Coronary artery disease related Mir-19A and Mir-26A are sensitive to simvastatin and ror-alpha ligands in macrophage cells

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H. Kubatova1_{, H.}_Cermakova2_{, I. Kralova Lesna}1_{, S.}_Cejkova1_{, J. Fronek}2_{, L.} Janousek2_{, R. Poledne}1_{, J. Pi}0

t ha1_.1_{Institute for Clinical and Experimental}

Medicine, Experimental Medicine Centre- Atherosclerosis Research Laboratory, Prague, Czech Republic;2_{Institute for Clinical and Experimental}

Medicine, Transplantation Centre- Transplantation Surgery Department, Prague, Czech Republic

Background and Aims: Characterization of plaque instability in vivo and detailed determination of macrophage phenotypes in endarterectomy specimens is challenging. We addressed this issue in a pilot study combining ultrasound evaluation of plaque morphology andﬂow cyto-metric analysis of endarterectomy specimens.

Methods: Patients indicated to carotid endarterectomy underwent detailed ultrasound scanning of the plaque before surgery. During a sub-sequent analysis, the gray scale median (GSM) value and the expected annual risk of stroke were estimated. Endarterectomy specimens were further processed for ﬂow cytometric analysis. Different macrophage phenotypes were characterized using CD14, CD16 and CD206 markers. The amount of different macrophage phenotypes in plaques was measured and compared to gray scale median value and other clinical parameters. Results: Twelve consecutive patients were involved in the pilot study. Of those, ten patients were asymptomatic and eleven were on statins prior to hospitalization. The value of GSM ranged from 12 to 97. A signiﬁcant negative correlation was found between the GSM value and the percentage of CD14+CD16+CD206- cells. A similar negative trend was observed for CD14+CD16+CD206+ cells.

Conclusions: Aflow cytometric protocol for endarterectomy specimens was developed. The GSM value seems to be connected to the percentage of different macrophage phenotypes. Therefore, the combination of ultra-sound evaluation andflow cytometric analysis could further refine the definition of the unstable carotid artery plaque.

Posters 26 - 29 May, 2019

02. Inﬂammation, immunity and macrophages - 02.02 Macrophages in lipid metabolism and atherosclerosis

EAS19-0369.

EICOSAPENTAENOIC ACID (EPA) DIFFERENTIALLY AFFECTS ABCA1-MEDIATED CHOLESTEROL EFFLUX FROM PRIMARY HUMAN MACROPHAGES OR THP-1 CELL LINE

N. Fournier1,2_{, H. Dakroub}1_{, J.F. Benoist}3_{, F. Allaoui}1_{, G. Sayet}4_{, J.L.} Paul1,2_.1_{Faculte de Pharmacie- Universite Paris Sud - Paris Saclay, LipSys2}

EA 7357- equipe "macrophages- cholesterol and atherosclerosis", Chatenay-Malabry, France;2_{Hopital Europeen Georges Pompidou, AP-HP, Laboratoire}

de Biochimie, Paris, France;3_{Hopital Robert Debre, AP-HP, Laboratoire de}

Biochimie Hormonologie, Paris, France;4_{Faculte de Pharmacie- Universite}

Paris Sud - Paris Saclay, LipSys2 EA 7357, equipe "chimie analytique pharmaceutique", Chatenay-Malabry, France

Background and Aims: A diet rich in n-3 polyunsaturated fatty acids (PUFAs), especially in eicosapentaenoic acid (EPA, C20:5 n-3) is car-dioprotective. PUFAs modulate cellular eicosanoids formed from arach-idonic acid (AA, C20:4 n-6) and EPA. We investigated the impact of EPA supplementation on ABCA1-mediated cholesterol efﬂux, an anti-atherogenic pathway.

Methods: Human monocyte-derived macrophages (HMDM) or human monocytic leukemia THP-1 cells differentiated into macrophages with PMA were incubated for 34 h without (control cells) or with 70

m

M EPA. After an overnight treatment with LXR/RXR agonists, the isotopic choles-terol efﬂux promoted by lipid-free apolipoprotein AI was determined after 4 h of incubation with macrophages.

