- Email: [email protected]
0134 : Atrial fibrillation is associated with a marker of endothelial function and oxidative stress in patients with acute myocardial infarction
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Archives of Cardiovascular Diseases Supplements (2016) 8, 216-218
Topic 10 – Oxidative Stress, NO April 07th, Thursday 2016
0274 Exosomes from metabolic syndrome patients induce endothelial dysfunction through mitochondrial-generated oxidative stress Marine Malloci * (1), Séverine Dubois (2), Ramaroson Andriantsitohaina (1), Gilles Simard (1), Carmen Martinez (1) (1) Université d’Angers – IBS, CHU Angers, INSERM U1063, Angers, France – (2) CHU Angers, Angers, France * Corresponding author: [email protected] (Marine Malloci) Metabolic syndrome (MetS) is characterized by a cluster of interrelated risk factors -hyperglycemia, dyslipidemia, hypertension and obesity-leading to an increased risk of cardiovascular events. Exosomes are nanoscaled vesicles (30100 nm) released by cells, that bear proteins and nucleic acids. It has been demonstrated that circulating exosome rate is positively correlated with (i) an increase of inflammation, (ii) a decrease of HDL cholesterol and (iii) prevalence of MetS. However, it is unknown whether exosomes from MetS patients can act as biological vectors through their interaction with target cells.We hypothesized that exosomes may be implicated in MetS-associated endothelial dysfunction. Circulating exosomes from healthy subjects and MetS patients have been isolated from plasma and, then, characterized. As expected, isolated exosomes have an average size of 63±15 nm and are enriched in exosomal specific proteins, tetraspanins such as CD81, CD9, CD63 and endolysosomal protein TSG101. Moreover, the majority of plasmatic exosomes are from leukocyte and platelet origins in both healthy subjects and MetS patients. MetS patients display increased levels of exosomes compared to healthy subjects. We demonstrated that treatment of human endothelial aortic cells with exosomes from MetS patients induce endothelial dysfunction associated with decreased nitric oxide and increased reactive oxygen species (ROS) production. The increase on fluorescence associated to Mitosox suggests an enhanced production of mitochondrial ROS following 4 hours of MetS exosome treatment. These data provide evidence that circulating exosomes from MetS patients induce endothelial dysfunction via both increased oxidative stress and reduced nitric oxide production. Altogether, we show exosomes as novel players implicated in endothelial dysfunction associated with MetS and consequently potential targets to prevent and treat vascular consequences of this syndrome. The author hereby declares no conflict of interest
0343 Importance of the membrane estrogen receptor alpha (ER ) in the vascular response to shear stress in mice Julie Favre * (1), Emilie Vessière (1), Anne-Laure Guihot (1), Linda Grimaud (1), Laurent Loufrani (1), Jean-François Arnal (2), Françoise Lenfant (2), Daniel Henrion (1) (1) Faculté de Médecine, CNRS UMR6214, INSERM UMR1083, Angers, France – (2) CHU Toulouse, Rangueil, INSERM/UPS UMR 1048, Toulouse, France * Corresponding author: [email protected] (Julie Favre) Resistance arteries are sensitive to mechanical forces exerted on the vessel wall. While endothelial shear stress triggers flow-mediated dilation (FMD), chronic increase in shear forces drives expansive arterial remodeling. We showed previously that endothelial estrogen receptor alpha (ERα) controlled this adaptive remodeling. We aimed to evaluate ERα contribution in acute response to flow, by using various genetic models of ERα deficiency in male mice, reducing estrogen hormonal influence. FMD was evaluated following step increase in flow applied to pressurized mesenteric arteries (arteriography). Arteries were isolated from wild-type (WT), (i) ERαKO, or mice invalidated for (ii) the ligand-dependent transactivation function AF2 of ERa (AF2°) and (iii) the plasma membrane-located ERα (mutated for the palmitoylation site C451A). In mice deficient in ERα, FMD was attenuated (dilation 50μl/min
© Elsevier Masson SAS. All rights reserved.
