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Plasma extracellular vesicles (EV) are an emerging source of biomarkers for diagnosis and prognosis of cardiovascular disease (CVD). Risk stratification for common adverse events such as major adverse limb events (MALE) and major adverse cardiovascular events (MACE) by an EV blood sample could improve healthcare management by individualising drug therapy or improving informed decision making regarding revascularisations in patients with peripheral artery disease (PAD). As such, this study investigated the associations between plasma EV proteins and prospectively registered MALE and MACE in consecutive patients undergoing femoral endarterectomy.Using the Athero-Express biobank study, four EV proteins (Cystatin C, CD14, Serpin C1, and Serpin G1) were measured in the high density lipoprotein subfraction isolated from plasma of 317 PAD patients undergoing arterial revascularisation. Multivariable Cox proportional hazard regression was used to investigate the association between plasma EV protein levels and MACE and MALE in the three year post-operative period.Most patients were treated for claudication (Fontaine II, 52.8%), although rest pain (Fontaine III, 30.1%) and ischaemic wounds (Fontaine IV, 17.1%) were common in this cohort. Within three years 51 patients died, amongst whom 25 deaths were due to CVD, 39 patients experienced a MACE, and 125 patients experienced a MALE. Multivariable regression models, based on statistically proven covariables and literature, showed a significant association of Serpin G1 (HR 1.49; 95% CI 1.08 - 2.06; p = .016) and CD14 (HR 1.40; 1.03 - 1.90; p = .029) with MACE, and of Serpin G1 (HR 1.29; 1.07 - 1.57; p = .009) with MALE.Serpin G1 and CD14 plasma EV protein levels are associated with future MACE and MALE in patients with severe PAD.

Aims Current risk scores do not accurately identify patients at highest risk of recurrent atherosclerotic cardiovascular disease (ASCVD) in need of more intensive therapeutic interventions. Advances in high-throughput plasma proteomics, analysed with machine learning techniques, may offer new opportunities to further improve risk stratification in these patients. Methods and results Targeted plasma proteomics was performed in two secondary prevention cohorts: the Second Manifestations of ARTerial disease (SMART) cohort (n = 870) and the Athero-Express cohort (n = 700). The primary outcome was recurrent ASCVD (acute myocardial infarction, ischaemic stroke, and cardiovascular death). Machine learning techniques with extreme gradient boosting were used to construct a protein model in the derivation cohort (SMART), which was validated in the Athero-Express cohort and compared with a clinical risk model. Pathway analysis was performed to identify specific pathways in high and low C-reactive protein (CRP) patient subsets. The protein model outperformed the clinical model in both the derivation cohort [area under the curve (AUC): 0.810 vs. 0.750; P < 0.001] and validation cohort (AUC: 0.801 vs. 0.765; P < 0.001), provided significant net reclassification improvement (0.173 in validation cohort) and was well calibrated. In contrast to a clear interleukin-6 signal in high CRP patients, neutrophil-signalling-related proteins were associated with recurrent ASCVD in low CRP patients. Conclusion A proteome-based risk model is superior to a clinical risk model in predicting recurrent ASCVD events. Neutrophil-related pathways were found in low CRP patients, implying the presence of a residual inflammatory risk beyond traditional NLRP3 pathways. The observed net reclassification improvement illustrates the potential of proteomics when incorporated in a tailored therapeutic approach in secondary prevention patients.

