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Arterial pericyte-like cells as the second line of defense
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Abstracts / Atherosclerosis 263 (2017) e111ee282
are unclear. The authors investigated the role played by SIRT1 in TLR2mediated monocyte adhesion to vascular endothelium; a key early event during the progression of atherosclerosis. Methods: SIRT1 TG and wild-type mice were purchased from Jackson Laboratories. Results: Atherosclerotic lesion formation in aortic sinuses was significantly enhanced in wild type (WT) mice fed with high fat diet (HFD) for 12 weeks, but was significantly inhibited in SIRT1 transgenic (TG) mice. En face immunohistochemistry of endothelial surfaces revealed a marked increase in monocyte adhesion to aortic endothelium in Pam3CSK4treated WT mice, and this was significantly attenuated in SIRT1 TG mice. Likewise, the adhesion capacity of primary monocytes isolated from Pam3CSK4-treated WT mice was higher than that of monocytes from SIRT1 TG mice. In in vitro study, Pam3CSK4 increased monocyte adhesion to endothelial cells and enhanced the expression of Mac-1 (an adhesion molecule). However, these effects of Pam3CSK4 were attenuated by recombinant SIRT1 and by the overexpression of SIRT1 in monocytes. In addition, Pam3CSK4 suppression of SIRT1 expression in monocytes was found to be regulated by the transcription factors NF-kB and CREB. The roles of NF-kB and CREB in this inhibition of SIRT1 were confirmed by the siRNA knockdowns of NF-kB and CREB. Conclusions: SIRT1 suppresses atherosclerosis progression induced by TLR2-mediated monocyte adhesion to endothelium through Mac-1 expression in monocytes. These findings suggest therapeutic interventions should be developed to promote SIRT1 expression in atherosclerosis.
PO011. RAET1E POLYMORPHISMS ARE ASSOCIATED WITH INCREASED RISK OF DEVELOPING CORONARY ARTERY DISEASE AND WITH SOME CARDIOMETABOLIC PARAMETERS. THE GENETICS OF ATHEROSCLEROTIC DISEASE (GEA) MEXICAN STUDY nchez2, Nonanzit Pe rez n1, Rosalinda Posadas-Sa Gilberto Vargas-Alarco Manuel Rodríguez-Pe rez1, Jose Manuel Fragoso1, Teresa ndez1, Jose Herna Villarreal-Molina3, Carlos Posadas-Romero2. 1 Department of Molecular vez, Mexico, Mexico; Biology, Instituto Nacional de Cardiología Ignacio Cha 2 Department of Endocrinology, Instituto Nacional de Cardiología Ignacio vez, Mexico, Mexico; 3 Cardiovascular Genomics Laboratory, Instituto Cha mica (INMEGEN), Mexico, Mexico Nacional de Medicina Geno Aim: Considering that Raet1e has been describes as a novel atherosclerosis gene, the objective of the present study was to establish whether the polymorphisms of this gene are associated with premature coronary artery disease (pCAD) and/or other cardiovascular risk factors. Methods: Using an informatics analysis, five Raet1e gene polymorphisms with possible functional effect (rs6925151, rs9371533, rs7756850, rs2151910 and rs9383921) were selected. The polymorphisms were genotyped in 1158 patients with pCAD and 1104 controls (calcium score¼0 assessed by computed tomography). Clinical, anthropometric and biochemical measurements were performed. The associations were evaluated using logistic regression analyses. Results: Under different models adjusted by age, gender, body mass index, current smoking, and type 2 diabetes mellitus, the rs6925151 (OR ¼ 1.25, Phet ¼ 0.026; OR ¼ 1.268, Pcod1 ¼ 0.034), rs9371533 (OR ¼ 1.255, Phet ¼ 0.024), rs7756850 (OR ¼ 1.274, Phet ¼ 0.016; OR ¼ 1.294, Pcod1 ¼ 0.031) and rs9383921 (OR ¼ 1.232, Phet ¼ 0.037) polymorphisms were associated with increased risk of developing pCAD. In pCAD patients some polymorphisms were associated with hypertension, hyperinsulinemia, insulin resistance, total cholesterol > 200 mg/dL, cholesterol no-HDL >/- 160 mg/ dL, gamma-glutamyl- transferasa, and low levels of adiponectin, whereas in controls, some polymorphisms were associated with total abdominal fat, subcutaneous abdominal fat, hipo-alpha-lipoproteinemia, hypertriglyceridemia, LDL pattern B and high levels of aspartate aminotransferase. Conclusions: For the first time, our study shows that the Raet1e polymorphisms are associated with increased risk of developing pCAD and with some clinical and metabolic parameters.
