Carotid Endarterectomy Plaques Research Articles

Myeloid cannabinoid CB1 receptor deletion confers atheroprotection in male mice by reducing macrophage proliferation in a sex-dependent manner.

Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. To address this, we generated a transgenic mouse model to study the role of myeloid CB1 signaling in atherosclerosis. Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1 deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences in proliferation were dependent on estrogen receptor (ER)α-estradiol signaling. Kinase activity profiling identified a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further revealed chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism and inflammatory markers, suggesting a possible implication of CB1-dependent regulation in human pathophysiology. Impaired macrophage CB1 signaling is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming in male mice. The importance of macrophage CB1 signaling appears to be sex-dependent.

Circulating LIGHT/TNFSF14 levels are negatively associated with cardiovascular mortality, while intra-plaque levels are associated with symptomatic cerebrovascular disease

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): The Swedish Heart and Lung Foundation, Åke Wiberg's Foundation Background Plaque rupture is the underlying cause of most cardiovascular events, such as stroke and myocardial infarction. Co-stimulatory molecules, the immune checkpoints, are emerging as strong candidates to battle atherosclerosis progression. The co-stimulatory molecule LIGHT (tumour necrosis factor 14; TNFSF14) exists in foam cell-rich regions of atherosclerotic plaques, but whether it is associated with a high-risk plaque phenotype and cardiovascular events is not known. Purpose This study investigates the association between intra-plaque and circulating LIGHT levels and cardiovascular disease. Method Plaque levels of LIGHT/TNFSF14 were measured in plasma and homogenised carotid endarterectomy plaques by Proximity Extension Assay in subjects from the Carotid Plaque Imaging Cohort (CPIP; N=557 and 201, respectively). Homogenates were further examined by multi-plex cytokine analysis and ELISA, and tissue plaque sections through immunohistochemistry. Multivariate Cox proportional hazards analysis was performed on normalized (logarithmically transformed) values. Results Plaque, but not circulating, levels of LIGHT were associated with the occurrence of pre-operative cerebrovascular symptoms (p=0.00014). Furthermore, intra-plaque levels of LIGHT correlated strongly with the histological plaque vulnerability index (p=5x10-15): a ratio between the sum of destabilising versus stabilising plaque components (i.e. macrophages, intra-plaque haemorrhage and lipids versus smooth muscle cells and collagen) as well as with necrotic core size and the inflammatory cytokines interleukin(IL)-1β, IL-6, chemokine (C-C motif) ligand (CCL)-2, CCL4 and CCL5. Moreover, plaque LIGHT levels correlated with several components of the extracellular matrix turnover machinery, including cleaved collagen, fibromodulin, ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs-7) and matrix metalloproteinases 1, 2, 9 and 10. While plaque LIGHT levels were not predictive of cardiovascular death, circulating LIGHT levels were negatively associated with risk of cardiovascular death (after 6 ½ years) after correcting for the possible confounders age, sex, cerebrovascular symptoms, diabetes, high-sensitivity C-reactive protein, inflammatory disease, hypertension and smoking (hazard ratio=0.032, confidence interval=0.420-0.951, p=0.028). Conclusion Though intra-plaque LIGHT levels are associated with a rupture-prone plaque phenotype and pre-operative symptoms, circulating levels are associated with a lower risk of future cardiovascular mortality. This suggest that upregulation of LIGHT in atherosclerotic plaques may occur as part of a reparative rupture response and, systemically, reflect efficient wound-healing capacity.

LIGHT/TNFSF14 levels in carotid atherosclerotic plaques are associated with symptomatic cerebrovascular disease

