. However, the role of mitochondria conveyed from somatic cells to MSC remains unknown. By using a co-culture system consisting of MSC and distressed somatic cells such as cardiomyocytes or endothelial cells, we showed that mitochondria from suffering cells acted as danger-signaling organelles that triggered the anti-apoptotic function of MSC. We demonstrated that foreign somatic-derived mitochondria were engulfed and degraded by MSC, leading to induction of the cytoprotective enzyme heme oxygenase-1 (HO-1) and stimulation of mitochondrial biogenesis. As a result, the capacity of MSC to donate their mitochondria to injured cells to combat oxidative stress injury was enhanced. We found that similar mechanisms - activation of autophagy, HO-1 and mitochondrial biogenesis - occurred after exposure of MSC to exogenous mitochondria isolated from somatic cells, strengthening the idea that somatic mitochondria alert MSC of a danger situation and subsequently promote an adaptive reparative response. In addition, the cascade of events triggered by the transfer of somatic mitochondria into MSC was recapitulated in a model of myocardial infarction in vivo. Specifically, MSC engrafted into infarcted hearts of mice reduced damage, upregulated HO-1 and increased mitochondrial biogenesis, while inhibition of mitophagy or HO-1 failed to protect against cardiac apoptosis. In conclusion, our study reveals a new facet about the role of mitochondria released from dying cells as a key environmental cue that controls the cytoprotective function of MSC and opens novel avenues to improve the effectiveness of MSC-based therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/cdd.2017.51DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520168PMC
July 2017

Detection and Removal of Endogenous Carbon Monoxide by Selective and Cell-Permeable Hemoprotein Model Complexes.

J Am Chem Soc 2017 04 17;139(16):5984-5991. Epub 2017 Apr 17.

Department of Molecular Chemistry and Biochemistry, Faculty of Science and Engineering, Doshisha University , Kyotanabe, Kyoto 610-0321, Japan.

Carbon monoxide (CO) is produced in mammalian cells during heme metabolism and serves as an important signaling messenger. Here we report the bioactive properties of selective CO scavengers, hemoCD1 and its derivative R8-hemoCD1, which have the ability to detect and remove endogenous CO in cells. HemoCD1 is a supramolecular hemoprotein-model complex composed of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(II) and a per-O-methylated β-cyclodextrin dimer having an pyridine linker. We demonstrate that hemoCD1 can be used effectively to quantify endogenous CO in cell lysates by a simple spectrophotometric method. The hemoCD1 assay detected ca. 260 pmol of CO in 10 hepatocytes, which was well-correlated with the amount of intracellular bilirubin, the final breakdown product of heme metabolism. We then covalently attached an octaarginine peptide to a maleimide-appended hemoCD1 to synthesize R8-hemoCD1, a cell-permeable CO scavenger. Indeed, R8-hemoCD1 was taken up by intact cells and captured intracellular CO with high efficiency. Moreover, we revealed that removal of endogenous CO by R8-hemoCD1 in cultured macrophages led to a significant increase (ca. 2.5-fold) in reactive oxygen species production and exacerbation of inflammation after challenge with lipopolysaccharide. Thus, R8-hemoCD1 represents a powerful expedient for exploring specific and still unidentified biological functions of CO in cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.7b02229DOI Listing
April 2017

Differential Effects of CORM-2 and CORM-401 in Murine Intestinal Epithelial MODE-K Cells under Oxidative Stress.

Front Pharmacol 2017 8;8:31. Epub 2017 Feb 8.

Heymans Institute of Pharmacology, Faculty of Medicine and Health Sciences, Ghent University Ghent, Belgium.