PO012. INTRACELLULAR TRAFFIC OF LDL ASSOCIATES Alexander Orekhov1, Alexandra Melnichenko2. 1 Institute of Generel Pathology and Pathophysiology, Moscow, Russia; 2 Institute for Atherosclerosis Research, Moscow, Russia Aim: Cholesterol accumulation in macrophages is the earliest manifestation of atherosclerosis at the arterial cell level. Cholesterol accumulation causes by associated LDL but not non-associated LDL. This may be due to the differences in intracellular metabolism. The present study characterizes intracellular traffic of LDL in cultured human monocytes-derived macrophages. Methods: LDL was fluorescently labeled by incubation with DiO or DiI. Association of LDL was induced by incubation at 37 C with constant stirring. Macrophages in primary cultures were incubated with 50 mg/mL LDL overnight followed by confocal microscopy. Results: Dramatic difference between intracellular distribution of associated and non-associated LDL was found. Non-associated LDL was evenly distributed in cell cytoplasm. Associated LDL was distributed as large homogenous conglomerates of vesicle-like form with intensive fluorescence. We used staining early endosome antigen 1 (EEA-1) and lysosomal associated membrane protein (LAMP-1) for identification of these vesicle-like structures. There was partial co-localization of associated LDL with both LAMP-1 and EEA-1. On the other hand, non-associated LDL was not colocalized with endosomal or lysosomal cell compartments. Different types of activated human macrophages were also used. Similarly to non-activated macrophages both M1 and M2 macrophages accumulated LDL associates but not non-associated LDL in endosomal and lysosomal cell compartments. Conclusions: Different pattern of intracellular distribution of associated and non-associated LDL suggests different pathways of intracellular lipoprotein traffic. We suppose that this may explain the mechanism of cholesterol accumulation caused by associated LDL. This work was supported by the Russian Foundation for Basic Research (Grant # 15-0409279).
PO013. ARTERIAL PERICYTE-LIKE CELLS AS THE SECOND LINE OF DEFENSE Alexander Orekhov1, Nikita Nikiforov2. 1 Institute of General Pathology and Pathophysiology, Moscow, Russia; 2 Institute for Atherosclerosis Research, Moscow, Russia Aim: We found a three-dimensional network formed by stellate cells recognized as pericytes by specific antibodies. Methods: In the present study we have investigated the involvement of pericytes in atherogenesis in human aortic intima. Results: Intimal pericyte-like cells expressed CD68, which is considered a macrophage-specific antigen. The majority of lipid-laden and foam cells were of pericyte origin. In primary culture, intimal pericytes exhibited high phagocytic activity. Intimal pericyte-like cells released proinflammatory cytokines and chemokines, inducing recruitment of inflammatory cells. Lipid deposition in the intima correlated with the expression of MHC class II molecule HLA-DR, which is a marker of antigen presenting cells. Immunofluorescent analysis of HLA-DR expression in the aortic intima revealed a large population of HLA-DR-positive pericyteelike cells. Some of these cells also contained apo-B lipoprotein in the perinuclear space. We incubated the primary culture of subendothelial human aortic cells with multiple modified LDL obtained from the blood of atherosclerotic patients and demonstrated that it induced intracellular cholesterol accumulation. In parallel, expression of proinflammatory molecules and HLA-DR was increased. Conclusions: Thus, pluripotent intimal pericytes possessed phagocytotic activity like macrophages and were capable of recruiting inflammatory cellular elements like immune-competent cells and expressing HLA-DR similarly to antigen presenting cells. Therefore, in addition to endothelium
Abstracts / Atherosclerosis 263 (2017) e111ee282
as the first line of defense, intimal pericytes forming three-dimensional network beneath endothelial lining may be regarded as the second line of defense assisting professional immune cells like macrophages and dendritic cells. The work was supported by Ministry of Education and Sciences, Russia (Grant # 14.W02.16.6995-Scientific_School).