BackgroundGenome-wide association studies have discovered hundreds of common genetic variants for atherosclerotic disease and cardiovascular risk factors. The translation of susceptibility loci into biological mechanisms and targets for drug discovery remains challenging. Intersecting genetic and gene expression data has led to the identification of candidate genes. However, previously studied tissues are often non-diseased and heterogeneous in cell composition, hindering accurate candidate prioritization. Therefore, we analyzed single-cell transcriptomics from atherosclerotic plaques for cell-type-specific expression to identify atherosclerosis-associated candidate gene-cell pairs.Methods and ResultsTo identify disease-associated genes, we applied gene-based analyses using GWAS summary statistics from 46 atherosclerotic and cardiovascular disease, risk factors, and other traits. We then intersected these candidates with scRNA-seq data to identify genes specific for individual cell (sub)populations in atherosclerotic plaques. The coronary artery disease loci demonstrated a prominent signal in plaque smooth muscle cells (SKI, KANK2, SORT1) p-adj. = 0.0012, and endothelial cells (SLC44A1, ATP2B1) p-adj. = 0.0011. Further sub clustering revealed genes in risk loci for coronary calcification specifically enriched in a synthetic smooth muscle cell population. Finally, we used liver-derived scRNA-seq data and showed hepatocyte-specific enrichment of genes involved in serum lipid levels.ConclusionWe discovered novel gene-cell pairs, on top of known pairs, pointing to new biological mechanisms of atherosclerotic disease. We highlight that loci associated with coronary artery disease reveal prominent association levels in mainly plaque smooth muscle and endothelial cell populations. We present an intuitive single-cell transcriptomics-driven workflow rooted in human large-scale genetic studies to identify putative candidate genes and affected cells associated with cardiovascular traits. Collectively, our workflow allows for the identification of cell-specific targets relevant for atherosclerosis and can be universally applied to other complex genetic diseases and traits.Translational perspectiveGWAS identified a large number of genomic loci associated with atherosclerotic disease. The translation of these results into drug development and faster diagnostics remains challenging. With our approach, we cross-reference the GWAS findings for atherosclerotic disease with scRNA-seq data of disease-relevant tissue and bring the GWAS findings closer to the functional and mechanistic studies.Abstract Figure

Histopathological studies have revealed key processes of atherosclerotic plaque thrombosis. However, the diversity and complexity of lesion types highlight the need for improved sub- phenotyping. We hypothesized that unbiased clustering of plaques based on gene expression results in an alternative categorization of late-stage atherosclerotic lesions.We analyzed the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These novel plaque phenotypes associated with clinical presentation (pIn conclusion, the definition of the plaque at risk for a thrombotic event can be fine-tuned by in- depth transcriptomic based phenotyping. These differential plaque phenotypes prove clinically relevant for both carotid and coronary artery plaques and point to differential underlying biology of symptomatic lesions.

Patients with symptomatic carotid artery stenosis have a high risk of future ipsilateral cerebrovascular events and this risk can be effectively reduced by a surgival removel of the carotid plaque (carotid endarterectomy, CEA). Despite CEA, these patients maintain at high risk for future major cardiovascular events (MACE) after CEA due to the systemic nature of atherosclerosis. Novel options for intensification of medical treatment (intensive lipid lowering or anticoagulants or new anti-inflammatory drugs) are emerging but have hazardous side effects and are expensive. Therefore, risk stratification tools for MACE are warranted. Clinical parameters fail to accurately discriminate between patients at high risk of MACE and those at low risk. Biomarkers could improve risk prediction models. This thesis investigates several types of biomarkers; circulating blood markers, radiographic markers on cerebral imaging and genetics, in relation to future cardiovascular events in patients undergoing CEA. These biomarkers were also associated to histological characteristics of the carotid plaque in order to increase our understanding of the complex mechanisms of atherosclerosis. In the future, these biomarkers can be useful to select high-risk patients that qualify for intensified medical treatment or follow-up. Ultimately, these biomarkers incorporated in risk prediction models could help physicians select appropriate patients for specific treatments.