Abstract Background Plaque rupture is the underlying cause of most cardiovascular events, such as stroke and myocardial infarction. Co-stimulatory molecules, the immune checkpoints, are emerging as strong candidates to battle atherosclerosis progression. The co-stimulatory molecule LIGHT (tumour necrosis factor 14; TNFSF14) exists in foam cell-rich regions of atherosclerotic plaques, but whether it has a driving role in promoting a high-risk plaque phenotype is not known. Purpose This study investigates the association between intra-plaque LIGHT and plaque vulnerability. Methods Plaque levels of LIGHT/TNFSF14 were measured in homogenised carotid endarterectomy plaques by Proximity Extension Assay (PEA) in subjects from the Carotid Plaque Imaging Cohort (CPIP; N=201). Homogenates were further examined by multi-plex cytokine analysis and ELISA, and tissue plaque sections through histology/immunohistochemistry. Results Plaque levels of LIGHT were associated with the occurrence of pre-operative cerebrovascular symptoms (p=0.00014). Furthermore, LIGHT correlated strongly with the histological plaque vulnerability index (p=5x10-15): a ratio between the sum of destabilising versus stabilising plaque components (i.e. macrophages, intra-plaque haemorrhage and lipids versus smooth muscle cells and collagen) as well as with necrotic core size and the inflammatory cytokines interleukin(IL)-1β, IL-6, chemokine (C-C motif) ligand (CCL)-2, CCL4 and CCL5. Moreover, plaque LIGHT levels correlated with several components of the extracellular matrix turnover machinery, including cleaved collagen, the collagen cross-linking proteoglycans fibromodulin and lumican, the matricellular protein COMP (cartilage oligomeric matrix protein), and the remodelling regulator ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs-7). Levels also correlated with matrix metalloproteinases (MMPs) 1, 2, 9 and 10, and tissue inhibitor of metalloproteinase (TIMP)-1. Conclusion Expression of LIGHT in atherosclerotic plaques does not only correlate with markers of plaque destabilisation¸ but is also significantly elevated in plaques from symptomatic, compared to asymptomatic, patients. These results connect LIGHT content to a rupture-prone plaque phenotype and suggest the LIGHT signalling pathway as a potential therapeutic target to promote atherosclerotic plaque stability.

Deep learning assisted classification of spectral photoacoustic imaging of carotid plaques

Spectral photoacoustic imaging (sPAI) is an emerging modality that allows real-time, non-invasive, and radiation-free assessment of tissue, benefiting from their optical contrast. sPAI is ideal for morphology assessment in arterial plaques, where plaque composition provides relevant information on plaque progression and its vulnerability. However, since sPAI is affected by spectral coloring, general spectroscopy unmixing techniques cannot provide reliable identification of such complicated sample composition. In this study, we employ a convolutional neural network (CNN) for the classification of plaque composition using sPAI. For this study, nine carotid endarterectomy plaques were imaged and were then annotated and validated using multiple histological staining. Our results show that a CNN can effectively differentiate constituent regions within plaques without requiring fluence or spectra correction, with the potential to eventually support vulnerability assessment in plaques.

Injury to the tunica media initiates atherogenesis in the presence of hyperlipidemia.

Fatty streaks initiating the formation of atheromatous plaque appear in the tunica intima. The tunica media is not known to be a nidus for lipid accumulation initiating atherogenesis. We assessed changes to the tunica media in response to a micro-injury produced in the pig aorta. In addition, we assessed human carotid endarterectomy plaques for indication of atheroma initiation in the tunica media. Three healthy landrace female pigs underwent laparotomy to inject autologous blood and create micro-hematomas at 6 sites within the tunica media of the infrarenal abdominal aorta. These pigs were fed a high-fat diet (HFD) for 4-12 weeks. Post-mortem aortas from all pigs, including a control group of healthy pigs, were serially stained to detect lipid deposits, vasa vasora (VV), immune cell infiltration and inflammatory markers, as well as changes to the vascular smooth muscle cell (vSMC) compartment. Moreover, 25 human carotid endarterectomy (CEA) specimens were evaluated for their lipid composition in the tunica media and intima. High lipid clusters, VV density, and immune cell infiltrates were consistently observed at 5 out of 6 injection sites under prolonged hyperlipidemia. The hyperlipidemic diet also affected the vSMC compartment in the tunica media adjacent to the tunica adventitia, which correlated with VV invasion and immune cell infiltration. Analysis of human carotid specimens post-CEA indicated that 32% of patients had significantly greater atheroma in the tunica media than in the arterial intima. The arterial intima is not the only site for atherosclerosis initiation. We show that injury to the media can trigger atherogenesis.

Carotid endarterectomy plaques of patients with elevated levels of lipoprotein(a) demonstrate increased intraplaque angiogenesis