PO014. DIAGNOSTIC VALUE OF MODIFIED IMMUNOGLOBULIN G DETERMINATION IN PATIENTS WITH CORONARY ATHEROSCLEROSIS Maryana Shogenova, Radima Zhetisheva, Aleksandr Karpov, Eugene Efremov, Valeriy Masenko, Vladimir Naumov. Russian Cardiology Research Center, Moscow, Russia Aim: To determine the diagnostic value of methylglyoxal-modified immunoglobulin G (MG-IgG) and malondialdehyde-modified immunoglobulin G (MDA-IgG) in the atherosclerotic process in patients (pts) with coronary atherosclerosis compared to methylglyoxal-modified low density lipoproteins (MG-LDL) and malondialdehyde-modified low density lipoproteins (MDA-LDL). Methods: Three groups of male pts aged from 28 to 68 years were included in study: group with angiographically documented severe coronary stenosis (> 50%, n ¼ 50), a group with initial coronary arteries' atherosclerotic lesions (<50%, n ¼ 20) and a healthy pts group without coronary heart disease (n ¼ 10). Serum MDA-LDL, MG-LDL, MDA-IgG, MG-IgG were identified by immune-enzyme analysis using monoclonal antibodies, results were presented in relative units (R) as a sample signal (A450) to cutoff value ratio. Results: There were not significant differences in MDA-LDL [p¼0,28] and MG-LDL [p¼0,08] levels between these groups of pts, but MDA-IgG [p¼0,01] and MG-IgG [p¼0,02] levels in healthy pts group were significantly higher compared to the pts with severe coronary stenosis. Conclusions: We didn't found significant evidence of association between MDA-LDL and MG-LDL levels and coronary atherosclerosis in our clinical study. However, results of the modified IgG determination have been more informative. MDA-IgG and MG-IgG levels in healthy pts group were higher than in pts with severe coronary atherosclerosis. Which indicates that contribution of modified IgG in atherogenesis and in cardiovascular risk requires further investigation.
PO016. INVOLVEMENT OF CHRONIC HERPESVIRAL INFECTION IN INTRAVASKULAR INFLAMMATORY RESPONSE AND DESTABILISATION OF CURRENT CAD Kvantaliani Tamar1, Akhvlediani Manana2. 1 Medical Centre Gidmedi Plus, Tbilisi, Georgia; 2 Research Institute of Clinical Medicine, Tbilisi, Georgia Aim: Among potential biomarkers eligible to identify patients at risk of coronary, carotid and peripheral plaque instability is reasonable to consider also chronic infections. Our previous studies confirmed that certain viral agents could contribute to chronic inflammatory response suggesting to be recognized as a potential pathogen of atherosclerosis (A). We attempted to investigate possible relationship between I-type Herpes Simplex Virus (HSV-I) infection and some intravascular risk factors precipitating dramatic changes in arterial wall. Methods: 185 CAD patients, seropositive (group I, n¼78) and seronegative (group II, n¼107) to HSV-I antibodies, 50 similarly infected patients without evidence of A (group III), underwent measurement of lipoprotein profile, lipid hydroperoxide (LPO), antiviral IgG antibodies (using ELISA), CRP, fibrinogen (F), parameters of coagulative metabolism. Results: Study revealed significantly high levels of inflammation markers, LPO activity and sizeable positive correlation between plasma LPO and antibody index in groups I and III, compared with data of seronegative patients. Correlation appeared substantial in cases of fourfold and more increase of IgG antibody levels, indicating recurrent stage of infection. Conclusions: Data confirm that HSV-I infection activates free-radical reactions, predisposing lipid oxidation despite alterations in cholesterol particles. HSV-I infection might cause combined toxic effect on vascular
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endothelium by direct impact of antiviral antibodies and modified lipids increasing proinflammatory and procoagulant activity expressed by infected endothelial cells. In cases of recurrent infection, especially in individuals with A vessel injury, it can precipitate development of oxidative stress and serve as additional triggering mechanism of further progression of A.