WT: 59+/–4% vs. ERαKO: 41+/–4% p<0.01) without significant modification in response to acetylcholine. Invalidation of AF2 (ERα nuclear transcriptional action) only partially impacted FMD (WT: 61+/–5% vs. AF2°: 51+/–5%). Ex vivo ligand-dependent activation of ERα with 17β estradiol or blockade by ICI 182,780 had no significant effect on FMD in WT arteries. Importantly, invalidation of membrane ERα (C451A) markedly altered FMD (WT: 56+/–7% vs. C451A: 34+/–6% p<0.05). In contrast to ERαKO and AF2°, C451A arteries showed a reduced dilating response to acetylcholine but only a decreased sensitivity to insulin, which shares common intracellular pathways triggered by shear stress, without altering maximal vasodilation (WT: 31+/–7% vs. C451A: 15+/–3% p<0.05). We show for the first time that membrane ERα contributes to arterial shear-sensing irrespective of the presence of its agonist. Cell membrane ERα participates to vascular homeostasis and could constitute a novel therapeutical target in peripheral vascular diseases. The author hereby declares no conflict of interest
0169 Effects of intermittent hypoxia on rat ophthalmic artery reactivity: role of endothelin 1, oxidative stress and endothelium derived hyperpolarizing factors Marielle Mentek *, Marie Baldazza, Jessica Morand, Gilles Faury, Florent Aptel, Diane Godin-Ribuot, Christophe Chiquet Université de Grenoble, INSERM U1042, Grenoble, France * Corresponding author: [email protected] (Marielle Mentek) Obstructive sleep apnea (OSA) is a common disease, characterized by recurrent desaturation-reoxygenation sequences. Intermittent hypoxia (IH) has been proposed as major component of OSA. OSA has been recently associated with a higher frequency of optic neuropathies. There are currently no pathophysiological data regarding the effect of HI or OSA on ocular vascular system. This study aims to characterize IH impact on rat ophthalmic artery (OA) reactivity. Particularly, the role of endothelin 1 (ET-1), oxidative stress and endothelium derived hyperpolarizing factors (EDHF) were studied. Rats were exposed to 14 days IH (IH rats) or normoxia (NX rats). Ophthalmic artery reactivity was studied using wire myography. Endothelin A receptor (ETRA) expression and superoxide anions production in OA were studied by immunohistolabelling and DHE labelling respectively. After 14 days IH, ETRA immunolabeling and superoxide anions expression in OA were increased by 22 % (p = 0.015) and 23 % (p = 0.04) respectively in IH rats. Ophthalmic artery contraction to 3.10-8 M ET-1 was increased by 16% (p<0.05) in IH rats. ETRA blockage (BQ-123) almost completely abolished contractile response to ET-1 in both groups. Endothelin B receptor (ETRB) blockage (BQ-788) increased response to ET-1 in NX rats but not in IH rats. Relaxation to acetylcholine (Ach) was significantly delayed in IH rats (p<0.0001). After NOS blockade with L-NAME, difference in Ach induced relaxation between groups was abolished. Cytochrome P450 inhibition by fluconazole had an opposite effect on response to Ach in NX and IH rats, increasing relaxation to Ach in IH rats and delaying it in NX rats. 14-day IH-exposure induces endothelial dysfunction in rat ophtalmic artery, associated with increase in oxidative stress and change in EDHF contribution to endothelial function. The increased contractile response to ET-1 in IH rats was due to an increase in ETRA -vasoconstriction and a decrease in ETRB vasorelaxation. The author hereby declares no conflict of interest
0134 Atrial fibrillation is associated with a marker of endothelial function and oxidative stress in patients with acute myocardial infarction Karim Stamboul (1), Julie Lorin (2), Luc Lorgis (1), Jean-Claude Beer (1), Claude Touzery (1), Luc Rochette (2), Catherine Vergely (2), Yves Cottin (1), Marianne Zeller * (2) (1) CHU Dijon, Bocage, Dijon, France – (2) Université de Bourgogne, INSERM U866, Dijon, France * Corresponding author: [email protected] (Marianne Zeller) Background Atrial fibrillation (AF), whether silent or symptomatic, is a frequent and severe complication of acute myocardial infarction (AMI). Asymmetric dimethylarginine (ADMA), an endogenous eNOS inhibitor, is a