Background and Aims : Elevated lipoprotein(a) [Lp(a)] levels have been demonstrated to be a causal risk factor for the development of cardiovascular disease. Previously, we described that patients with elevated Lp(a) levels have an increased risk of major adverse cardiovascular events (MACE), mostly stroke, 30 days after undergoing carotid endarterectomy (CEA). Aim: Unravelling the mechanism underlying this increase in MACE.Methods: We performed targeted plasma proteomics analysis using the proximity extension assay for 276 proteins on complete plasma from patients undergoing CEA with high levels (>195 mg/dl; N=39) and low levels (<7 mg/dl; N=73) of Lp(a).Results: Surprisingly, no differences were observed in the expression of systemic plasma proteins. Hence, we started to assess the local effects of Lp(a) on plaque morphology. Plaque phenotyping demonstrated that patients with high levels (>195 mg/dl; N=57) of Lp(a) had a 33% increase in mean vessel density compared to patients with low levels (<7 mg/dl; N=106), as attested by semi-quantitatively scoring of the plaques. This increase in neovascularization correlates with decreased plaque stability, as was indicated by increased intraplaque haemorrhage. In-vitro stimulation of Human Arterial Endothelial Cells with physiologically relevant levels of Lp(a) (100 mg/dl), results in enhanced collagen degradation capacity, hyper-sprouting in a 3D spheroid assay and increased blood vessel invasion using a 3D collagen-invasiveness assay. Currently, we are elucidating the signalling pathways underlying this Lp(a)-induced angiogenesis, which we strive to present at the EAS 2022.Conclusions: Lp(a)-induced intraplaque angiogenesis can contribute to the increased risk of major adverse cardiovascular events in patients with elevated levels of Lp(a). Background and Aims : Elevated lipoprotein(a) [Lp(a)] levels have been demonstrated to be a causal risk factor for the development of cardiovascular disease. Previously, we described that patients with elevated Lp(a) levels have an increased risk of major adverse cardiovascular events (MACE), mostly stroke, 30 days after undergoing carotid endarterectomy (CEA). Aim: Unravelling the mechanism underlying this increase in MACE. Methods: We performed targeted plasma proteomics analysis using the proximity extension assay for 276 proteins on complete plasma from patients undergoing CEA with high levels (>195 mg/dl; N=39) and low levels (<7 mg/dl; N=73) of Lp(a). Results: Surprisingly, no differences were observed in the expression of systemic plasma proteins. Hence, we started to assess the local effects of Lp(a) on plaque morphology. Plaque phenotyping demonstrated that patients with high levels (>195 mg/dl; N=57) of Lp(a) had a 33% increase in mean vessel density compared to patients with low levels (<7 mg/dl; N=106), as attested by semi-quantitatively scoring of the plaques. This increase in neovascularization correlates with decreased plaque stability, as was indicated by increased intraplaque haemorrhage. In-vitro stimulation of Human Arterial Endothelial Cells with physiologically relevant levels of Lp(a) (100 mg/dl), results in enhanced collagen degradation capacity, hyper-sprouting in a 3D spheroid assay and increased blood vessel invasion using a 3D collagen-invasiveness assay. Currently, we are elucidating the signalling pathways underlying this Lp(a)-induced angiogenesis, which we strive to present at the EAS 2022. Conclusions: Lp(a)-induced intraplaque angiogenesis can contribute to the increased risk of major adverse cardiovascular events in patients with elevated levels of Lp(a).

Abstract 137: Role Of Cellular Communication Network Factor 2 (ccn2) And Male Sex On Flow-mediated Arterial Remodeling And Atherosclerotic Plaque Formation

CCN2 or connective tissue growth factor is up-regulated in response to disturbed flow and arterial stiffening. Carotid artery disease and peripheral arterial disease (PAD) are associated with disturbed flow and arterial stiffening. We have identified a key role for CCN2 in stiffened arterial remodeling and flow-mediated focal atherosclerotic plaque formation in translational models of carotid and PAD and in patient carotid plaques/PAD arteries. The objective of this project was to determine the modifiability of CCN2 pathways in pathologic arterial remodeling and atherosclerosis. Methods: To test our hypothesis that CCN2 was an important mediator of pathology arterial remodeling in vascular disease , we utilized murine models of stiffened arteries (fibulin-5 KO) in atherogenic conditions (PCSK9 + HFD) and clinical arterial tissue from patients with PAD and carotid artery disease. Results: Disturbed flow increases EC expression of CCN2 3.5 fold in WT carotid arteries. This is doubled again in Fibulin 5 KO ( KO ) carotid arteries. These KO carotid arteries are ~3x stiffer than littermate control arteries, which is a similar ratio of PAD compared to healthy control arteries ( 1A ). Under disturbed flow and atherogenic conditions KO arteries had more elastin breaks (P=.04), greater plaque area (P=.007) and more lipid deposition (P=.02), and Glagov’s outward remodeling (P=.02) ( 1B/C ). This was most pronounced in male mice. CCN2 staining is significantly increased in PAD arteries and carotid endarterectomy plaques (P&lt;.05) ( 1D/E ). To evaluate the modifiability of CCN2, we utilized CCN2 ECKO animals. We found that male (but not female) CCN2 ECKO animals had significantly less atherosclerotic plaque burden (P=.001) and similar elastin breaks/lipid deposition compared to littermate controls ( 1F/G ). Conclusion: CCN2 may play an important and modifiable role in flow-mediated pathologic arterial remodeling and focal atherosclerotic plaque burden.