PO017. LDLR INFLUENCE ON CD4 T CELLS DIFFERENTIATION IN MICE AND IN FAMILIAL HYPERCHOLESTEROLEMIA PATIENTS Annalisa Moregola1, Fabrizia Bonacina1, David Coe4, Fabio Pellegatta2, Liliana Grigore2, Katia Garlaschelli2, Andrea Baragetti1, 2, Alerico Luigi Catapano1, 3, Federica Marelli-Berg4, Giuseppe Danilo Norata1, 2, 4. 1 Department of Pharmacological and Biomolecular Sciences, Milan, Italy; 2 Centro SISA per lo studio dell'aterosclerosi, Ospedale Bassini, Cinisello Balsamo, Italy; 3 IRCCS Multimedica, Milan, Italy; 4 William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University, London, United Kingdom Aim: Expansion of CD4 T cells of the effector memory phenotype is known to correlate with atherosclerosis and disease progression. Here we investigate whether key players linking systemic and cellular lipid metabolism, such as the LDL-R, affect the differentiation and functionality of CD4+ T cells. Methods: Phenotypical and functional characterization of CD4+ T cells by flow cytometry analysis. Results: Under resting condition the differentiation of CD4+ T cells into secondary lymphoid organs was similar in LDLR KO and WT mice,while after stimulation in vitro CD4+T cells of LDLR KO proliferated more rapidly as compared to WT(p<0.05). In an in vivo setting of immune cells response(skin allograft transplantation), the draining lymph nodes of the graft in the LDLR KO presented increased levels of TEM(p<0.05) and an increased expansion of Treg compared to WT(p<0.05). Similar results were obtained in heterozygote familiar hypercholesterolemia (FH) patients. FH patients presented with significantly increased levels of circulating TEM(p<0.01), an increased proliferation of PBMC in vitro after antigenic stimulation and significantly increased levels of circulating Treg(p<0.01) compared to age and sex matched controls. To investigate whether these observations could be the consequences of an impaired Treg functionality we performed a suppression assay: Treg from FH patients were less effective in suppressing CD4 proliferation. Conclusions: All together our data suggest a novel connection between LDL receptors and immune response, showing that LDLR deficiency in CD4+ T cells leads to an expansion of TEM as a consequence of dysfunctional Treg cells thus supporting the increased immune response observed during atherosclerosis.
