- Email: [email protected]
Impact of erythrocyte trafficking on early stages of atheroma
Abstracts / Atherosclerosis 241 (2015) e72ee148
EAS-0806. IMPACT OF ERYTHROCYTE TRAFFICKING ON EARLY STAGES OF ATHEROMA , V. Ollivier, L. Louedec, J. Laschet, L. R. Bayles*, S. Delbosc, B. Ho-Tin-Noe Gouya, A. Nicoletti, J.B. Michel. U1148, INSERM, Paris Cedex 18, France
* Corresponding author. Background: Red Blood Cells (RBC) are a major source of cholesterol (cell membrane), and of pro-oxidant iron-loaded heme. This has been shown to be clinically important in intraplaque haemorrhage. We hypothesise that RBC trafficking participates in the early stages of atheroma. Methods: To further explore the role of RBC we have used a descriptive approach, examining human tissue, vascular smooth muscle cell (VSMC) biology and experimental animal models. Human aortic tissues (healthy, fatty streak or fibrolipidic plaques) were collected from deceased organ donors. In vitro experiments demonstrating erythrophagocytosis were performed using primary VSMC. Atheroma development was investigated in Lewis rats under various conditions. Results: In humans, Perl’s/DAB precipitation highlighted the presence of iron in 67% of lesions. Frequency was significantly different between groups, with low frequency of iron detection in normal aorta (24%) as compared to fatty streaks (73%), and fibrolipidic plaques (90%). Iron tissue concentration and expression of iron transporters were significantly increased in early atherosclerotic lesions. In vitro experiments showed that sRBC (not fresh RBC) were engulfed by VSMC and this was associated with a significant increase in VSMC ROS production, and in the expression of metabolically relevant genes/ proteins. In rats, iron was detectable by histochemistry in all aortas from rats which received sRBC injections and were submitted to balloon injury. Analysis of iron concentration in the aortic wall showed a significant effect of sRBC injections, ballooning and splenectomy. Conclusion: Our preliminary results suggest that RBC phagocytosis may play an important role in early stages of atheroma. EAS-0816. MITOCHONDRIAL DYSFUNCTION IN CAROTID PLAQUE ATHEROSCLEROSIS A PREDICTOR OF PLAQUE VULNERABILITY J.R. Mercer*. University of Glasgow, Institute of Cardiovascular and Medical Sciences, Glasgow, United Kingdom
* Corresponding author. Introduction: DNA damage is a common finding in atherosclerotic plaques but the full consequences to disease aetiology remains incomplete. Previously we have shown excess DNA damage and/or reduced repair combines to degrade the energetic capacity of mitochondria. This effect results in a decline in synthetic and replicative capacity of plaque cells and increased propensity for plaque rupture. In addition to its role as a mediator of cell death and calcium homeostasis, mitochondria provide the energetic capacity of the cell as an ATP generator. Recent work has assessed the function of the respiratory chain in isolated human atherosclerotic carotid plaque components and their contribution to the concept of vulnerable plaque. Method: Human carotid plaques were micro-dissected into cap, core, shoulder and wall. Using polargraphic oxygen respirometry we assessed respiratory chain capacity. In addition to using standard histological stains for smooth muscle actin (SMA) and macrophages (Mac-3) we determined the relative contribution of each cell type to the energetic capacity of the plaque. Result: A significant 80.4% reduction in plaque cap respiration compared to underlying vessel wall was observed. While plaque cap and wall was almost exclusively SMAþ (99.4þ/-1.9 and 99.4%þ/-0.5), cellular density was significantly reduced in plaque caps, showing a 60.9% decline in SMAþ cellularity. Conclusion: This data suggests that as SMAþ vascular cellularity declines in the mature cap there is a concurrent down regulation in mitochondrial capacity equating to a significant 20.1% decrease in respiratory chain
e79
efficiency. Together these data confirm mitochondrial dysfunction as a key marker of plaque vulnerability. EAS-0833. EPIGENETIC REGULATION OF GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE CELLS J. Harman, R. Madsen, A. Dalby, M. Davies, H. Jørgensen*. Cardiovascular Medicine, University of Cambridge, Cambridge, United Kingdom
* Corresponding author. Vascular smooth muscle cell (VSMC) phenotypic switching is a hallmark of atherosclerosis, UK’s main cause of death. During phenotypic switching, the gene expression profile of VSMCs changes dramatically. The resulting loss of contractility and increased migration, proliferation and secretion of proinflammatory cytokines contribute directly to atherosclerotic lesion growth and vascular inflammation. We study how regulation of gene expression by epigenetic mechanisms impact on VSMC phenotypic switching. Using a stem cell model we identified methylation of Histone H3 Lysine 9 (H3K9) as a regulatory pathway of VSMC gene expression. Subsequent studies using ex vivo and cultured VSMCs isolated from mouse aorta demonstrated that contractile VSMC genes display increased levels of methylated H3K9 during in vitro induced phenotypic switching. We have mapped the levels of methylated H3K9 genome-wide and find that this repressive epigenetic mark is also found at the promoters of inflammation-associated genes, including a subset of matrix metallo proteases in VSMCs. Using a small molecule inhibitor of the G9a/GLP methyl transferases, we furthermore demonstrated that this pathway is functionally relevant for regulating gene expression in VSMC. Our studies demonstrate that H3K9 methylation represses the expression of disease-related genes in VSMCs and suggest that this pathway might function to prevent unsolicited vascular inflammation.