Phenotyping carotid endarterectomy plaques of patients with elevated levels of lipoprotein(A)

Background and Aims: Elevated lipoprotein(a) [Lp(a)] levels have been demonstrated to be a causal risk factor for the development of cardiovascular disease. While previous studies have shown the atherogenicity of Lp(a) on monocyte activation and migration as well by activation of the vessel wall, the atherosclerotic plaque phenotype of individuals with elevated Lp(a) levels have not been studied up till now.

Phenotyping carotid endarterectomy plaques of patients with elevated levels of lipoprotein(A)

Phenotyping carotid endarterectomy plaques of patients with elevated levels of lipoprotein(A)

Differences in Pathological Composition Among Large Artery Occlusion Cerebral Thrombi, Valvular Heart Disease Atrial Thrombi and Carotid Endarterectomy Plaques.

Background and Purpose: Acute ischemic stroke (AIS) with large artery occlusion (LAO) may lead to severe disability or death if not promptly treated. To determine the source of cerebral artery occlusion thrombosis, we studied the pathological components of cerebral artery thrombosis with different etiological classifications to guide clinical formulation of preventive treatment.Materials and Methods: Eighty-eight thrombi from AIS patients with LAO, 12 atrial thrombi from patients with valvular heart disease (VHD), and 11 plaques obtained by carotid endarterectomy (CEA) from patients with carotid artery stenosis were included in this retrospective study. The hematoxylin and eosin–stained specimens were quantitatively analyzed for erythrocytes, white blood cells (WBCs) and fibrin; platelets were shown by immunohistochemistry for CD31.Results: The thrombi of VHD showed the highest percentage of fibrin, followed by those of cardioembolism (CE) and stroke of undetermined etiology (SUE), and these values were higher than those of the other groups. Plaques obtained by CEA showed the highest erythrocyte number, followed by the large artery atherosclerosis (LAA) thrombi, and showed significantly noticeable differences between other stroke subtypes. The proportions of fibrin and erythrocytes in the thrombi of CE and SUE were most similar to those in the thrombi of VHD, and the LAA thrombi were the closest to those obtained by CEA. CE thrombi and CEA plaques had a higher percentage of WBCs than thrombi of other stroke thrombus subtypes and VHD.Conclusions: CE and most cryptogenic thrombi may originate from the heart, and the formation of carotid atherosclerotic plaques may be related to atherosclerotic cerebral embolism. Inflammation may be involved in their formation.

A Novel Putative Role of TNK1 in Atherosclerotic Inflammation Implicating the Tyk2/STAT1 Pathway.

Objective Atherosclerosis is a chronic inflammatory disease which is responsible for many clinical manifestations. The present study was to investigate the anti-inflammatory functions and mechanisms of TNK1 in atherosclerosis. Methods The ApoE(-/-) mice and human carotid endarterectomy (CEA) atherosclerotic plaques were used to investigate the differential expression of TNK1. The ApoE(-/-) mice were fed with high-fat diet (HFD) or normal-fat diet (NFD) for 8 weeks; the aorta was separated and stained with oil red O to evaluate the formation of atherosclerosis. TNK1 in mice aorta was measured by qPCR. The human CEA were obtained and identified as ruptured and stable plaques. The level of TNK1 was measured by qPCR and Western-blot staining. Further studies were conducted in THP-1 cells to explore the anti-inflammatory effects of TNK1. We induced the formation of macrophages by incubating THP-1 cells with PMA (phorbol 12-myristate 13-acetate). Afterwards, oxidized low-density lipoprotein (oxLDL) was used to stimulate the inflammation, and the secretion of inflammatory factors was measured by ELISA and qPCR. The levels of TNK1, total STAT1 and Tyk2, and the phosphorylation of STAT1 and Tyk2 were measured by western blot to uncover the mechanisms of TNK1. Results The oil red O staining indicated obvious deposition of lipid on the aorta of ApoE(-/-) mice after 8-week HFD treatment. The TNK1 level was much higher in both the HFD-fed ApoE(-/-) mice aorta arch and the ruptured human CEA plaques. We found that TNK1 was highly expressed in THP-1 cells, compared to other atherosclerotic related cells (HUVEC, HBMEC, and HA-VSMC), indicating TNK1 might be involved in the inflammation. Suppressing the expression of TNK1 by shTNK1 inhibited the oxLDL-induced secretion of inflammatory factors, such as IL-12, IL-6, and TNF-α. ShTNK1 also inhibited the uptake of lipid and decreased the cellular cholesterol content in THP-1 cells. Furthermore, the shTNK1 suppressed the oxLDL-induced phosphorylation of Tyk2 and STAT1. Conclusion TNK1 participated in the inflammation in atherosclerosis. shTNK1 suppressed the oxLDL-induced inflammation and lipid deposition in THP-1 cells. The mechanism might be related to the Tyk2/STAT signal pathway.