Carbon monoxide (CO)-releasing molecules (CO-RMs) are intensively studied to provide cytoprotective and anti-inflammatory effects of CO in inflammatory conditions including intestinal inflammation. The water-soluble CORM-A1 reduced apoptosis and NADPH oxidase (NOX)-derived reactive oxygen species (ROS) induced by tumor necrosis factor (TNF)-α/cycloheximide (CHX) in mouse MODE-K intestinal epithelial cells (IECs), without influencing TNF-α/CHX-induced mitochondrial superoxide anion ([Formula: see text]). The aim of the present study in the same model was to comparatively investigate the influence of lipid-soluble CORM-2 and water-soluble CORM-401, shown to release more CO under oxidative conditions. CORM-2 abolished TNF-α/CHX-induced total cellular ROS whereas CORM-401 partially reduced it, both partially reducing TNF-α/CHX-induced cell death. Only CORM-2 increased mitochondrial [Formula: see text] production after 2 h of incubation. CORM-2 reduced TNF-α/CHX-, rotenone- and antimycin-A-induced mitochondrial [Formula: see text] production; CORM-401 only reduced the effect of antimycin-A. Co-treatment with CORM-401 during 1 h exposure to HO reduced HO (7.5 mM)-induced ROS production and cell death, whereas CORM-2 did not. The study illustrates the importance of the chemical characteristics of different CO-RMs. The lipid solubility of CORM-2 might contribute to its interference with TNF-α/CHX-induced mitochondrial ROS signaling, at least in mouse IECs. CORM-401 is more effective than other CO-RMs under HO-induced oxidative stress conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphar.2017.00031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296622PMC
February 2017

Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus.

Free Radic Biol Med 2017 03 18;104:311-323. Epub 2017 Jan 18.

Inserm U955, Equipe 12, Créteil 94000, France; University Paris-Est, Faculty of Medicine, Créteil 94000, France. Electronic address:

Microglia fulfill important immunological functions in the brain by responding to pathological stresses and modulating their activities according to pro- or anti-inflammatory stimuli. Recent evidence indicates that changes in metabolism accompany the switch in microglia activation state, favoring glycolysis over oxidative phosphorylation when cells exhibit a pro-inflammatory phenotype. Carbon monoxide (CO), a byproduct of heme breakdown by heme oxygenase, exerts anti-inflammatory action and affects mitochondrial function in cells and tissues. In the present study, we analyzed the metabolic profile of BV2 and primary mouse microglia exposed to the CO-releasing molecules CORM-401 and CORM-A1 and investigated whether CO affects the metabolic adaptation of cells to the inflammatory stimulus lipopolysaccharide (LPS). Microglia respiration and glycolysis were measured using an Extracellular Flux Analyzer to provide a real-time bioenergetic assessment, and biochemical parameters were evaluated to define the metabolic status of the cells under normal or inflammatory conditions. We show that CO prevents LPS-induced depression of microglia respiration and reduction in ATP levels while altering the early expression of inflammatory markers, suggesting the metabolic changes induced by CO are associated with control of inflammation. CO alone affects microglia respiration depending on the concentration, as low levels increase oxygen consumption while higher amounts inhibit respiration. Increased oxygen consumption was attributed to an uncoupling activity observed in cells, at the molecular level (respiratory complex activities) and during challenge with LPS. Thus, application of CO is a potential countermeasure to reverse the metabolic changes that occur during microglia inflammation and in turn modulate their inflammatory profile.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2017.01.022DOI Listing
March 2017

Biological signaling by carbon monoxide and carbon monoxide-releasing molecules.

Am J Physiol Cell Physiol 2017 Mar 11;312(3):C302-C313. Epub 2017 Jan 11.

Inserm U955, Team 12, Créteil, France; and Faculty of Medicine, Université Paris Est, Créteil, France

Carbon monoxide (CO) is continuously produced in mammalian cells during the degradation of heme. It is a stable gaseous molecule that reacts selectively with transition metals in a specific redox state, and these characteristics restrict the interaction of CO with defined biological targets that transduce its signaling activity. Because of the high affinity of CO for ferrous heme, these targets can be grouped into heme-containing proteins, representing a large variety of sensors and enzymes with a series of diverse function in the cell and the organism. Despite this notion, progress in identifying which of these targets are selective for CO has been slow and even the significance of elevated carbonmonoxy hemoglobin, a classical marker used to diagnose CO poisoning, is not well understood. This is also due to the lack of technologies capable of assessing in a comprehensive fashion the distribution and local levels of CO between the blood circulation, the tissue, and the mitochondria, one of the cellular compartments where CO exerts its signaling or detrimental effects. Nevertheless, the use of CO gas and CO-releasing molecules as pharmacological approaches in models of disease has provided new important information about the signaling properties of CO. In this review we will analyze the most salient effects of CO in biology and discuss how the binding of CO with key ferrous hemoproteins serves as a posttranslational modification that regulates important processes as diverse as aerobic metabolism, oxidative stress, and mitochondrial bioenergetics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpcell.00360.2016DOI Listing
March 2017

Carbon monoxide shifts energetic metabolism from glycolysis to oxidative phosphorylation in endothelial cells.