Sex differences are evident in the clinical presentation and underlying histology of atherosclerotic disease with women developing more stable atherosclerotic lesions than men. It is unknown whether this is explained by sex differences in gene regulation in cellular compartments of atherosclerotic plaques. To study sex differences in gene regulation we performed genome-wide DNA methylation and transcriptomics analysis on plaques of 485 carotid endarterectomy patients (31% female). Sex-differential DNA methylation at 4,848 sites in the autosome was enriched for cell-fate commitment and developmental processes, and its deconvolution predicted more smooth muscle cells in females, as compared to more immune cells in males. RNA-sequencing of the same plaques corroborated the sex differences in DNA methylation predicted cell-types, in which genes that were higher expressed in females were enriched for TGF-beta signaling and extracellular matrix biology. In addition, female-biased genes were enriched for targeting by regulatory loci based on sex differential methylation. Lastly, by using single-cell RNA sequencing we showed that these female-biased genes are mostly expressed in smooth muscle cells, and higher expressed in smooth muscle cells from female (predominantly stable) plaques as compared to male (relatively unstable) plaques. Our approach identified female-biased genes in smooth muscle cells in fibrous atherosclerotic plaques. This points towards new mechanisms in smooth muscle cell biology of stable atherosclerotic plaques and offers new directions for research to develop new sex-specific therapeutics for atherosclerotic disease.

Mast cells (MCs) are important contributors to atherosclerotic plaque progression. For prospective studies on mast cell contributions to plaque instability, the distribution of intraplaque MCs needs to be elucidated. Plaque stability is generally histologically assessed by dividing the plaque specimen into segments to be scored on an ordinal scale. However, owing to competitive use, studies may have to deviate to adjacent segments, yet intersegment differences of plaque characteristics, especially MCs, are largely unknown. Therefore, the hypothesis that there is no segment to segment difference in MC distribution between atherosclerotic plaque segments was tested, and intersegment associations between MCs and other plaque characteristics was investigated.\nTwenty-six carotid atherosclerotic plaques from patients undergoing carotid endarterectomy included in the Athero-Express Biobank were analysed. The plaque was divided in 5 mm segments, differentiating between the culprit lesion (segment 0), adjacent segments (-1/+1) and more distant segments (-2/+2) for the presence of MCs. The associations between the intersegment distribution of MCs and smooth muscle cells, macrophage content, and microvessel density in the culprit lesion were studied.\nA statistically significant difference in MCs/mm2 between the different plaque segments (p < .001) was found, with a median of 2.79 (interquartile range [IQR] 1.63 - 7.10) for the culprit lesion, 1.34 (IQR 0.26 - 4.45) for the adjacent segment, and 0.62 (0.14 - 2.07) for the more distant segment. Post hoc analyses showed that intersegment differences were due to differences in MCs/mm2 between the culprit and adjacent segment (p = .037) and between the culprit lesion and the more distant segment (p < .001). MCs/mm2 in multiple different segments were positively correlated with microvessel density and macrophage content in the culprit lesion.\nMC numbers reveal significant intersegment differences in human carotid plaques. Future histological studies on MCs should use a standardised segment for plaque characterisation as plaque segments cannot be used interchangeably for histological MC analyses.

Background and Purpose: General population studies have shown that elevated Lp(a) (lipoprotein[a]) levels are an emerging risk factor for cardiovascular disease and subsequent cardiovascular events. The role of Lp(a) for the risk of secondary MACE in patients undergoing carotid endarterectomy (CEA) is unknown. Our objective is to assess the association of elevated Lp(a) levels with the risk of secondary MACE in patients undergoing CEA. Methods: Lp(a) concentrations were determined in preoperative blood samples of 944 consecutive patients with CEA included in the Athero-Express Biobank Study. During 3-year follow-up, major adverse cardiovascular events (MACE), consisting of myocardial infarction, stroke, and cardiovascular death, were documented. Results: After 3 years follow-up, Kaplan-Meier cumulative event rates for MACE were 15.4% in patients with high Lp(a) levels (>137 nmol/L; >80th cohort percentile) and 10.2% in patients with low Lp(a) levels (≤137 nmol/L; ≤80th cohort percentile; log-rank test: P =0.047). Cox regression analyses adjusted for conventional cardiovascular risk factors revealed a significant association between high Lp(a) levels and 3-year MACE with an adjusted hazard ratio of 1.69 (95% CI, 1.07–2.66). One-third of MACE occurred within 30 days after CEA, with an adjusted hazard ratio for the 30-day risk of MACE of 2.05 (95% CI, 1.01–4.17). Kaplan-Meier curves from time point 30 days to 3 years onward revealed no significant association between high Lp(a) levels and MACE. Lp(a) levels were not associated with histological carotid plaque characteristics. Conclusions: High Lp(a) levels (>137 nmol/L; >80th cohort percentile) are associated with an increased risk of 30-day MACE after CEA. This identifies elevated Lp(a) levels as a new potential risk factor for secondary cardiovascular events in patients after carotid surgery. Future studies are required to investigate whether Lp(a) levels might be useful in guiding treatment algorithms for carotid intervention.