PO018. INDIVIDUAL DIFFERENCE IN ACTIVATION OF HUMAN MONOCYTES Alexander Orekhov1, Nikita Nikiforov2. 1 Institute of Generel Pathology and Pathophysiology, Moscow, Russia; 2 Institute for Atherosclerosis Research, Moscow, Russia Aim: The recruitment of monocytes occurs from the very earlier stage of the development of atherosclerosis and involves attachment of monocytes to endothelial cells, followed by the migration of monocytes into the subendothelial layer of the arterial wall and their differentiation of into macrophages. In this study, we have evaluated the susceptibility to M1 and M2 activation of monocytes circulating in the blood of healthy individuals and patients with asymptomatic carotid atherosclerosis. Methods: Monocyte activation included the measurement of concentrations of cytokines produced by cells in response to pro-inflammatory stimulation with interferon-gamma in a concentration of 100 ng/ml or anti-inflammatory stimulation with interleukin-4 at 10 ng/ml. Secretion of TNF-alpha was considered to be a marker of pro-inflammatory activity of macrophages, while secretion of CCL18 chemokine as a marker of antiinflammatory activity. Concentrations of TNF-alpha and CCL18 in the
Abstracts / Atherosclerosis 263 (2017) e111ee282
are unclear. The authors investigated the role played by SIRT1 in TLR2mediated monocyte adhesion to vascular endothelium; a key early event during the progression of atherosclerosis. Methods: SIRT1 TG and wild-type mice were purchased from Jackson Laboratories. Results: Atherosclerotic lesion formation in aortic sinuses was significantly enhanced in wild type (WT) mice fed with high fat diet (HFD) for 12 weeks, but was significantly inhibited in SIRT1 transgenic (TG) mice. En face immunohistochemistry of endothelial surfaces revealed a marked increase in monocyte adhesion to aortic endothelium in Pam3CSK4treated WT mice, and this was significantly attenuated in SIRT1 TG mice. Likewise, the adhesion capacity of primary monocytes isolated from Pam3CSK4-treated WT mice was higher than that of monocytes from SIRT1 TG mice. In in vitro study, Pam3CSK4 increased monocyte adhesion to endothelial cells and enhanced the expression of Mac-1 (an adhesion molecule). However, these effects of Pam3CSK4 were attenuated by recombinant SIRT1 and by the overexpression of SIRT1 in monocytes. In addition, Pam3CSK4 suppression of SIRT1 expression in monocytes was found to be regulated by the transcription factors NF-kB and CREB. The roles of NF-kB and CREB in this inhibition of SIRT1 were confirmed by the siRNA knockdowns of NF-kB and CREB. Conclusions: SIRT1 suppresses atherosclerosis progression induced by TLR2-mediated monocyte adhesion to endothelium through Mac-1 expression in monocytes. These findings suggest therapeutic interventions should be developed to promote SIRT1 expression in atherosclerosis.
PO011. RAET1E POLYMORPHISMS ARE ASSOCIATED WITH INCREASED RISK OF DEVELOPING CORONARY ARTERY DISEASE AND WITH SOME CARDIOMETABOLIC PARAMETERS. THE GENETICS OF ATHEROSCLEROTIC DISEASE (GEA) MEXICAN STUDY nchez2, Nonanzit Pe rez n1, Rosalinda Posadas-Sa Gilberto Vargas-Alarco Manuel Rodríguez-Pe rez1, Jose Manuel Fragoso1, Teresa ndez1, Jose Herna Villarreal-Molina3, Carlos Posadas-Romero2. 1 Department of Molecular vez, Mexico, Mexico; Biology, Instituto Nacional de Cardiología Ignacio Cha 2 Department of Endocrinology, Instituto Nacional de Cardiología Ignacio vez, Mexico, Mexico; 3 Cardiovascular Genomics Laboratory, Instituto Cha mica (INMEGEN), Mexico, Mexico Nacional de Medicina Geno Aim: Considering that Raet1e has been describes as a novel atherosclerosis gene, the objective of the present study was to establish whether the polymorphisms of this gene are associated with premature coronary artery disease (pCAD) and/or other cardiovascular risk factors. Methods: Using an informatics analysis, five Raet1e gene polymorphisms with possible functional effect (rs6925151, rs9371533, rs7756850, rs2151910 and rs9383921) were selected. The polymorphisms were genotyped in 1158 patients with pCAD and 1104 controls (calcium score¼0 assessed by computed tomography). Clinical, anthropometric and biochemical measurements were performed. The associations were evaluated using logistic regression analyses. Results: Under different models adjusted by age, gender, body mass index, current smoking, and type 2 diabetes mellitus, the rs6925151 (OR ¼ 1.25, Phet ¼ 0.026; OR ¼ 1.268, Pcod1 ¼ 0.034), rs9371533 (OR ¼ 1.255, Phet ¼ 0.024), rs7756850 (OR ¼ 1.274, Phet ¼ 0.016; OR ¼ 1.294, Pcod1 ¼ 0.031) and rs9383921 (OR ¼ 1.232, Phet ¼ 0.037) polymorphisms were associated with increased risk of developing pCAD. In pCAD patients some polymorphisms were associated with hypertension, hyperinsulinemia, insulin resistance, total cholesterol > 200 mg/dL, cholesterol no-HDL >/- 160 mg/ dL, gamma-glutamyl- transferasa, and low levels of adiponectin, whereas in controls, some polymorphisms were associated with total abdominal fat, subcutaneous abdominal fat, hipo-alpha-lipoproteinemia, hypertriglyceridemia, LDL pattern B and high levels of aspartate aminotransferase. Conclusions: For the first time, our study shows that the Raet1e polymorphisms are associated with increased risk of developing pCAD and with some clinical and metabolic parameters.