FEBS Lett 2016 Oct 11;590(20):3469-3480. Epub 2016 Oct 11.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland.

Carbon monoxide (CO) modulates mitochondrial respiration, but the mechanisms involved are not completely understood. The aim of the present study was to investigate the acute effects of CO on bioenergetics and metabolism in intact EA.hy926 endothelial cells using live cell imaging techniques. Our findings indicate that CORM-401, a compound that liberates CO, reduces ATP production from glycolysis, and induces a mild mitochondrial depolarization. In addition, CO from CORM-401 increases mitochondrial calcium and activates complexes I and II. The subsequent increase in mitochondrial respiration leads to ATP production through oxidative phosphorylation. Thus, our results show that nonactivated endothelial cells rely primarily on glycolysis, but in the presence of CO, mitochondrial Ca increases and activates respiration that shifts the metabolism of endothelial cells from glycolysis- to oxidative phosphorylation-dependent ATP production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12434DOI Listing
October 2016

Unusual Dynamics of Ligand Binding to the Heme Domain of the Bacterial CO Sensor Protein RcoM-2.

J Phys Chem B 2016 Oct 6;120(41):10686-10694. Epub 2016 Oct 6.

LOB, Ecole Polytechnique, CNRS, INSERM, Université Paris-Saclay , 91128 Palaiseau Cedex, France.

The aerobic Gram-negative bacterium Burkholderia xenovorans expresses two highly homologous carbon monoxide (CO)-responsive transcriptional regulators, RcoM-1 and RcoM-2, which display extraordinarily high CO affinities, even under oxygenic conditions. To gain insight into the origin and perspectives of this feature, we characterized the ligand-binding properties of the N-terminal, heme-binding Per/Arnt/Sim sensor domain of RcoM-2 by time-resolved spectroscopy. We show that upon photodissociation of the heme-ligand bond, CO geminately rebinds to the heme with picosecond time constants and more than 99% rebinding yield, an unprecedented property of native heme proteins. Remarkably, the rebinding kinetics speeds up when the protein motions are slowed by cooling or solvent viscosity. This indicates that the origin of the observed efficient rebinding is a protein-imposed CO configuration in the heme pocket that is highly favorable for binding, a feature strongly in contrast to that of hemoglobins. The binding of CO to the ferrous heme from the solvent requires dissociation of the methionine axial heme ligand. From the kinetics of ligand binding and the extreme stability of the CO complex, we deduce that the dissociation constant for CO is lower than 100 pM. Finally, we show that when the ferric complex is exposed to CO gas or a CO-releasing molecule under oxygenic conditions formation of the ferrous carbonyl complex can occur on a time scale of minutes in the presence of a redox mediator. These findings pave the way for possible applications of the RcoM-2 heme domain as a CO sensor and/or scavenger.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jpcb.6b08160DOI Listing
October 2016

Heme Oxygenase-1 and Carbon Monoxide in the Heart: The Balancing Act Between Danger Signaling and Pro-Survival.

Circ Res 2016 06;118(12):1940-1959

Inserm, U955, Equipe 12, Créteil, 94000, France.