Identification of key genes in ruptured atherosclerotic plaques by weighted gene correlation network analysis

The rupture of atherosclerotic plaques is essential for cardiovascular and cerebrovascular events. Identification of the key genes related to plaque rupture is an important approach to predict the status of plaque and to prevent the clinical events. In the present study, we downloaded two expression profiles related to the rupture of atherosclerotic plaques (GSE41571 and GSE120521) from GEO database. 11 samples in GSE41571 were used to identify the differentially expressed genes (DEGs) and to construct the weighted gene correlation network analysis (WGCNA) by R software. The gene oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment tool in DAVID website, and the Protein-protein interactions in STRING website were used to predict the functions and mechanisms of genes. Furthermore, we mapped the hub genes extracted from WGCNA to DEGs, and constructed a sub-network using Cytoscape 3.7.2. The key genes were identified by the molecular complex detection (MCODE) in Cytoscape. Further validation was conducted using dataset GSE120521 and human carotid endarterectomy (CEA) plaques. Results: In our study, 868 DEGs were identified in GSE41571. Six modules with 236 hub genes were identified through WGCNA analysis. Among these six modules, blue and brown modules were of the highest correlations with ruptured plaques (with a correlation of 0.82 and −0.9 respectively). 72 hub genes were identified from blue and brown modules. These 72 genes were the most likely ones being related to cell adhesion, extracellular matrix organization, cell growth, cell migration, leukocyte migration, PI3K-Akt signaling, focal adhesion, and ECM-receptor interaction. Among the 72 hub genes, 45 were mapped to the DEGs (logFC > 1.0, p-value < 0.05). The sub-network of these 45 hub genes and MCODE analysis indicated 3 clusters (13 genes) as key genes. They were LOXL1, FBLN5, FMOD, ELN, EFEMP1 in cluster 1, RILP, HLA-DRA, HLA-DMB, HLA-DMA in cluster 2, and SFRP4, FZD6, DKK3 in cluster 3. Further expression detection indicated EFEMP1, BGN, ELN, FMOD, DKK3, FBLN5, FZD6, HLA-DRA, HLA-DMB, HLA-DMA, and RILP might have potential diagnostic value.

Abstract 139: Promoter Methylation Changes in ALOX12, AIRE1, and NETO1 : Novel Epigenetic Markers for Atherosclerosis

Background and Objective: We aimed to identify and validate specific genes developing changes in promoter methylation in atherosclerotic plaques. Methods: We initially profiled DNA methylation signatures via a high-throughput sequencing platform from 5 carotid endarterectomy (CEA) plaques to identify genes with altered promoter methylation. The significances of the identified gene-specific promoter methylations were validated by the comparison between 1) plaques and non-plaque intima of the common carotid artery harvested from 20 cadavers, 2) cellular components composing atherosclerosis including plaques and buffy coats of blood of 27 CEA patients, 5 human umbilical vein endothelial cells (HUVEC), 2 human aortic endothelial (HAEC) and 2 smooth muscle (HASMC) cell lines, and, 3) monocytes, T- and B-cell circulating in blood of 19 ischemic stroke patients. Finally, the localization pattern the genes was compared by the inflammatory cell types infiltrated into atherosclerotic plaque and non-plaque intima using immunofluorescence stain. Results: Upon the profiling, five genes ( AIRE1 , ALOX12 , FANK1 , NETO1 , and SERHL2 ) displayed changes in promoter methylation. Of these, AIRE1 , ALOX12, and NETO1 displayed higher methylation in plaques than in non-plaque intima, but lower than buffy coat of blood. In vascular cells, AIRE1 and NETO1 methylation was lower in HUVEC, HAEC and HASMC, but, ALOX12 methylation was higher in HUVEC and HAEC, but, lower in HASMC than CEA plaques. Of inflammatory cells, methylation of the three genes was significantly lower in monocytes than T- and B-cells. On immunofluorescence statin for ALOX12, CD14(+)-monocytes was 3 times more frequent than CD4(+)-T-cell of total ALOX12-stained cells in plaques than non-plaque intima. Conclusions: The present study profiled and validated the promoter methylation changes of AIRE1 , ALOX12, and NETO1 as novel epigenetic markers related with atherosclerosis. In particular, epigenetic augmentation of the 3 genes occurred in monocytes of the cellular components composing atherosclerotic plaques.