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Archives of Cardiovascular Diseases Supplements (2016) 8, 216-218
risk factor for endothelial dysfunction. We addressed the relationship between ADMA plasma levels and AF occurrence in AMI. Methods 273 patients hospitalized for AMI were included. Continuous electrocardiographic monitoring (CEM) ≥48 hours was recorded and ADMA was measured by High Performance Liquid Chromatography on admission blood sample. Results The incidence of silent and symptomatic AF was 39(14%) and 29 (11%), respectively. AF patients were markedly older than patients without AF (≈ 20 y). There was a trend towards higher ADMA levels in patients with symptomatic AF than in patients with silent AF or no AF (0.53 vs 0.49 and 0.49 μmol/L, respectively). After matching on age, we found that patients with symptomatic AF had a higher heart rate on admission and a higher rate of patients with LV dysfunction (28% vs. 3%, p=0.025). Patients who developed symptomatic AF had a higher ADMA level (0.53 vs. 0.43 μmol/L; p=0.001). Multivariate logistic regression analysis to estimate symptomatic AF occurrence showed that ADMA was independently associated with symptomatic AF (OR: 2.46 [1.21-5.00], p=0.013) beyond history of AF, LVEF<40% and elevated HR. Conclusion We show that high ADMA level is associated with the occurrence of AF. Although no causative role can be concluded from our observational study, our work further supports the hypothesis that endothelial dysfunction is involved in the pathogenesis of AF in AMI. The author hereby declares no conflict of interest
0333 Tissue specificity of the membrane vs nuclear actions of estrogen receptor alpha: insights from targeted mutations in mouse models Jean-François Arnal *, Coralie Fontaine, Marie-Cecile Valera, Marine Adlanmerini, Pierre Gourdy, Françoise Lenfant CHU Toulouse, Rangueil, INSERM U1048 – I2MR, Toulouse, France * Corresponding author: [email protected] (Jean-Francois Arnal) Thanks to unique mouse models, we demonstrated that the estrogen receptor alpha (ERa), but not ERb, is absolutely necessary for most of the arterial and metabolic actions of 17b-estradiol (E2). Estrogens also elicit deleterious effects on the uterus and breast (partly via their proliferative effect which increases the risk of cancer) as well as increased risk of venous thromboembolism, which are the two main limitations of classic estrogen therapies. ER contains two independent transactivation functions AF-1 and AF-2, and our team dissected for the first time in vivo the roles of functions ERaAF-1 and AF-2, allowing to define the key role of these nuclear actions in several tissues. More recently, we reported the key role of the ERa fraction localized to the plasma membrane which is absolutely necessary for the protective endothelial actions of E2 as well as, more unexpectedly, for female fertility. One mystery of estrogens biology is that selective ER modulators (SERMs, such as tamoxifen, raloxifen) have a highly tissue-specific action, behaving as agonists in some tissues (such as bone) and as antagonist in others (breast). We will report how these mouse models allowed us to characterize: 1) the tissue-specific roles of ERa : membrane ERa mediates the effect of E2 in the endothelium (acceleration of reendothelialization, activation of endothelial NO synthase), whereas nuclear ERa mediates the effect of E2 in the uterus. 2) a new natural SERM (Estetrol) currently tested in a phase III clinical trial as a safer oral contraception or hormonal treatment of menopause, as this molecule does not alter the hepatic-derived coagulation factors, and thereby could not increase the venous thrombosis risk. 3) an Estrogen Dendrimer Conjugate, a selective activator of membrane ERa, which could be a new endothelial protective SERM. The author declares a conflict of interest: JF Arnal'team received a financial support from Uteron/Mithra to study the mechanisms of action of Estetrol.