Objective Intraplaque haemorrhage (IPH) has been independently associated with a higher risk of future ipsilateral stroke in patients with carotid artery stenosis. Evaluation of plaque characteristics may contribute to risk assessment of recurrent (silent) cerebrovascular events in order to prioritise patients for timing of treatment. It is unknown if patients showing histologically apparent IPH also have increased risk of silent ischaemic brain lesions in the waiting period between index event and revascularisation. Methods A retrospective analysis was performed based on prospectively collected data of patients included simultaneously in the magnetic resonance imaging (MRI) substudy of the International Carotid Stenting Study and Athero-Express biobank. Patients randomised for carotid endarterectomy (CEA) underwent surgery between 2003 and 2008. Brain MRI was performed one to seven days prior to CEA. Plaques were histologically examined for presence of IPH. The primary outcome parameter was presence of silent ipsilateral brain ischaemia on magnetic resonance diffusion weighted imaging (MR-DWI) appearing hypo or isointense on apparent diffusion coefficient. Results Fifty-three patients with symptomatic carotid stenosis meeting the study criteria were identified, of which 13 showed one or more recent ipsilateral DWI lesion on pre-operative scan. The median time between latest ipsilateral neurological event and revascularisation was 45 days (range 6–200) in DWI negative patients vs. 34 days (range 6–74, p = .16) in DWI positive patients. IPH was present in 24/40 (60.0%) DWI negative patients vs. 12/13 (92.3%) DWI positive patients (OR 8.00; 95% CI 0.95–67.7, p = .06). Multivariable logistic regression analysis correcting for age and type of index event revealed that IPH was independently associated with DWI lesions in the waiting period till surgery (OR 10.8; 95% CI 1.17–99.9, p = .04). Conclusion Symptomatic patients with ipsilateral carotid stenosis and silent brain ischaemia on pre-operative MR-DWI, more often showed pathological evidence of IPH compared with those without ischaemic lesions. This identifies carotid IPH as a marker for patients at risk of silent brain ischaemia and possibly for future stroke and other arterial disease complications. Such patients may be more likely to benefit from CEA than those without evidence of ipsilateral carotid IPH.

Background and aims The sex- and age-related differences in the composition of iliofemoral atherosclerotic plaques are largely unknown. Therefore, the aim of the current study is to gain insight into plaque composition across strata of age and sex in a large cohort of vascular surgery patients. Methods Peripheral atherosclerotic plaques of patients who underwent iliofemoral endarterectomy (n = 790) were harvested between 2002 and 2014. The plaques were semi-quantitatively analyzed for the presence of lipid cores, calcifications, plaque hemorrhages (PH), collagen, macrophage and smooth muscle cell (SMC) content, and quantitatively for microvessel density. Patients were stratified by age tertiles and sex. Results Ageing was independently associated with rupture-prone iliofemoral plaque characteristics, such as higher prevalence of plaque calcifications (OR 1.52 (95%CI:1.03–2.24) p = 0.035) and PH (OR 1.46 (95%CI:1.01–2.09) p = 0.042), and lower prevalence of collagen (OR 0.52 (95%CI:0.31–0.86) p = 0.012) and SMCs (OR 0.59 (95%CI:0.39–0.90) p = 0.015). Sex-stratified data showed that men had a higher prevalence of lipid cores (OR 1.62 (95%CI:1.06–2.45) p = 0.025) and PH (OR 1.62 (95%CI:1.16–2.54) p = 0.004) compared to women. These sex-differences attenuated with increasing age, with women showing an age-related increase in calcifications (p = 0.002), PH (p = 0.015) and decrease in macrophages (p = 0.005). In contrast, men only showed a decrease in collagen (p = 0.043). Conclusions Atherosclerotic iliofemoral plaques derived from men display more rupture-prone characteristics compared to women. Yet, this difference is attenuated with an increase in age, with older women having more rupture-prone characteristics compared to younger women.