Start of Antiplatelet Therapy Increases the Prevalence of Intraplaque Hemorrhage in Patients With Advanced Carotid Artery Lesions: A Longitudinal MR Imaging Study

Introduction: Intraplaque hemorrhage (IPH) is an important feature of plaque vulnerability. Patients with a history of transient ischemic attack or stroke are treated with platelet aggregation inhibitors to prevent secondary myocardial infarction or ischemic stroke. Although antiplatelet medication is effective in reduction of clot formation, it also has adverse effects. It has therefore been hypothesized that platelet aggregation inhibitors could also initiate the onset or progression of IPH. A histopathological study on 154 carotid endarterectomy plaques found that preoperative antiplatelet therapy was associated with an increase in the prevalence of repeated IPH in symptomatic carotid plaques. This longitudinal study investigates the effect of antiplatelet agents on the prevalence of IPH. Methods: 63 symptomatic patients underwent carotid MRI at baseline and after two years of follow-up. Presence of IPH in both carotid arteries was scored. Medication prescription before and after the index event and after follow-up was collected.The presence of IPH was determined using MP-RAGE images and analysed blinded for the clinical data using dedicated vessel wall analysis software (VesselMass). IPH was considered present when a hyperintense signal, compared to the sternocleidomastoid muscle, was visible in the bulk of the plaque. Results: A significant higher prevalence of any IPH was found two years after the index event (42.9% vs. 55.6%; p = 0.008) (Figure 1) Patients already using antiplatelet agents before the index event (n=19) did not show significantly more IPH (52.6% vs. 63.2%, p = 0.50) at follow-up. Patients who did not use platelet aggregation inhibitors before the index event (n=44) and were prescribed medication after the event, showed a significantly higher prevalence of IPH (38.6% vs. 52.3%, p=0.031) at follow-up (Figure 2) Conclusion: Patients who started antiplatelet therapy after the index event show a significant increase in bilateral IPH two years after inclusion, while no significant changes in IPH status were found in the patients that already use antiplatelet therapy before the index event. Future studies are warranted to investigate causal relationships. Disclosure: JE Wildberger reports institutional grants from AGFA, Bard, Bayer, GE, Optimed, Philips, Siemens outside the submitted work; and speakers Bureau: Bayer, Siemens outside the submitted work. This research was performed within the framework of the Center for Translational Molecular Medicine (www.ctmm.nl), project PARISk (grant 01C-202), and supported by the Dutch Heart Foundation. This project has received funding from the European Union Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 722609. ME Kooi and JE Wildberger are supported by Stichting de Weijerhorst. ME Kooi is supported by an Aspasia Grant 2018/SGw/00460457 from NWO. J Hendrikse has received funding from the European Research Council under the European Union's Horizon 2020 Programme (H2020) / ERC grant agreement n°637024. FHBM Schreuder is supported by the Dutch Heart Foundation (2012T077).

Abstract WP541: Profiling of Microrna From Atherosclerotic Tissues and Validation of Their Significance From Peripheral Blood of Ischemic Stroke Patients Having Atherosclerosis

Background and Objectives: To search circulating microRNAs related to atherosclerosis, we first performed a microRNA expression profiling using atherosclerotic plaques. Then, their expression significance was validated using the serum of stroke patients having severe atherosclerosis on cranial vessels. Methods: We performed miRNA expressions from 10 carotid endarterectomy (CEA) plaques and 2 human umbilical vein endothelial cell (HUVEC) lines using a microRNA microarray kit (TaqMan® Array MicroRNA Cards, Applied Biosystems®, Life Technologies). The expression differences were compared using relative quantitation (RQ=2 -(miRNA expression -reference RNA expression) ) between the CEA plaques and the HUVECs following a criteria including over 4-fold-up or less than 0.25-fold-down regulations after log2 transformation of the expression difference. Among the microRNAs showing significant expression differences, target microRNAs were selected using an internal clustering validation measurement (Xie-Beni index). The expression of the target microRNAs was validated with the serum of 32 patients with severe atherosclerosis and that of 33 patients with no atherosclerosis in intra- and extra-cranial vessels. Results: A total of 151 microRNAs were identified after the comparison of the microRNA expressions between CEA and HUVECs. Among them, eight miRNAs (miR199b, miR142-3p, miR145, miR145, miR204, miR9, miR370, miR23b, and miR363) were selected as the final target microRNAs after the clustering validation analysis. As for the validation experiments with the serum of the atherosclerosis and non-atherosclerosis patients, the RQ of miR142-3p (atherosclerosis, 3.79±7.57; no-atherosclerosis patients, 1.10±1.45, p=0.043), among the 8 target miRNAs, showed significant differences in the serum of patients with atherosclerosis. Conclusion: The present study profiled miRNAs from CEA plaques and validated their significance within the serum of atherosclerosis patients. These findings suggest that miR142-3P could be a potential marker for atherosclerosis that is detectable from the circulating blood.