0546 Mitochondrial induced oxidative stress triggers Ca2+-dependent proteolysis and underlie VIDD Haikel Dridi * Université de Montpellier, INSERM U1046, Montpellier, France * Corresponding author: [email protected] (Haikel Dridi) Rationale In rodents, prolonged controlled mechanical ventilation (CMV) and associated diaphragm inactivity elicits complex pathologic changes
referred to as ventilator-induced diaphragm dysfunction (VIDD). Previous workers have demonstrated that VIDD is characterized by increased mitochondrial generation of reactive oxygen species (ROS) and redox-induced dysfunction in calcium release. Powers et al., (2011) have demonstrated that SS-31, a mitochondrial targeted antioxidant, markedly attenuates mitochondrial ROS generation. Since, the calcium-release pathology associated with VIDD is thought to be secondary to myofiber redox changes, we hypothesized that SS-31 should markedly attenuate these abnormalities in myofiber calcium regulation. Objective To test the hypothesis that SS-31, a mitochondrial-targeted antioxidant, attenuates the effect of prolonged CMV on diaphragm myofiber calcium homeostasis. Measurements and main results Specific force of diaphragm fibers decreased significantly 28 ±5%, (n=10) following 12hrs of mechanical ventilation compared to fibers from unventilated control mice. These changes were associated with a significant decrease in cross-sectional area (574 ±19 versus 673± 21 μm m2), an increase in calpain activity, the oxidation of the RyR1 macromolecular complexed, RyR1 phosphorylation at Ser-2844 and the depletion of the stabilizing subunit calstabin1 resulting in an increase in sarcoplasmic reticulum (SR) Ca2+ leak. Diaphragm from mice treated with SS-31 were protected against all of these changes. On the basis of these findings we propose that SR Ca2+ leak via RyR1 due to mitochondrial induced oxidative stress, triggers Ca2+-dependent proteolysis and underlie VIDD. These results emphasize the need to develop therapeutic interventions that mitigate mitochondrial dysfunctional thereby reduce mechanical ventilation-induced diaphragmatic weakness. The author hereby declares no conflict of interest
0460 Nitric oxide and hemoglobin form a paramagnetic compound quantifiable by Electron Paramagnetic Resonance (EPR) spectroscopy in venous erythrocytes that reflects vascular NO bioavailability in vivo Flavia Dei Zotti *, Irina Lobysheva, Jean-Luc Balligand Université Catholique de Louvain, Bruxelles, Belgique * Corresponding author: [email protected] (Flavia Dei Zotti) Reduced bioavailability of nitric oxide is a hallmark of endothelial dysfunction in metabolic and cardiovascular diseases, but its quantification in circulating blood remains a challenge.NO can form iron-nitrosyl complexes with hemoglobin(5-coordinate-α-HbNO) in erythrocytes(RBCs).We hypothesized that this complex reflects bioavailability of vascular NO and endothelial function in vivo.We developed a modified subtraction method using EPR to quantify it in RBCs from mouse,rat and human venous blood. NO could be supplied from vascular or erythrocytic NOS.We detected eNOS proteins in rodent and human RBC.To test eNOS functionality, we measured nitrite/ nitrate production and HbNO formation in human RBCs and from eNOS(+/+) and eNOS(-/-)mice in vitro and its sensitivity to NOS or arginase inhibitors.Nitrite and HbNO signals increased after arginase inhibition and were abrogated upon NOS inhibition in human and eNOS(+/+) but insensitive to these modulators in eNOS(-/-)RBCs. We found that upon exposure to exogenous NO-donor,the formation of HbNO was higher in hypoxic conditions(1%O2:0.018+/–0.002 compared to room air 0.0036+/–0.0004 μmol HbNO/μmolNO-donor).Accordingly, the stability of pre-formed HbNO was higher under hypoxia(17% degradation after 30 min vs 49% at room air),and preserved at 21% of O2 by incubation with catalase(CAT:2μM HbNO vs 0.5μM HbNO in untreated controls).Conversely,CAT inhibition increased ROS formation in RBCs, measured by FACS analysis of DCFDA fluorescence.This suggested that HbNO formation is sensitive to oxidative degradation, possibly by H2O2.We compared circulating HbNO levels in venous RBCs from normal volunteers or patients with cardiovascular diseases and found decreased HbNO in patients(0.141+/–11μM vs 0.22+/–12μM in volunteers;N=38 and48).We conclude that HbNO reflects exposure of RBCs to NO in vivo and is sensitive to oxidative degradation by H2O2.HbNO could be developed as a biomarker of NO bioavailability and/or oxidative stress ex vivo. The author hereby declares no conflict of interest
© Elsevier Masson SAS. All rights reserved.