Introduction Knowledge on factors that influence atherosclerotic plaque composition may allow improved risk stratification and treatment selection in iliofemoral and carotid atherosclerotic disease, since outcomes of different treatment strategies are influenced by the type of underlying lesions. The sex- and age-related differences in iliofemoral atherosclerotic plaque composition is largely unknown. Unravelling the intertwined relation between sex, age and plaque composition might provide important implications for the treatment of peripheral artery disease. Purpose We aimed to elucidate the associations between sex, age and plaque composition in a histopathological analysis of plaque specimens of patients undergoing iliofemoral endarterectomy. Given the systemic nature of atherosclerosis, analyses are replicated in atherosclerotic plaque specimens obtained from patients undergoing carotid surgery. Methods Peripheral atherosclerotic plaques of 790 patients who underwent iliofemoral endarterectomy were harvested between 2002 and 2014. A cohort of patients (n=2006) who underwent carotid endarterectomy was used to replicate analyses on sex-specific aging effects in plaques from a different vascular bed. The atherosclerotic plaques were semi-quantitatively analyzed for the presence of lipid cores, plaque calcifications, plaque hemorrhages and collagen, macrophage and smooth muscle cell content, and quantitatively for microvessel density. Patients were stratified by age tertiles and by sex. Results Men had a higher prevalence of lipid cores (25.7% versus 20.5%, odds ratio [OR] 1.62 and 95% confidence interval [CI] 1.06–2.45, P=0.025) and plaque hemorrhage (54.3% versus 42.9%, OR 1.62 and 95% CI 1.16–2.54, P=0.004) when compared to women. Women showed an increase in plaque calcifications, plaque hemorrhage and a decrease in macrophages with increasing age (figure panel B, D, E), whereas men only showed a decrease in collagen content (figure panel C). These sex-specific aging effects were replicated in plaques obtained from the carotid arteries. s Conclusion Atherosclerotic iliofemoral plaques derived from men display more rupture-prone characteristics compared to women. However, advanced age was more often associated with an increase in the presence of vulnerable plaque characteristics in women as compared to men in both the iliofemoral and carotid atherosclerotic plaque.

BackgroundFamily history (FHx) of cardiovascular disease (CVD) is a risk factor for CVD and a proxy for cardiovascular heritability. Polygenic risk scores (PRS) summarizing >1 million variants for coronary artery disease (CAD) are associated with incident and recurrent CAD events. However, little is known about the influence of FHx or PRS on secondary cardiovascular events (sCVE) in patients undergoing carotid endarterectomy (CEA).MethodsWe included 1,788 CEA patients from the Athero-Express Biobank. A weighted PRS for CAD including 1.7 million variants was calculated (MetaGRS). The composite endpoint of sCVE during three years follow-up included coronary, cerebrovascular and peripheral events and cardiovascular death. We assessed the impact of FHx and MetaGRS on sCVE and carotid plaque composition.ResultsPositive FHx was associated with a higher 3-year risk of sCVE independent of cardiovascular risk factors and MetaGRS (adjusted HR 1.40, 95%CI 1.07-1.82, p=0.013). Patients in the highest MetaGRS quintile had a higher 3-year risk of sCVE compared to the rest of the cohort independent of cardiovascular risk factors including FHx (adjusted HR 1.35, 95%CI 1.01-1.79, p=0.043), and their atherosclerotic plaques contained more fat (adjusted OR 1.59, 95%CI, 1.11-2.29, p=0.013) and more macrophages (OR 1.49, 95%CI 1.12-1.99, p=0.006).ConclusionIn CEA patients, both positive FHx and higher MetaGRS were independently associated with increased risk of sCVE. Moreover, higher MetaGRS was associated with vulnerable plaque characteristics. Future studies should unravel underlying mechanisms and focus on the added value of PRS and FHx in individual risk prediction for sCVE.