Abstract WP546: Changes in Promoter Methylation in AIRE1 and ALOX12: Novel Epigenetic Markers in Atherosclerotic Tissues

Objective: The present study aimed to identify and validate specific genes developing changes in promoter methylation in atherosclerotic plaques. Approach and Results: We initially comprehensively profiled DNA methylation signatures via Solexa sequencing, using amplicons prepared via methylated-CpG island amplification (MCA) in genomic DNA obtained from 5 plaques acquired through carotid endarterectomy (CEA) to identify genes with altered promoter methylation. The changes in promoter methylation of these genes were validated via bisulfite pyrosequencing from plaques and non-plaque intima of the common carotid artery (CCA) harvested from 20 cadavers as well as from 27 CEA plaques and 5 human umbilical vein endothelial cell (HUVEC) lines. Expression changes based on changes in promoter methylation in these genes were evaluated in 20 CEA plaques and in 2 HUVEC cell lines after demethylation with 5-aza-2'-deoxycytidine. Upon MCA-Solexa sequencing, five genes ( AIRE1 , ALOX12 , FANK1 , NETO1 , and SERHL2 ) displayed changes in promoter methylation. Of these, AIRE1 and ALOX12 displayed significantly greater promoter methylation in plaques rather than in the non-plaque intima of the CCA. Furthermore, promoter methylation was greater in AIRE1 in CEA plaques than in HUVECs, while that in ALOX12 was greater in HUVEC cells than in CEA plaques. Concurrently, gene expression levels decreased with an increase in promoter methylation in these genes in CEA plaques. After DNA demethylation in HUVEC cells, AIRE1 and ALOX12 expression levels increased by 23 and 52 folds. Conclusions: Changes in promoter methylation in AIRE1 and ALOX12 were identified and validated in atherosclerotic plaques, thereby establishing novel epigenetic markers for atherosclerosis.

Abstract WP551: Senescence-Induced Methylation Changes in Global DNA and Atherosclerosis-Related Genes in Human Vascular Endothelial Cells

Background: Endothelial senescence has been known as a pathophysiological alteration related to atherosclerosis development. We investigated whether methylation changes in global DNA and atherosclerosis-related genes are developed in in-vitro senescence of human vascular endothelial cells. Methods: We induced in-vitro replicative senescence by subculturing 5 human umbilical vein endothelial cell (HUVEC) lines from three to fifteen passages. Using DNA extracted from the 5th, 10th, and 15th passages of the 5 HUVECs, we evaluated LINE1 methylation for global methylation status as well as promoter methylation status for 5 genes ( AIRE1, ALOX12, FANK1, NETO1 , and SERHL2 ) profiled from carotid endarterectomy plaques for gene-specific methylation evaluations. Differences in LINE1 methylations and promoter methylations of the 5 genes were evaluated using pyrosequencing and compared among the 5th, 10th, and 15th passages of the 5 HUVECs using analysis of variance (ANOVA) tests. Results: LINE1 global methylation subsequently decreased as passages were advanced from 5 to 15 (p&lt;0.001). On gene-specific promoter methylation analysis, ALOX12 (p&lt;0.001) and SERHL2 (p&lt;0.001) showed significant passage-dependent increase of promoter methylation. AIRE1 and NETO1 also showed an increase of promoter methylation as passages increased, but without statistical significance. FANK1 exhibited an increase-decrease pattern of methylation changes showing an increase of methylation at passage 10 compared to passage 5, but showing a decrease in passage 15 (p=0.005). Conclusion: After replicative senescence for vascular endothelial cells, global methylation subsequently decreased. However, atherosclerosis-related genes showed either a increase or an increase-decrease of promoter methylations. In particular, the increase of ALOX12 methylation showed as a possible epigenetic marker related to vascular endothelial cell senescence.

Multiple Non-Essential Transition Metals Are Accumulated in Carotid Atherosclerotic Plaques: Missing Link in Atherosclerosis?