Understanding the processes governing the ability of the heart to repair and regenerate after injury is crucial for developing translational medical solutions. New avenues of exploration include cardiac cell therapy and cellular reprogramming targeting cell death and regeneration. An attractive possibility is the exploitation of cytoprotective genes that exist solely for self-preservation processes and serve to promote and support cell survival. Although the antioxidant and heat-shock proteins are included in this category, one enzyme that has received a great deal of attention as a master protective sentinel is heme oxygenase-1 (HO-1), the rate-limiting step in the catabolism of heme into the bioactive signaling molecules carbon monoxide, biliverdin, and iron. The remarkable cardioprotective effects ascribed to heme oxygenase-1 are best evidenced by its ability to regulate inflammatory processes, cellular signaling, and mitochondrial function ultimately mitigating myocardial tissue injury and the progression of vascular-proliferative disease. We discuss here new insights into the role of heme oxygenase-1 and heme on cardiovascular health, and importantly, how they might be leveraged to promote heart repair after injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.116.306588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905590PMC
June 2016

Diverse Nrf2 Activators Coordinated to Cobalt Carbonyls Induce Heme Oxygenase-1 and Release Carbon Monoxide in Vitro and in Vivo.

J Med Chem 2016 Jan 15;59(2):756-62. Epub 2016 Jan 15.

Equipe 12, Inserm U955 , 8 Rue du Général Sarrail, Créteil, 94000, France.

The Nrf2/heme oxygenase-1 (HO-1) axis affords significant protection against oxidative stress and cellular damage. We synthesized a series of cobalt-based hybrid molecules (HYCOs) that combine an Nrf2 inducer with a releaser of carbon monoxide (CO), an anti-inflammatory product of HO-1. Two HYCOs markedly increased Nrf2/HO-1 expression, liberated CO and exerted anti-inflammatory activity in vitro. HYCOs also up-regulated tissue HO-1 and delivered CO in blood after administration in vivo, supporting their potential use against inflammatory conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.5b01509DOI Listing
January 2016

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

Biochem Pharmacol 2016 Feb 22;102:64-77. Epub 2015 Dec 22.

Université Paris-Est, Faculté de Medicine, Créteil 94000, France; INSERM, U955, Equipe 12, Créteil 94000, France. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bcp.2015.12.014DOI Listing
February 2016

Nrf2 activators modulate oxidative stress responses and bioenergetic profiles of human retinal epithelial cells cultured in normal or high glucose conditions.

Pharmacol Res 2015 Sep 15;99:296-307. Epub 2015 Jul 15.

Université Paris-Est, Faculty of Medicine, Créteil, 94000, France; Inserm U955, Equipe 12, 94000 Créteil, France. Electronic address:

Retinal pigment epithelial cells exert an important supporting role in the eye and develop adaptive responses to oxidative stress or high glucose levels, as observed during diabetes. Endogenous antioxidant defences are mainly regulated by Nrf2, a transcription factor that is activated by naturally-derived and electrophilic compounds. Here we investigated the effect of the Nrf2 activators dimethylfumarate (DMF) and carnosol on antioxidant pathways, oxygen consumption rate and wound healing in human retinal pigment epithelial cells (ARPE-19) cultured in medium containing normal (NG, 5mM) or high (HG, 25 mM) glucose levels. We also assessed wound healing using an in vivo corneal epithelial injury model. We found that Nrf2 nuclear translocation and heme oxygenase activity increased in ARPE cells treated with 10 μM DMF or carnosol irrespective of glucose culture conditions. However, HG rendered retinal cells more sensitive to regulators of glutathione synthesis or inhibition and caused a decrease of both cellular and mitochondrial reactive oxygen species. Culture in HG also reduced ATP production and mitochondrial function as measured with the Seahorse XF analyzer and electron microscopy analysis revealed morphologically damaged mitochondria. Acute treatment with DMF or carnosol did not restore mitochondrial function in HG cells; conversely, the compounds reduced cellular maximal respiratory and reserve capacity, which were completely prevented by N-acetylcysteine thus suggesting the involvement of thiols in this effect. Interestingly, the scratch assay showed that wound closure was faster in cells cultured in HG than NG and was accelerated by carnosol. This effect was reversed by an inhibitor of heme oxygenase activity. Moreover, topical application of carnosol to the cornea of diabetic rats significantly accelerated wound healing. In summary, these data indicate that culture of retinal epithelial cells in HG does not affect the activation of the Nrf2/heme oxygenase axis but influences other crucial oxidative and mitochondrial-dependent cellular functions. The additional effect on wound closure suggests that results obtained in in vitro experimental settings need to be carefully evaluated in the context of the glucose concentrations used in cell culture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phrs.2015.07.006DOI Listing
September 2015

Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells.