Archives of Cardiovascular Diseases Supplements (2016) 8, 216-218
Topic 10 – Oxidative Stress, NO April 07th, Thursday 2016
0274 Exosomes from metabolic syndrome patients induce endothelial dysfunction through mitochondrial-generated oxidative stress Marine Malloci * (1), Séverine Dubois (2), Ramaroson Andriantsitohaina (1), Gilles Simard (1), Carmen Martinez (1) (1) Université d’Angers – IBS, CHU Angers, INSERM U1063, Angers, France – (2) CHU Angers, Angers, France * Corresponding author: [email protected] (Marine Malloci) Metabolic syndrome (MetS) is characterized by a cluster of interrelated risk factors -hyperglycemia, dyslipidemia, hypertension and obesity-leading to an increased risk of cardiovascular events. Exosomes are nanoscaled vesicles (30100 nm) released by cells, that bear proteins and nucleic acids. It has been demonstrated that circulating exosome rate is positively correlated with (i) an increase of inflammation, (ii) a decrease of HDL cholesterol and (iii) prevalence of MetS. However, it is unknown whether exosomes from MetS patients can act as biological vectors through their interaction with target cells.We hypothesized that exosomes may be implicated in MetS-associated endothelial dysfunction. Circulating exosomes from healthy subjects and MetS patients have been isolated from plasma and, then, characterized. As expected, isolated exosomes have an average size of 63±15 nm and are enriched in exosomal specific proteins, tetraspanins such as CD81, CD9, CD63 and endolysosomal protein TSG101. Moreover, the majority of plasmatic exosomes are from leukocyte and platelet origins in both healthy subjects and MetS patients. MetS patients display increased levels of exosomes compared to healthy subjects. We demonstrated that treatment of human endothelial aortic cells with exosomes from MetS patients induce endothelial dysfunction associated with decreased nitric oxide and increased reactive oxygen species (ROS) production. The increase on fluorescence associated to Mitosox suggests an enhanced production of mitochondrial ROS following 4 hours of MetS exosome treatment. These data provide evidence that circulating exosomes from MetS patients induce endothelial dysfunction via both increased oxidative stress and reduced nitric oxide production. Altogether, we show exosomes as novel players implicated in endothelial dysfunction associated with MetS and consequently potential targets to prevent and treat vascular consequences of this syndrome. The author hereby declares no conflict of interest
0343 Importance of the membrane estrogen receptor alpha (ER ) in the vascular response to shear stress in mice Julie Favre * (1), Emilie Vessière (1), Anne-Laure Guihot (1), Linda Grimaud (1), Laurent Loufrani (1), Jean-François Arnal (2), Françoise Lenfant (2), Daniel Henrion (1) (1) Faculté de Médecine, CNRS UMR6214, INSERM UMR1083, Angers, France – (2) CHU Toulouse, Rangueil, INSERM/UPS UMR 1048, Toulouse, France * Corresponding author: [email protected] (Julie Favre) Resistance arteries are sensitive to mechanical forces exerted on the vessel wall. While endothelial shear stress triggers flow-mediated dilation (FMD), chronic increase in shear forces drives expansive arterial remodeling. We showed previously that endothelial estrogen receptor alpha (ERα) controlled this adaptive remodeling. We aimed to evaluate ERα contribution in acute response to flow, by using various genetic models of ERα deficiency in male mice, reducing estrogen hormonal influence. FMD was evaluated following step increase in flow applied to pressurized mesenteric arteries (arteriography). Arteries were isolated from wild-type (WT), (i) ERαKO, or mice invalidated for (ii) the ligand-dependent transactivation function AF2 of ERa (AF2°) and (iii) the plasma membrane-located ERα (mutated for the palmitoylation site C451A). In mice deficient in ERα, FMD was attenuated (dilation 50μl/min
© Elsevier Masson SAS. All rights reserved.