Population studies revealed that metal exposure through food, environment, and smoking was related with increased risk of cardiovascular disease. In this study, we determined complex chemical elements in surgical carotid endarterectomy specimens and carotid tissues from autopsies without atherosclerosis. Atherosclerotic plaques from 41 endarterectomies and normal carotid tissue from 30 autopsies were collected and elemental composition was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) method. Eleven (26.8%) patients never smoked in carotid endarterectomy group. One patient was brass souvenir worker and one was goldsmith and others did not have direct contact with metals in the carotid endarterectomy group. Na, Cu, Mn, Bi, Co, Mo, Ni, Pb, Sb, Se, Sn, Ti, and W levels were not different between two groups. Bi, Co, Mo, Pb, Ti, and W were below the detection limit of ICP-OES in both groups. Concentrations of Mg, K, Ca, P, Fe, B, Zn, Al, As, Cr, Pt, and Hg were significantly higher in carotid endarterectomies than normal carotid tissue samples. Cd and S values were significantly higher in autopsy samples. There is significant multiple non-essential transition metal accumulation in atherosclerotic carotid endarterectomy plaques. The cardiovascular consequences of metal toxicity have not been researched adequately due to large emphasis on the role of cholesterol in atherosclerosis. High level of non-essential transition metal elements in the carotid atherosclerotic plaques may add the missing link of atherogenesis and may necessitate new treatment and prevention strategies in carotid disease if confirmed by further research.

Tropoelastin: A novel marker for plaque progression and instability.

Elastolysis and ineffective elastogenesis favor the accumulation of tropoelastin, rather than cross-linked elastin, in atherosclerotic plaques. We developed gadolinium-labeled tropoelastin-specific magnetic resonance contrast agents (Gd-TESMAs) for tropoelastin imaging in animal models. Two peptides, VVGSPSAQDEASPLS and YPDHVQYTHY were selected to target tropoelastin. In vitro binding, relaxivity, and biodistribution experiments enabled characterization of the probes and selecting the best candidate for in vivo MRI. MRI was performed in atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice and New Zealand white rabbits with stable and rupture-prone plaques using Gd-TESMA. Additionally, human carotid endarterectomy specimens were imaged ex vivo. The VVGSPSAQDEASPLS-based probe discriminated between tropoelastin and cross-linked elastin (64±7% vs 1±2%, P=0.001), had high in vitro relaxivity in solution (r1-free=11.7±0.6mM-1s-1, r1-bound to tropoelastin = 44±1mM-1s-1) and favorable pharmacokinetics. In vivo mice vascular enhancement (4wks=0.13±0.007mm2, 8wks=0.22±0.01mm2, 12wks=0.33±0.01mm2, P<0.001) and R1 relaxation rate (4wks=0.90±0.01 s-1, 8wks=1.40±0.03 s-1, 12wks=1.87±0.04s-1, P<0.001) increased with atherosclerosis progression after Gd-TESMA injection. Conversely, statin-treated (0.13±0.01mm2, R1 =1.37±0.03s-1) and control (0.10±0.005mm2, R1 =0.87±0.05s-1) mice showed less enhancement. Rupture-prone rabbit plaques had higher R1 relaxation rate compared with stale plaques (R1=2.26±0.1s-1vs R1=1.43±0.02s-1, P=0.001), after administration of Gd-TESMA that allowed detection of rupture-prone plaques with high sensitivity (84.4%) and specificity (92.3%). Increased vascular R1 relaxation rate was observed in carotid endarterectomy plaques after soaking (R1pre= 1.1±0.26 s-1 vs R1post= 3.0±0.1s-1, P=0.01). Ex vivo analyses confirmed the MRI findings and showed uptake of the contrast agent to be specific for tropoelastin. MRI of tropoelastin provides a novel biomarker for atherosclerotic plaque progression and instability.

Diabetes adversely affects phospholipid profiles in human carotid artery endarterectomy plaques

Patients with diabetes are at higher risk of developing carotid artery stenosis and resultant stroke. Arachidonoyl phospholipids affect plaque inflammation and vulnerability, but whether diabetic patients have unique carotid artery phospholipidomic profiles is unknown. We performed a comprehensive paired analysis of phospholipids in extracranial carotid endarterectomy (CEA) plaques of matched diabetic and nondiabetic patients and analyzed mass spectrometry-derived profiles of three phospholipids, plasmenyl-phosphatidylethanolamine (pPE), phosphatidylserine (PS), and phosphatidylinositol (PI), in maximally (MAX) and minimally (MIN) diseased CEA segments. We also measured levels of arachidonic acid (AA), produced by pPE hydrolysis, and choline-ethanolamine phosphotransferase 1 (CEPT1), responsible for most pPE de novo biosynthesis. In paired analysis, MIN CEA segments had higher levels than MAX segments of pPE (P < 0.001), PS (P < 0.001), and PI (P < 0.03). MIN diabetic plaques contained higher levels than MAX diabetic plaques of arachidonoyl pPE38:4 and pPE38:5 and CEPT1 was upregulated in diabetic versus nondiabetic plaques. AA levels were relatively greater in MIN versus MAX segments of all CEA segments, and were higher in diabetic than nondiabetic plaques. Our findings suggest that arachidonoyl phospholipids are more likely to be abundant in the extracranial carotid artery plaque of diabetic rather than nondiabetic patients.