Objective Cerebral white matter lesions (WMLs) and lacunar infarcts are surrogates of cerebral small vessel disease (SVD). WML severity as determined by trained radiologists predicts post-operative stroke or death in patients undergoing carotid endarterectomy (CEA). It is unknown whether routine pre-operative brain imaging reports as part of standard clinical practice also predict short and long term risk of stroke and death after CEA. Methods Consecutive patients from the Athero-Express biobank study that underwent CEA for symptomatic high degree stenosis between March 2002 and November 2014 were included. Pre-operative brain imaging (computed tomography [CT] or magnetic resonance imaging [MRI]) reports were reviewed for reporting of SVD, defined as WMLs or any lacunar infarcts. The primary outcome was defined as any stroke or any cardiovascular death over three year follow up. The secondary outcome was defined as the 30 day peri-operative risk of stroke or cardiovascular death. Results A total of 1038 patients were included (34% women), of whom 659 (63.5%) had CT images and 379 (36.5%) MRI images available. Of all patients, 697 (67%) had SVD reported by radiologists. Patients with SVD had a higher three year risk of cardiovascular death than those without (6.5% vs. 2.1%, adjusted HR 2.52 [95% CI 1.12–5.67]; p = .026) but no association was observed for the three year risk of stroke (9.0% vs. 6.7%, for patients with SVD vs. those without, adjusted HR 1.24 [95% CI 0.76–2.02]; p = .395). No differences in 30 day peri-operative risk were observed for stroke (4.4% vs. 2.9%, for patients with vs. those without SVD; adjusted HR 1.49 [95% CI 0.73–3.05]; p = .28), and for the combined stroke/cardiovascular death risk (4.4% vs. 3.5%, adjusted HR 1.20 [95% CI 0.61–2.35]; p = .59). Conclusion Presence of SVD in pre-operative brain imaging reports can serve as a predictor for the three year risk of cardiovascular death in symptomatic patients undergoing CEA but does not predict peri-operative or long term risk of stroke.

BACKGROUND AND AIMS: Both hypertension and atherosclerotic plaque characteristics such as intraplaque hemorrhage (IPH) are associated with cardiovascular events (CVE). It is unknown if hypertension is associated with IPH. Therefore, we studied if hypertension is associated with unstable atherosclerotic plaque characteristics in patients undergoing carotid endarterectomy (CEA). METHODS: Prospectively collected data of CEA-patients (2002-2014) were retrospectively analyzed. Blood pressure (BP) was the mean of 3 preoperative measurements. Preoperative hypertension was defined as systolic BP ≥ 160 mmHg. Post-CEA, carotid atherosclerotic plaques were analyzed for the presence of calcifications, collagen, smooth muscle cells, macrophages, lipid core, IPH and microvessel density. Associations between BP (systolic and diastolic), patient characteristics and carotid plaque characteristics were assessed with univariate and multivariate analyses with correction for potential confounders. Results were replicated in a cohort of patients that underwent iliofemoral endarterectomy. RESULTS: Within CEA-patients (n = 1684), 708 (42%) had preoperative hypertension. Increased systolic BP was associated with the presence of plaque calcifications (adjusted OR1.11 [95% CI 1.01-1.22], p = 0.03), macrophages (adjusted OR1.12 [1.04-1.21], p < 0.01), lipid core >10% of plaque area (adjusted OR1.15 [1.05-1.25], p < 0.01), IPH (adjusted OR1.12 [1.03-1.21], p = 0.01) and microvessels (adjusted beta 0.04 [0.00-0.08], p = 0.03). Increased diastolic BP was associated with macrophages (adjusted OR1.36 [1.17-1.58], p < 0.01), lipid core (adjusted OR1.29 [1.10-1.53], p < 0.01) and IPH (adjusted OR1.25 [1.07-1.46], p < 0.01) but not with microvessels nor plaque calcifications. Replication in an iliofemoral-cohort (n = 657) showed that increased diastolic BP was associated with the presence of macrophages (adjusted OR1.78 [1.13-2.91], p = 0.01), lipid core (adjusted OR1.45 [1.06-1.98], p = 0.02) and IPH (adjusted OR1.48 [1.14-1.93], p < 0.01). CONCLUSIONS: Preoperative hypertension in severely atherosclerotic patients is associated with the presence of carotid plaque macrophages, lipid core and IPH. IPH, as a plaque marker for CVE, is associated with increased systolic and diastolic BP in both the CEA and iliofemoral population.