WT: 59+/–4% vs. ERαKO: 41+/–4% p<0.01) without significant modification in response to acetylcholine. Invalidation of AF2 (ERα nuclear transcriptional action) only partially impacted FMD (WT: 61+/–5% vs. AF2°: 51+/–5%). Ex vivo ligand-dependent activation of ERα with 17β estradiol or blockade by ICI 182,780 had no significant effect on FMD in WT arteries. Importantly, invalidation of membrane ERα (C451A) markedly altered FMD (WT: 56+/–7% vs. C451A: 34+/–6% p<0.05). In contrast to ERαKO and AF2°, C451A arteries showed a reduced dilating response to acetylcholine but only a decreased sensitivity to insulin, which shares common intracellular pathways triggered by shear stress, without altering maximal vasodilation (WT: 31+/–7% vs. C451A: 15+/–3% p<0.05). We show for the first time that membrane ERα contributes to arterial shear-sensing irrespective of the presence of its agonist. Cell membrane ERα participates to vascular homeostasis and could constitute a novel therapeutical target in peripheral vascular diseases. The author hereby declares no conflict of interest
0169 Effects of intermittent hypoxia on rat ophthalmic artery reactivity: role of endothelin 1, oxidative stress and endothelium derived hyperpolarizing factors Marielle Mentek *, Marie Baldazza, Jessica Morand, Gilles Faury, Florent Aptel, Diane Godin-Ribuot, Christophe Chiquet Université de Grenoble, INSERM U1042, Grenoble, France * Corresponding author: [email protected] (Marielle Mentek) Obstructive sleep apnea (OSA) is a common disease, characterized by recurrent desaturation-reoxygenation sequences. Intermittent hypoxia (IH) has been proposed as major component of OSA. OSA has been recently associated with a higher frequency of optic neuropathies. There are currently no pathophysiological data regarding the effect of HI or OSA on ocular vascular system. This study aims to characterize IH impact on rat ophthalmic artery (OA) reactivity. Particularly, the role of endothelin 1 (ET-1), oxidative stress and endothelium derived hyperpolarizing factors (EDHF) were studied. Rats were exposed to 14 days IH (IH rats) or normoxia (NX rats). Ophthalmic artery reactivity was studied using wire myography. Endothelin A receptor (ETRA) expression and superoxide anions production in OA were studied by immunohistolabelling and DHE labelling respectively. After 14 days IH, ETRA immunolabeling and superoxide anions expression in OA were increased by 22 % (p = 0.015) and 23 % (p = 0.04) respectively in IH rats. Ophthalmic artery contraction to 3.10-8 M ET-1 was increased by 16% (p<0.05) in IH rats. ETRA blockage (BQ-123) almost completely abolished contractile response to ET-1 in both groups. Endothelin B receptor (ETRB) blockage (BQ-788) increased response to ET-1 in NX rats but not in IH rats. Relaxation to acetylcholine (Ach) was significantly delayed in IH rats (p<0.0001). After NOS blockade with L-NAME, difference in Ach induced relaxation between groups was abolished. Cytochrome P450 inhibition by fluconazole had an opposite effect on response to Ach in NX and IH rats, increasing relaxation to Ach in IH rats and delaying it in NX rats. 14-day IH-exposure induces endothelial dysfunction in rat ophtalmic artery, associated with increase in oxidative stress and change in EDHF contribution to endothelial function. The increased contractile response to ET-1 in IH rats was due to an increase in ETRA -vasoconstriction and a decrease in ETRB vasorelaxation. The author hereby declares no conflict of interest
0134 Atrial fibrillation is associated with a marker of endothelial function and oxidative stress in patients with acute myocardial infarction Karim Stamboul (1), Julie Lorin (2), Luc Lorgis (1), Jean-Claude Beer (1), Claude Touzery (1), Luc Rochette (2), Catherine Vergely (2), Yves Cottin (1), Marianne Zeller * (2) (1) CHU Dijon, Bocage, Dijon, France – (2) Université de Bourgogne, INSERM U866, Dijon, France * Corresponding author: [email protected] (Marianne Zeller) Background Atrial fibrillation (AF), whether silent or symptomatic, is a frequent and severe complication of acute myocardial infarction (AMI). Asymmetric dimethylarginine (ADMA), an endogenous eNOS inhibitor, is a