Toxicol Appl Pharmacol 2015 Oct 14;288(2):161-78. Epub 2015 Jul 14.

Heymans Institute of Pharmacology, Faculty of Medicine and Health Sciences, Ghent University, Belgium.

Targeting excessive production of reactive oxygen species (ROS) could be an effective therapeutic strategy to prevent oxidative stress-associated gastrointestinal inflammation. NADPH oxidase (NOX) and mitochondrial complexes (I and II) are the major sources of ROS production contributing to TNF-α/cycloheximide (CHX)-induced apoptosis in the mouse intestinal epithelial cell line, MODE-K. In the current study, the influence of a polyphenolic compound (resveratrol) and a water-soluble carbon monoxide (CO)-releasing molecule (CORM-A1) on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells was assessed. This was compared with H2O2-, rotenone- or antimycin-A-induced ROS-generating systems. Intracellular total ROS, mitochondrial-derived ROS and mitochondrial superoxide anion (O2(-)) production levels were assessed. Additionally, the influence on TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψm) and mitochondrial function was studied. In basal conditions, CORM-A1 did not affect intracellular total or mitochondrial ROS levels, while resveratrol increased intracellular total ROS but reduced mitochondrial ROS production. TNF-α/CHX- and H2O2-mediated increase in intracellular total ROS production was reduced by both resveratrol and CORM-A1, whereas only resveratrol attenuated the increase in mitochondrial ROS triggered by TNF-α/CHX. CORM-A1 decreased antimycin-A-induced mitochondrial O2(-) production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O2(-) levels, while resveratrol abolished all three effects. Finally, resveratrol greatly reduced and abolished TNF-α/CHX-induced mitochondrial depolarization and mitochondrial dysfunction, while CORM-A1 only mildly affected these parameters. These data indicate that the cytoprotective effect of resveratrol is predominantly due to mitigation of mitochondrial ROS, while CORM-A1 acts solely on NOX-derived ROS to protect MODE-K cells from TNF-α/CHX-induced cell death. This might explain the more pronounced cytoprotective effect of resveratrol.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.taap.2015.07.007DOI Listing
October 2015

Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels.

Biochim Biophys Acta 2015 Oct 14;1847(10):1297-309. Epub 2015 Jul 14.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow 30-348, Poland. Electronic address:

Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10-100μM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbabio.2015.07.004DOI Listing
October 2015

Heme oxygenase-1: an emerging therapeutic target to curb cardiac pathology.

Basic Res Cardiol 2014 25;109(6):450. Epub 2014 Oct 25.

INSERM U955, Equipe 8, Faculty of Medicine, DHU A-TVB, Hôpital Henri Mondor, APHP, Creteil, University of Paris-Est, 3rd Floor, room 3006, Paris, France,

Activation of heme oxygenase-1 (HO-1), a heme-degrading enzyme responsive to a wide range of cellular stress, is traditionally considered to convey adaptive responses to oxidative stress, inflammation and vasoconstriction. These diversified effects are achieved through the degradation of heme to carbon monoxide (CO), biliverdin (which is rapidly converted to bilirubin by biliverdin reductase) and ferric iron. Recent findings have added antiproliferative and angiogenic effects to the list of HO-1/CO actions. HO-1 along with its reaction products bilirubin and CO are protective against ischemia-induced injury (myocardial infarction, ischemia-reperfusion (IR)-injury and post-infarct structural remodelling). Moreover, HO-1, and CO in particular, possess acute antihypertensive effects. As opposed to these curative potentials, the long-believed protective effect of HO-1 in cardiac remodelling in response to pressure overload and type 2 diabetes mellitus (DM) has been questioned by recent work. These challenges, coupled with emerging regulatory mechanisms, motivate further in-depth studies to help understand untapped layers of HO-1 regulation and action. The outcomes of these efforts may shed new light on critical mechanisms that could be used to harness the protective potential of this enzyme for the therapeutic benefit of patients suffering from such highly prevalent cardiovascular disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00395-014-0450-9DOI Listing
May 2015