PO012. INTRACELLULAR TRAFFIC OF LDL ASSOCIATES Alexander Orekhov1, Alexandra Melnichenko2. 1 Institute of Generel Pathology and Pathophysiology, Moscow, Russia; 2 Institute for Atherosclerosis Research, Moscow, Russia Aim: Cholesterol accumulation in macrophages is the earliest manifestation of atherosclerosis at the arterial cell level. Cholesterol accumulation causes by associated LDL but not non-associated LDL. This may be due to the differences in intracellular metabolism. The present study characterizes intracellular traffic of LDL in cultured human monocytes-derived macrophages. Methods: LDL was fluorescently labeled by incubation with DiO or DiI. Association of LDL was induced by incubation at 37 C with constant stirring. Macrophages in primary cultures were incubated with 50 mg/mL LDL overnight followed by confocal microscopy. Results: Dramatic difference between intracellular distribution of associated and non-associated LDL was found. Non-associated LDL was evenly distributed in cell cytoplasm. Associated LDL was distributed as large homogenous conglomerates of vesicle-like form with intensive fluorescence. We used staining early endosome antigen 1 (EEA-1) and lysosomal associated membrane protein (LAMP-1) for identification of these vesicle-like structures. There was partial co-localization of associated LDL with both LAMP-1 and EEA-1. On the other hand, non-associated LDL was not colocalized with endosomal or lysosomal cell compartments. Different types of activated human macrophages were also used. Similarly to non-activated macrophages both M1 and M2 macrophages accumulated LDL associates but not non-associated LDL in endosomal and lysosomal cell compartments. Conclusions: Different pattern of intracellular distribution of associated and non-associated LDL suggests different pathways of intracellular lipoprotein traffic. We suppose that this may explain the mechanism of cholesterol accumulation caused by associated LDL. This work was supported by the Russian Foundation for Basic Research (Grant # 15-0409279).
PO013. ARTERIAL PERICYTE-LIKE CELLS AS THE SECOND LINE OF DEFENSE Alexander Orekhov1, Nikita Nikiforov2. 1 Institute of General Pathology and Pathophysiology, Moscow, Russia; 2 Institute for Atherosclerosis Research, Moscow, Russia Aim: We found a three-dimensional network formed by stellate cells recognized as pericytes by specific antibodies. Methods: In the present study we have investigated the involvement of pericytes in atherogenesis in human aortic intima. Results: Intimal pericyte-like cells expressed CD68, which is considered a macrophage-specific antigen. The majority of lipid-laden and foam cells were of pericyte origin. In primary culture, intimal pericytes exhibited high phagocytic activity. Intimal pericyte-like cells released proinflammatory cytokines and chemokines, inducing recruitment of inflammatory cells. Lipid deposition in the intima correlated with the expression of MHC class II molecule HLA-DR, which is a marker of antigen presenting cells. Immunofluorescent analysis of HLA-DR expression in the aortic intima revealed a large population of HLA-DR-positive pericyteelike cells. Some of these cells also contained apo-B lipoprotein in the perinuclear space. We incubated the primary culture of subendothelial human aortic cells with multiple modified LDL obtained from the blood of atherosclerotic patients and demonstrated that it induced intracellular cholesterol accumulation. In parallel, expression of proinflammatory molecules and HLA-DR was increased. Conclusions: Thus, pluripotent intimal pericytes possessed phagocytotic activity like macrophages and were capable of recruiting inflammatory cellular elements like immune-competent cells and expressing HLA-DR similarly to antigen presenting cells. Therefore, in addition to endothelium
Abstracts / Atherosclerosis 263 (2017) e111ee282
as the first line of defense, intimal pericytes forming three-dimensional network beneath endothelial lining may be regarded as the second line of defense assisting professional immune cells like macrophages and dendritic cells. The work was supported by Ministry of Education and Sciences, Russia (Grant # 14.W02.16.6995-Scientific_School).