Objectives: The metabolic syndrome (MetS) is a cluster of risk factors for cardiovascular disease. The effect of MetS on clinical outcome in patients with cerebrovascular disease remains largely unknown because conflicting results have been published. This study aimed to determine the influence of MetS on the occurrence of restenosis after carotid endarterectomy (CEA).Methods: All patients who underwent CEA between June 2003 and December 2014 in two tertiary academic referral centres in The Netherlands were included. MetS was defined if three or more of the following criteria were present: hypertension, obesity, high fasting serum blood glucose, high serum triglycerides, or low serum high density lipoprotein cholesterol. The primary outcome measure was the occurrence of ipsilateral restenosis after index surgery. The secondary outcome measure was (all cause) mortality during follow up. For the primary analysis, missing data were multiply imputed using multivariable imputation by chained equations. A Cox proportional hazards model was used to perform an adjusted analysis on the multiply imputed data sets.Results: A total of 1668 CEA procedures (in 1577 patients) were performed. The presence or absence of MetS could not be determined in 263 patients because of missing data. There was no significant difference in freedom from restenosis in the MetS group vs. the no-MetS group (hazard ratio [HR], 1.10; 95% confidence interval [CI] 0.98-1.23; p = .10) or in all cause mortality (HR 1.20; 95% CI 0.94-1.54; p = .14).Conclusion: This study shows that MetS does not predict restenosis after CEA. Also, the presence of MetS did not influence patient survival negatively.

Background and aims Family history (FHx) of cardiovascular disease (CVD) is a risk factor for CVD and a proxy for cardiovascular heritability. Polygenic risk scores (PRS) summarizing >1 million variants for coronary artery disease (CAD) are associated with incident and recurrent CAD events. However, little is known about the influence of FHx or PRS on secondary cardiovascular events (sCVE) in patients undergoing carotid endarterectomy (CEA). Methods We included 1788 CEA patients from the Athero-Express Biobank. A weighted PRS for CAD including 1.7 million variants was calculated (MetaGRS). The composite endpoint of sCVE during three years of follow-up included coronary, cerebrovascular and peripheral events and cardiovascular death. We assessed the impact of FHx and MetaGRS on sCVE and carotid plaque composition. Results Positive FHx was associated with a higher 3-year risk of sCVE independent of cardiovascular risk factors and MetaGRS (adjusted HR 1.40, 95%CI 1.07–1.82, p = 0.013). Patients in the highest MetaGRS quintile had a higher 3-year risk of sCVE compared to the rest of the cohort independent of cardiovascular risk factors including FHx (adjusted HR 1.35, 95%CI 1.01–1.79, p = 0.043), and their atherosclerotic plaques contained more fat (adjusted OR 1.59, 95%CI, 1.11–2.29, p = 0.013) and more macrophages (OR 1.49, 95%CI 1.12–1.99, p = 0.006). Conclusions In CEA patients, both positive FHx and higher MetaGRS were independently associated with increased risk of sCVE. Moreover, higher MetaGRS was associated with vulnerable plaque characteristics. Future studies should unravel underlying mechanisms and focus on the added value of PRS and FHx in individual risk prediction for sCVE.