Monocytes/macrophages EAS-0108. EICOSAPENTAENOIC ACID (EPA) MEMBRANE INCORPORATION IMPAIRED ABCA1-DEPENDENT CHOLESTEROL EFFLUX THROUGH A PKA PATHWAY IN PRIMARY HUMAN MACROPHAGES N. Fournier 1, *, S. Tardivel 1, J.F. Benoist 2, B. Vedie 3, D. Rousseaue Paris-Sud 11, Ralliard 4, J.L. Paul 1. 1 EA 4529, UFR de Pharmacie Universit Chatenay-Malabry, France; 2 Laboratoire de Biochimie-Hormonologie, ^pital Robert Debr Ho e AP-HP, Paris, France; 3 Laboratoire de Biochimie, ^pital europ Ho een Georges Pompidou AP-HP, Paris, France; 4 UMR 1198, INRA, Jouy-en-Josas, France
* Corresponding author. Dietary polyunsaturated fatty acids (PUFA) affect the cellular phospholipids composition which may influence the function of membrane proteins. We investigated the impact of the incorporation of several PUFA on the ABCA1mediated cholesterol efflux, an anti-atherogenic process. The arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) supplementation decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, the membrane incorporation of eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70 mM of EPA in HMDM). The EPA addition did not alter the ABCA1 expression (mRNA or protein) in mouse or human macrophages. The EPAtreated HMDM exhibited strong phospholipids composition changes with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3) associated to a decreased level of AA. In human macrophages, cellular ATP was required for the effect of EPA. In addition, we observed that addition of forskolin, a direct activator of adenylate cyclase, and isobutylmethylxanthine, an inhibitor of cAMP phosphodiesterase restored ABCA1 cholesterol efflux in EPA-treated human macrophages. Moreover, the cyclooxygenase inhibition by indomethacin abolished the impact of EPA whereas the lipoxygenase inhibition by
EAS-0806. IMPACT OF ERYTHROCYTE TRAFFICKING ON EARLY STAGES OF ATHEROMA , V. Ollivier, L. Louedec, J. Laschet, L. R. Bayles*, S. Delbosc, B. Ho-Tin-Noe Gouya, A. Nicoletti, J.B. Michel. U1148, INSERM, Paris Cedex 18, France
* Corresponding author. Background: Red Blood Cells (RBC) are a major source of cholesterol (cell membrane), and of pro-oxidant iron-loaded heme. This has been shown to be clinically important in intraplaque haemorrhage. We hypothesise that RBC trafficking participates in the early stages of atheroma. Methods: To further explore the role of RBC we have used a descriptive approach, examining human tissue, vascular smooth muscle cell (VSMC) biology and experimental animal models. Human aortic tissues (healthy, fatty streak or fibrolipidic plaques) were collected from deceased organ donors. In vitro experiments demonstrating erythrophagocytosis were performed using primary VSMC. Atheroma development was investigated in Lewis rats under various conditions. Results: In humans, Perl’s/DAB precipitation highlighted the presence of iron in 67% of lesions. Frequency was significantly different between groups, with low frequency of iron detection in normal aorta (24%) as compared to fatty streaks (73%), and fibrolipidic plaques (90%). Iron tissue concentration and expression of iron transporters were significantly increased in early atherosclerotic lesions. In vitro experiments showed that sRBC (not fresh RBC) were engulfed by VSMC and this was associated with a significant increase in VSMC ROS production, and in the expression of metabolically relevant genes/ proteins. In rats, iron was detectable by histochemistry in all aortas from rats which received sRBC injections and were submitted to balloon injury. Analysis of iron concentration in the aortic wall showed a significant effect of sRBC injections, ballooning and splenectomy. Conclusion: Our preliminary results suggest that RBC phagocytosis may play an important role in early stages of atheroma. EAS-0816. MITOCHONDRIAL DYSFUNCTION IN CAROTID PLAQUE ATHEROSCLEROSIS A PREDICTOR OF PLAQUE VULNERABILITY J.R. Mercer*. University of Glasgow, Institute of Cardiovascular and Medical Sciences, Glasgow, United Kingdom