217
Archives of Cardiovascular Diseases Supplements (2016) 8, 216-218
risk factor for endothelial dysfunction. We addressed the relationship between ADMA plasma levels and AF occurrence in AMI. Methods 273 patients hospitalized for AMI were included. Continuous electrocardiographic monitoring (CEM) ≥48 hours was recorded and ADMA was measured by High Performance Liquid Chromatography on admission blood sample. Results The incidence of silent and symptomatic AF was 39(14%) and 29 (11%), respectively. AF patients were markedly older than patients without AF (≈ 20 y). There was a trend towards higher ADMA levels in patients with symptomatic AF than in patients with silent AF or no AF (0.53 vs 0.49 and 0.49 μmol/L, respectively). After matching on age, we found that patients with symptomatic AF had a higher heart rate on admission and a higher rate of patients with LV dysfunction (28% vs. 3%, p=0.025). Patients who developed symptomatic AF had a higher ADMA level (0.53 vs. 0.43 μmol/L; p=0.001). Multivariate logistic regression analysis to estimate symptomatic AF occurrence showed that ADMA was independently associated with symptomatic AF (OR: 2.46 [1.21-5.00], p=0.013) beyond history of AF, LVEF<40% and elevated HR. Conclusion We show that high ADMA level is associated with the occurrence of AF. Although no causative role can be concluded from our observational study, our work further supports the hypothesis that endothelial dysfunction is involved in the pathogenesis of AF in AMI. The author hereby declares no conflict of interest
0333 Tissue specificity of the membrane vs nuclear actions of estrogen receptor alpha: insights from targeted mutations in mouse models Jean-François Arnal *, Coralie Fontaine, Marie-Cecile Valera, Marine Adlanmerini, Pierre Gourdy, Françoise Lenfant CHU Toulouse, Rangueil, INSERM U1048 – I2MR, Toulouse, France * Corresponding author: [email protected] (Jean-Francois Arnal) Thanks to unique mouse models, we demonstrated that the estrogen receptor alpha (ERa), but not ERb, is absolutely necessary for most of the arterial and metabolic actions of 17b-estradiol (E2). Estrogens also elicit deleterious effects on the uterus and breast (partly via their proliferative effect which increases the risk of cancer) as well as increased risk of venous thromboembolism, which are the two main limitations of classic estrogen therapies. ER contains two independent transactivation functions AF-1 and AF-2, and our team dissected for the first time in vivo the roles of functions ERaAF-1 and AF-2, allowing to define the key role of these nuclear actions in several tissues. More recently, we reported the key role of the ERa fraction localized to the plasma membrane which is absolutely necessary for the protective endothelial actions of E2 as well as, more unexpectedly, for female fertility. One mystery of estrogens biology is that selective ER modulators (SERMs, such as tamoxifen, raloxifen) have a highly tissue-specific action, behaving as agonists in some tissues (such as bone) and as antagonist in others (breast). We will report how these mouse models allowed us to characterize: 1) the tissue-specific roles of ERa : membrane ERa mediates the effect of E2 in the endothelium (acceleration of reendothelialization, activation of endothelial NO synthase), whereas nuclear ERa mediates the effect of E2 in the uterus. 2) a new natural SERM (Estetrol) currently tested in a phase III clinical trial as a safer oral contraception or hormonal treatment of menopause, as this molecule does not alter the hepatic-derived coagulation factors, and thereby could not increase the venous thrombosis risk. 3) an Estrogen Dendrimer Conjugate, a selective activator of membrane ERa, which could be a new endothelial protective SERM. The author declares a conflict of interest: JF Arnal'team received a financial support from Uteron/Mithra to study the mechanisms of action of Estetrol.