Results: Using HMDM, EPA reduced ABCA1 functionality by 28% through a PKA-dependent mechanism. The membrane incorporation of EPA (24.5% of total FA) induced an important decrease in AA (6.6% and 15.5% for EPA-cells and control cells, respectively). The inhibition of cyclooxygenases (COX) activity abolished the impact of EPA. Using THP-1 macrophages, EPA induced a slight and not significant increase in ABCA1-mediated choles-terol efflux (+12%). The modifications of the FA profiles of membrane

phospholipids are currently under investigation (GC-MS). We previously observed that control THP-1 cells display much lower AA content (1.3% of total FA) than control HMDM.

Conclusions: In conclusion, the in vitro EPA membrane incorporation does not have the same consequences on the ABCA1-mediated cholesterol efﬂux from HMDM or THP-1 macrophages, highlighting the importance of considering the cellular model used for such studies. We hypothesize that changes in cellular eicosanoid production through COX activity are responsible for the impact of EPA in HMDM.

EAS19-0646.

NADPH OXIDASE e DERIVED REACTIVE OXYGEN SPECIES AUGMENT INFLAMMATORY MACROPHAGE RESPONSES VIA REDOX-SENSITIVE HISTONE DEACETYLASE-DEPENDENT EPIGENETIC MECHANISMS IN EXPERIMENTAL ATHEROSCLEROSIS

M. Vlad1, S.A. Manea1, M. Raicu1, H. Muresian2, M. Simionescu1, A. Manea1_.1_{Institute of Cellular Biology and Pathology 'Nicolae Simionescu',}

Molecular and Cellular Pharmacology, Bucharest, Romania; 2_University

Hospital of Bucharest, Cardiovascular Surgery Department, Bucharest, Romania

Background and Aims: Oxidative stress microenvironment shapes the phenotype of monocyte (Mon)-derived macrophage (Mac) in atheroscle-rosis. Histone acetylation-based epigenetic mechanisms have been implicated in Mac polarization. Thus far, the role of redox signaling in the regulation of inﬂammatory Mac (M1) functions remains elusive. The aim of this study was to explore the potential role of NADPH oxidase (Nox)-derived reactive oxygen species (ROS) and histone acetylation-related enzymes crosstalk in mediating Mac pro-inﬂammatory effects in experi-mental atherosclerosis.

Methods: Human non-atherosclerotic (superior thyroid artery) and atherosclerotic (carotid artery) tissues, and ApoE-/- mice maintained on normal/high-fat cholesterol rich diet were used. In vitro studies were done on resting and polarized (M1/M2) human THP-1 Mac and freshly isolated mouse Mon-derived Mac. Immunohistochemistry (IHC), real-time PCR and western blot techniques were employed.

Results: IHC staining revealed that HDAC class (I, II, IV) and Nox subtypes are colocalized with infiltrated Mac and lipid-rich deposits within human/ mouse atherosclerotic lesions. Pharmacological inhibition of histone deacetylase (HDAC) by SAHA decreased Nox expression and immune cell infiltration in atherosclerotic aorta of hypercholesterolemic ApoE-/- mice. Enhanced expression of HDAC (class I, II, IV) and Nox1-5 was detected in M1-Mac as compared to resting/anti-inflammatory (M2)-Mac. SAHA down-regulated the production of inflammatory cytokines/chemokines and nitric oxide in human/mouse M1-Mac. Exposure of Mac to Nox in-hibitor, GKT137831, significantly reduced the expression of HDAC isoforms and M1-produced inflammatory mediators.

Conclusions: These data indicate the existence of new redox-sensitive mechanisms whereby Nox-derived ROS regulate atherogenic Mac func-tions via HDAC-dependent/independent pathways.

Acknowledgements: Work supported by UEFISCDI (PN-III-P4-ID-PCE-2016-0665, PN-III-P1-1.1-TE-2016-0851, PN-III-P4-ID-PCCF-2016-0172). Posters 26 - 29 May, 2019

EAS19-0671.