* Corresponding author. Introduction: DNA damage is a common finding in atherosclerotic plaques but the full consequences to disease aetiology remains incomplete. Previously we have shown excess DNA damage and/or reduced repair combines to degrade the energetic capacity of mitochondria. This effect results in a decline in synthetic and replicative capacity of plaque cells and increased propensity for plaque rupture. In addition to its role as a mediator of cell death and calcium homeostasis, mitochondria provide the energetic capacity of the cell as an ATP generator. Recent work has assessed the function of the respiratory chain in isolated human atherosclerotic carotid plaque components and their contribution to the concept of vulnerable plaque. Method: Human carotid plaques were micro-dissected into cap, core, shoulder and wall. Using polargraphic oxygen respirometry we assessed respiratory chain capacity. In addition to using standard histological stains for smooth muscle actin (SMA) and macrophages (Mac-3) we determined the relative contribution of each cell type to the energetic capacity of the plaque. Result: A significant 80.4% reduction in plaque cap respiration compared to underlying vessel wall was observed. While plaque cap and wall was almost exclusively SMAþ (99.4þ/-1.9 and 99.4%þ/-0.5), cellular density was significantly reduced in plaque caps, showing a 60.9% decline in SMAþ cellularity. Conclusion: This data suggests that as SMAþ vascular cellularity declines in the mature cap there is a concurrent down regulation in mitochondrial capacity equating to a significant 20.1% decrease in respiratory chain
e79
efficiency. Together these data confirm mitochondrial dysfunction as a key marker of plaque vulnerability. EAS-0833. EPIGENETIC REGULATION OF GENE EXPRESSION IN VASCULAR SMOOTH MUSCLE CELLS J. Harman, R. Madsen, A. Dalby, M. Davies, H. Jørgensen*. Cardiovascular Medicine, University of Cambridge, Cambridge, United Kingdom
* Corresponding author. Vascular smooth muscle cell (VSMC) phenotypic switching is a hallmark of atherosclerosis, UK’s main cause of death. During phenotypic switching, the gene expression profile of VSMCs changes dramatically. The resulting loss of contractility and increased migration, proliferation and secretion of proinflammatory cytokines contribute directly to atherosclerotic lesion growth and vascular inflammation. We study how regulation of gene expression by epigenetic mechanisms impact on VSMC phenotypic switching. Using a stem cell model we identified methylation of Histone H3 Lysine 9 (H3K9) as a regulatory pathway of VSMC gene expression. Subsequent studies using ex vivo and cultured VSMCs isolated from mouse aorta demonstrated that contractile VSMC genes display increased levels of methylated H3K9 during in vitro induced phenotypic switching. We have mapped the levels of methylated H3K9 genome-wide and find that this repressive epigenetic mark is also found at the promoters of inflammation-associated genes, including a subset of matrix metallo proteases in VSMCs. Using a small molecule inhibitor of the G9a/GLP methyl transferases, we furthermore demonstrated that this pathway is functionally relevant for regulating gene expression in VSMC. Our studies demonstrate that H3K9 methylation represses the expression of disease-related genes in VSMCs and suggest that this pathway might function to prevent unsolicited vascular inflammation.
Monocytes/macrophages EAS-0108. EICOSAPENTAENOIC ACID (EPA) MEMBRANE INCORPORATION IMPAIRED ABCA1-DEPENDENT CHOLESTEROL EFFLUX THROUGH A PKA PATHWAY IN PRIMARY HUMAN MACROPHAGES N. Fournier 1, *, S. Tardivel 1, J.F. Benoist 2, B. Vedie 3, D. Rousseaue Paris-Sud 11, Ralliard 4, J.L. Paul 1. 1 EA 4529, UFR de Pharmacie Universit Chatenay-Malabry, France; 2 Laboratoire de Biochimie-Hormonologie, ^pital Robert Debr Ho e AP-HP, Paris, France; 3 Laboratoire de Biochimie, ^pital europ Ho een Georges Pompidou AP-HP, Paris, France; 4 UMR 1198, INRA, Jouy-en-Josas, France
* Corresponding author. Dietary polyunsaturated fatty acids (PUFA) affect the cellular phospholipids composition which may influence the function of membrane proteins. We investigated the impact of the incorporation of several PUFA on the ABCA1mediated cholesterol efflux, an anti-atherogenic process. The arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) supplementation decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, the membrane incorporation of eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70 mM of EPA in HMDM). The EPA addition did not alter the ABCA1 expression (mRNA or protein) in mouse or human macrophages. The EPAtreated HMDM exhibited strong phospholipids composition changes with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3) associated to a decreased level of AA. In human macrophages, cellular ATP was required for the effect of EPA. In addition, we observed that addition of forskolin, a direct activator of adenylate cyclase, and isobutylmethylxanthine, an inhibitor of cAMP phosphodiesterase restored ABCA1 cholesterol efflux in EPA-treated human macrophages. Moreover, the cyclooxygenase inhibition by indomethacin abolished the impact of EPA whereas the lipoxygenase inhibition by