PO014. DIAGNOSTIC VALUE OF MODIFIED IMMUNOGLOBULIN G DETERMINATION IN PATIENTS WITH CORONARY ATHEROSCLEROSIS Maryana Shogenova, Radima Zhetisheva, Aleksandr Karpov, Eugene Efremov, Valeriy Masenko, Vladimir Naumov. Russian Cardiology Research Center, Moscow, Russia Aim: To determine the diagnostic value of methylglyoxal-modified immunoglobulin G (MG-IgG) and malondialdehyde-modified immunoglobulin G (MDA-IgG) in the atherosclerotic process in patients (pts) with coronary atherosclerosis compared to methylglyoxal-modified low density lipoproteins (MG-LDL) and malondialdehyde-modified low density lipoproteins (MDA-LDL). Methods: Three groups of male pts aged from 28 to 68 years were included in study: group with angiographically documented severe coronary stenosis (> 50%, n ¼ 50), a group with initial coronary arteries' atherosclerotic lesions (<50%, n ¼ 20) and a healthy pts group without coronary heart disease (n ¼ 10). Serum MDA-LDL, MG-LDL, MDA-IgG, MG-IgG were identified by immune-enzyme analysis using monoclonal antibodies, results were presented in relative units (R) as a sample signal (A450) to cutoff value ratio. Results: There were not significant differences in MDA-LDL [p¼0,28] and MG-LDL [p¼0,08] levels between these groups of pts, but MDA-IgG [p¼0,01] and MG-IgG [p¼0,02] levels in healthy pts group were significantly higher compared to the pts with severe coronary stenosis. Conclusions: We didn't found significant evidence of association between MDA-LDL and MG-LDL levels and coronary atherosclerosis in our clinical study. However, results of the modified IgG determination have been more informative. MDA-IgG and MG-IgG levels in healthy pts group were higher than in pts with severe coronary atherosclerosis. Which indicates that contribution of modified IgG in atherogenesis and in cardiovascular risk requires further investigation.
PO016. INVOLVEMENT OF CHRONIC HERPESVIRAL INFECTION IN INTRAVASKULAR INFLAMMATORY RESPONSE AND DESTABILISATION OF CURRENT CAD Kvantaliani Tamar1, Akhvlediani Manana2. 1 Medical Centre Gidmedi Plus, Tbilisi, Georgia; 2 Research Institute of Clinical Medicine, Tbilisi, Georgia Aim: Among potential biomarkers eligible to identify patients at risk of coronary, carotid and peripheral plaque instability is reasonable to consider also chronic infections. Our previous studies confirmed that certain viral agents could contribute to chronic inflammatory response suggesting to be recognized as a potential pathogen of atherosclerosis (A). We attempted to investigate possible relationship between I-type Herpes Simplex Virus (HSV-I) infection and some intravascular risk factors precipitating dramatic changes in arterial wall. Methods: 185 CAD patients, seropositive (group I, n¼78) and seronegative (group II, n¼107) to HSV-I antibodies, 50 similarly infected patients without evidence of A (group III), underwent measurement of lipoprotein profile, lipid hydroperoxide (LPO), antiviral IgG antibodies (using ELISA), CRP, fibrinogen (F), parameters of coagulative metabolism. Results: Study revealed significantly high levels of inflammation markers, LPO activity and sizeable positive correlation between plasma LPO and antibody index in groups I and III, compared with data of seronegative patients. Correlation appeared substantial in cases of fourfold and more increase of IgG antibody levels, indicating recurrent stage of infection. Conclusions: Data confirm that HSV-I infection activates free-radical reactions, predisposing lipid oxidation despite alterations in cholesterol particles. HSV-I infection might cause combined toxic effect on vascular
e115
endothelium by direct impact of antiviral antibodies and modified lipids increasing proinflammatory and procoagulant activity expressed by infected endothelial cells. In cases of recurrent infection, especially in individuals with A vessel injury, it can precipitate development of oxidative stress and serve as additional triggering mechanism of further progression of A.