CORONARY ARTERY DISEASE RELATED MIR-19A AND MIR-26A ARE SENSITIVE TO SIMVASTATIN AND ROR-ALPHA LIGANDS IN MACROPHAGE CELLS

N. Coban1, C. Gulec1, A.S. Ozuynuk1, N. Erginel-Unaltuna1, A.F. Erkan2_. 1_{Aziz Sancar Institute for Experimental Medicine- Istanbul}

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University, Department of Genetic, _Istanbul, Turkey;2_{Medical Faculty-Ufuk}

University, Department of Cardiology, Ankara, Turkey

Background and Aims: ROR-alpha is a member of ligand-activated nu-clear receptor superfamily of transcription factors. Melatonin and choles-terol are two known ligands of ROR-alpha, both of which were shown to be important for atherosclerosis. The aim of this study was to determine differentially expressed miRNAs in coronary artery disease (CAD) patients, and to test the dependence of their expression on ROR-alpha activity in THP-1 macrophage cells.

Methods: The Agilent’s microarray analyses were performed to compare plasma miRNA proﬁles of selected individuals with CAD (n¼4) and non-CAD (n¼3). Promo transcription factor binding site prediction tool was used to identify ROR-alpha response elements (ROREs) in promoter of selected miRNAs genes. Furthermore, THP-1 macrophage cells were treated with simvastatin and ROR-alpha speciﬁc ligands (CPG52608 and SR1001) and the expressions of differentially expressed miRNAs were analyzed.

Results: Microarray results indicate that miR-19a and miR-26a were signiﬁcantly up-regulated in CAD patients compared to non-CAD group (fold change>1.5, p<0.05). We identiﬁed ROREs within the promoter re-gions of miR-19a host gene MIR17HG and miR-26a host gene CTDSP2/ CTDSPL. In addition, we observed that simvastatin repressed the expres-sion of miR-19a and miR-26a, and this represexpres-sion was partially increased by SR1001 for miR-26a in macrophage cells. While repressor effect of simvastatin was more clear in miR-26a, its prevention by CPG52608 was observed to be more effective in miR-19a and miR-26a.

Conclusions: Our results suggest that miR-19a host gene MIR17HG and miR-26a host gene CTDSP2/CTDSPL are potential target genes of ROR-alpha. It seems also that ROR-alpha may control various genes indirectly through miRNA clusters.

EAS19-0693.

EZETIMIBE PROTECTS THP-1 CELLS FROM ISCHEMIA-REPERFUSION INJURY REDUCING OXIDATIVE STRESS AND UP-REGULATING NRF2/ ARE GENE EXPRESSION

D. Peserico, C. Stranieri, U. Garbin, A.M. Fratta Pasini. University of Verona, Department of Medicine- Section of Internal Medicine and Atherothrombotic and Degenerative Diseases, Verona, Italy

Background and Aims: We demonstrated that physical training, charac-terized by repeated ischemia-reperfusion (I-R) episodes (ischemic condi-tioning, IC), protects circulating cells from peripheral artery disease (PAD) patients against ischemic harms by reducing oxidative stress (OS) and by up-regulating nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxi-dant response element (ARE) pathway expression. Ezetimibe (Eze) has been shown to alleviate OS enhancing Nrf2 nuclear translocation in an AMPK/p62-dependent manner. In a cellular I-R and IC model, we aimed to investigate: 1) the effect of Eze on OS and Nrf2/ARE gene expression 2) whether Eze could have a synergistic effect on IC.

Methods: THP-1 cells were treated with or without Eze (50

m

M) overnight, then subjected to 1 or 6 repetitive I-R cycles using EVOS FL Auto Imaging System. Reactive oxygen species (ROS) formation was evaluated with DCF in cytoﬂuorimetry. Nrf2/ARE and p62 gene expression were evaluated by RT-PCR and western blotting.