Objective Patients undergoing carotid endarterectomy (CEA) maintain a substantial residual risk of major cardiovascular events (MACE). Improved risk stratification is warranted to select high risk patients qualifying for secondary add on therapy. Plasma extracellular vesicles (EVs) are involved in atherothrombotic processes and their content has been related to the presence and recurrence of cardiovascular events. The association between pre-operative levels of five cardiovascular disease related proteins in plasma EVs and the post-operative risk of MACE was assessed. Methods In 864 patients undergoing CEA from 2002 to 2016 included in the Athero-Express biobank, three plasma EV subfractions (low density lipoprotein [LDL], high density lipoprotein [HDL], and tiny extracellular vesicles [TEX]) were isolated from pre-operative blood samples. Using an electrochemiluminescence immunoassay, five proteins were quantified in each EV subfraction: cystatin C, serpin C1, serpin G1, serpin F2, and CD14. The association between EV protein levels and the three year post-operative risk of MACE (any stroke, myocardial infarction, or cardiovascular death) was evaluated using multivariable Cox proportional hazard regression analyses. Results During a median follow up of three years (interquartile range 2.2 – 3.0), 137 (16%) patients developed MACE. In the HDL-EV subfraction, increased levels of CD14, cystatin C, serpin F2, and serpin C1 were associated with an increased risk of MACE (adjusted hazard ratios per one standard deviation increase of 1.30, 95% confidence interval [CI] 1.15–1.48; 1.22, 95% CI 1.06–1.42; 1.36, 95% CI 1.16–1.61; and 1.29, 95% CI 1.10–1.51; respectively), independently of cardiovascular risk factors. No significant associations were found for serpin G1. CD14 improved the predictive value of the clinical model encompassing cardiovascular risk factors (net re-classification index = 0.16, 95% CI 0.08–0.21). Conclusion EV derived pre-operative plasma levels of cystatin C, serpin C1, CD14, and serpin F2 were independently associated with an increased long term risk of MACE after CEA and are thus markers for residual cardiovascular risk. EV derived CD14 levels could improve the identification of high risk patients who may benefit from secondary preventive add on therapy in order to reduce future risk of MACE.

Ceramides and phosphatidylcholines (PCs) are bioactive lipids and lipid bilayer membrane components. Distinct ceramides/PCs (ratios) predict cardiovascular outcome in patients with coronary artery disease. Extracellular vesicles (EVs) are proposed biomarkers for cardiovascular disease and contain ceramides/PCs. Ceramides/PCs have not been studied in patients undergoing carotid endarterectomy (CEA) nor in EVs. We therefore investigated whether levels of ceramides/PCs in plasma and EVs are associated with postoperative risk of major adverse cardiovascular events (MACE) following CEA. In 873 patients undergoing CEA of the Athero-Express biobank, we quantitatively measured seven ceramides/PCs in preoperative blood samples: Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), Cer(d18:1/24:1), PC(14:0/22:6), PC(16:0/16:0) and PC(16:0/22:5) in plasma and two plasma EV-subfractions (LDL and TEX). We analyzed the association of ceramides, PCs and their predefined ratios with the three-year postoperative risk of MACE (including stroke, myocardial infarction and cardiovascular death). A total of 138 patients (16%) developed MACE during the three-year follow-up. In the LDL-EV subfraction, higher levels of Cer(d18:1/24:1) and Cer(d18:1/16:0)/PC(16:0/22:5) ratio were significantly associated with an increased risk of MACE (adjusted HR per SD [95% CI] 1.24 [1.01–1.53] and 1.26 [1.04–1.52], respectively). In the TEX-EV subfraction, three ratios Cer(d18:1/16:0)/Cer(d18:1/24:0), Cer(d18:1/18:0)/Cer(d18:1/24:0) and Cer(d18:1/24:1)/Cer(d18:1/24:0) were positively associated with MACE (adjusted HR per SD 1.34 [1.06–1.70], 1.24 [1.01–1.51] and 1.31 [1.08–1.58], respectively). In conclusion, distinct ceramides and PCs in plasma EVs determined in preoperative blood were independently associated with an increased 3-year risk of MACE after CEA. These lipids are therefore potential markers to identify high-risk CEA patients qualifying for secondary preventive add-on therapy.