PO017. LDLR INFLUENCE ON CD4 T CELLS DIFFERENTIATION IN MICE AND IN FAMILIAL HYPERCHOLESTEROLEMIA PATIENTS Annalisa Moregola1, Fabrizia Bonacina1, David Coe4, Fabio Pellegatta2, Liliana Grigore2, Katia Garlaschelli2, Andrea Baragetti1, 2, Alerico Luigi Catapano1, 3, Federica Marelli-Berg4, Giuseppe Danilo Norata1, 2, 4. 1 Department of Pharmacological and Biomolecular Sciences, Milan, Italy; 2 Centro SISA per lo studio dell'aterosclerosi, Ospedale Bassini, Cinisello Balsamo, Italy; 3 IRCCS Multimedica, Milan, Italy; 4 William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University, London, United Kingdom Aim: Expansion of CD4 T cells of the effector memory phenotype is known to correlate with atherosclerosis and disease progression. Here we investigate whether key players linking systemic and cellular lipid metabolism, such as the LDL-R, affect the differentiation and functionality of CD4+ T cells. Methods: Phenotypical and functional characterization of CD4+ T cells by flow cytometry analysis. Results: Under resting condition the differentiation of CD4+ T cells into secondary lymphoid organs was similar in LDLR KO and WT mice,while after stimulation in vitro CD4+T cells of LDLR KO proliferated more rapidly as compared to WT(p<0.05). In an in vivo setting of immune cells response(skin allograft transplantation), the draining lymph nodes of the graft in the LDLR KO presented increased levels of TEM(p<0.05) and an increased expansion of Treg compared to WT(p<0.05). Similar results were obtained in heterozygote familiar hypercholesterolemia (FH) patients. FH patients presented with significantly increased levels of circulating TEM(p<0.01), an increased proliferation of PBMC in vitro after antigenic stimulation and significantly increased levels of circulating Treg(p<0.01) compared to age and sex matched controls. To investigate whether these observations could be the consequences of an impaired Treg functionality we performed a suppression assay: Treg from FH patients were less effective in suppressing CD4 proliferation. Conclusions: All together our data suggest a novel connection between LDL receptors and immune response, showing that LDLR deficiency in CD4+ T cells leads to an expansion of TEM as a consequence of dysfunctional Treg cells thus supporting the increased immune response observed during atherosclerosis.
PO018. INDIVIDUAL DIFFERENCE IN ACTIVATION OF HUMAN MONOCYTES Alexander Orekhov1, Nikita Nikiforov2. 1 Institute of Generel Pathology and Pathophysiology, Moscow, Russia; 2 Institute for Atherosclerosis Research, Moscow, Russia Aim: The recruitment of monocytes occurs from the very earlier stage of the development of atherosclerosis and involves attachment of monocytes to endothelial cells, followed by the migration of monocytes into the subendothelial layer of the arterial wall and their differentiation of into macrophages. In this study, we have evaluated the susceptibility to M1 and M2 activation of monocytes circulating in the blood of healthy individuals and patients with asymptomatic carotid atherosclerosis. Methods: Monocyte activation included the measurement of concentrations of cytokines produced by cells in response to pro-inflammatory stimulation with interferon-gamma in a concentration of 100 ng/ml or anti-inflammatory stimulation with interleukin-4 at 10 ng/ml. Secretion of TNF-alpha was considered to be a marker of pro-inflammatory activity of macrophages, while secretion of CCL18 chemokine as a marker of antiinflammatory activity. Concentrations of TNF-alpha and CCL18 in the