0546 Mitochondrial induced oxidative stress triggers Ca2+-dependent proteolysis and underlie VIDD Haikel Dridi * Université de Montpellier, INSERM U1046, Montpellier, France * Corresponding author: [email protected] (Haikel Dridi) Rationale In rodents, prolonged controlled mechanical ventilation (CMV) and associated diaphragm inactivity elicits complex pathologic changes
referred to as ventilator-induced diaphragm dysfunction (VIDD). Previous workers have demonstrated that VIDD is characterized by increased mitochondrial generation of reactive oxygen species (ROS) and redox-induced dysfunction in calcium release. Powers et al., (2011) have demonstrated that SS-31, a mitochondrial targeted antioxidant, markedly attenuates mitochondrial ROS generation. Since, the calcium-release pathology associated with VIDD is thought to be secondary to myofiber redox changes, we hypothesized that SS-31 should markedly attenuate these abnormalities in myofiber calcium regulation. Objective To test the hypothesis that SS-31, a mitochondrial-targeted antioxidant, attenuates the effect of prolonged CMV on diaphragm myofiber calcium homeostasis. Measurements and main results Specific force of diaphragm fibers decreased significantly 28 ±5%, (n=10) following 12hrs of mechanical ventilation compared to fibers from unventilated control mice. These changes were associated with a significant decrease in cross-sectional area (574 ±19 versus 673± 21 μm m2), an increase in calpain activity, the oxidation of the RyR1 macromolecular complexed, RyR1 phosphorylation at Ser-2844 and the depletion of the stabilizing subunit calstabin1 resulting in an increase in sarcoplasmic reticulum (SR) Ca2+ leak. Diaphragm from mice treated with SS-31 were protected against all of these changes. On the basis of these findings we propose that SR Ca2+ leak via RyR1 due to mitochondrial induced oxidative stress, triggers Ca2+-dependent proteolysis and underlie VIDD. These results emphasize the need to develop therapeutic interventions that mitigate mitochondrial dysfunctional thereby reduce mechanical ventilation-induced diaphragmatic weakness. The author hereby declares no conflict of interest
0460 Nitric oxide and hemoglobin form a paramagnetic compound quantifiable by Electron Paramagnetic Resonance (EPR) spectroscopy in venous erythrocytes that reflects vascular NO bioavailability in vivo Flavia Dei Zotti *, Irina Lobysheva, Jean-Luc Balligand Université Catholique de Louvain, Bruxelles, Belgique * Corresponding author: [email protected] (Flavia Dei Zotti) Reduced bioavailability of nitric oxide is a hallmark of endothelial dysfunction in metabolic and cardiovascular diseases, but its quantification in circulating blood remains a challenge.NO can form iron-nitrosyl complexes with hemoglobin(5-coordinate-α-HbNO) in erythrocytes(RBCs).We hypothesized that this complex reflects bioavailability of vascular NO and endothelial function in vivo.We developed a modified subtraction method using EPR to quantify it in RBCs from mouse,rat and human venous blood. NO could be supplied from vascular or erythrocytic NOS.We detected eNOS proteins in rodent and human RBC.To test eNOS functionality, we measured nitrite/ nitrate production and HbNO formation in human RBCs and from eNOS(+/+) and eNOS(-/-)mice in vitro and its sensitivity to NOS or arginase inhibitors.Nitrite and HbNO signals increased after arginase inhibition and were abrogated upon NOS inhibition in human and eNOS(+/+) but insensitive to these modulators in eNOS(-/-)RBCs. We found that upon exposure to exogenous NO-donor,the formation of HbNO was higher in hypoxic conditions(1%O2:0.018+/–0.002 compared to room air 0.0036+/–0.0004 μmol HbNO/μmolNO-donor).Accordingly, the stability of pre-formed HbNO was higher under hypoxia(17% degradation after 30 min vs 49% at room air),and preserved at 21% of O2 by incubation with catalase(CAT:2μM HbNO vs 0.5μM HbNO in untreated controls).Conversely,CAT inhibition increased ROS formation in RBCs, measured by FACS analysis of DCFDA fluorescence.This suggested that HbNO formation is sensitive to oxidative degradation, possibly by H2O2.We compared circulating HbNO levels in venous RBCs from normal volunteers or patients with cardiovascular diseases and found decreased HbNO in patients(0.141+/–11μM vs 0.22+/–12μM in volunteers;N=38 and48).We conclude that HbNO reflects exposure of RBCs to NO in vivo and is sensitive to oxidative degradation by H2O2.HbNO could be developed as a biomarker of NO bioavailability and/or oxidative stress ex vivo. The author hereby declares no conflict of interest
© Elsevier Masson SAS. All rights reserved.