Results: When THP-1 cells were exposed to 1 I-R cycle, the preincubation with Eze signiﬁcantly reduced ROS formation (p<0.01) and up-regulated Nrf2/ARE pathway expression and p62 phosphorylation (p<0.001). Mul-tiple I-R cycles, acting as IC, signiﬁcantly reduced ROS formation and up-regulated Nrf2/ARE gene expression (p<0.001); in these conditions, Eze preincubation was able not only to almost abolish ROS formation (p<0.01) but also further up-regulate Nrf2/ARE expression.

Conclusions: In our I-R model, Eze not only restores I-R-induced oxidative damages through Nrf2/ARE signaling up-regulation but also has a syner-gistic effect on IC. This new“pleiotropic” effect, if conﬁrmed in vivo, may strengthen the use of Eze in PAD patients.

EAS19-0741.

DEVELOPMENT OF TRANSGENIC MOUSE MODEL SUPERPRODUCER SEALIDASE WITH SECRETION INTO THE BLOOD

V. Kalmykov, K. Vinogradov, M. Zamkova. Institute of Biology Gene Russian Academy of Sciences, Centre of collective usage, Moscow, Russia

Background and Aims: Create a mouse model with inducible over-expression of neuraminidase (sialidase) in the blood. Neuraminidase (sialidase) is an enzyme that performs the desialylation of low density li-poproteins. Increased sialidase activity is observed in the blood of patients with atherosclerosis.

Methods: To obtain the superproducer of neuraminidase, we plan to use the Mus musculus Neu3 gene cDNA with a secretion signal from the cell, which will be inserted into the vector with a stop tape surrounded by loxP sites under the b-actin promoter. Genetic construction involves the random insertion of the transgene. The export sequence of the albumin export site, 1 to 18 amino acids of the N domain of albumin, will be used as a secretion signal to the blood. Tissue-speciﬁc lysozyme M promoter will provide Neu3 expression in macrophages / neutrophils after crossing recombinase-producing Cre mice with Neu3 mice with a stop tape sur-rounded by loxP sites. Primary transgenes will be obtained by microin-jection of the genetic construct into the pronucleus of the ovum. Results: After crossing the obtained line of transgenic animals with a transgenic line of mice expressing the Cre recombinase under the lysozyme M promoter, we obtain the line mouse superproducers of neuraminidase expressed in the blood.

Conclusions: Our approach will allow investigating the role of free siali-dase in the blood for the development of atherogenesis, including against the background of the Apoe mutation - -. This work was sapportid by grand RSF 17-75-20249

EAS19-0800.

A NOVEL HYPOTHESIS: ERYTHROCYTE SENESCENCE PLAYS A KEY ROLE IN THE ATHEROSCLEROSIS DEVELOPMENT

A. Deykin1, Y. Silaeva1, E. Leonova2.1Institute of Gene Biology Russian Academy of Sciences, Сentre of collective usage, Moscow, Russia; 2_St.

Petersburg State University, Institute of Translational Biomedicine, St. Petersburg, Russia

Background and Aims: Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by chronic inflammation, high blood pressure, oxidative stress, and progressive loss of cell and organ function with aging. We propose a hypothesis that the development of athero-sclerosis is based on oxidative stress of erythrocytes and their senescence. Methods: Our hypothesis is based on several facts. At thefirst, it is known that cholesterol content is increased in membrane of senescent erythro-cytes. The second, senescent erythrocytes lose their plasticity and ability of deformation, which affects the rheological blood properties, that can injure the vessel wall. The third, macrophages are involved in all stages of atherogenesis. They can undergo polarization by shifting between M1 and M2 functional phenotypes.

Results: It is known that efferocytosis, or ingestion of apoptotic cells, is stimulated by M2 macrophage polarization and macrophage polarization toward the pro-inﬂammatory M1 macrophage is a major promoter to atheroma formation. It is known that efferocytosis, or ingestion of apoptotic cells, is stimulated by M2 macrophage polarization. A failure of efferocytosis leads to a prolongation of chronic pathology in tissue. In addition, fat-laden macrophages contribute to plague progression by transforming into foam cells in response to excess lipid deposition in arteries. We postulate that

Abstracts / Atherosclerosis 287 (2019) e123ee288 e240