The accumulation of erythrocytes quantified and visualized by Glycophorin C in carotid atherosclerotic plaque reflects intraplaque hemorrhage and pre-procedural neurological symptoms.

The accumulation of erythrocyte membranes within an atherosclerotic plaque may contribute to the deposition of free cholesterol and thereby the enlargement of the necrotic core. Erythrocyte membranes can be visualized and quantified in the plaque by immunostaining for the erythrocyte marker glycophorin C. Hence, we theorized that the accumulation of erythrocytes quantified by glycophorin C could function as a marker for plaque vulnerability, possibly reflecting intraplaque hemorrhage (IPH), and offering predictive value for pre-procedural neurological symptoms. We employed the CellProfiler-integrated slideToolKit workflow to visualize and quantify glycophorin C, defined as the total plaque area that is positive for glycophorin C, in single slides of culprit lesions obtained from the Athero-Express Biobank of 1819 consecutive asymptomatic and symptomatic patients who underwent carotid endarterectomy. Our assessment included the evaluation of various parameters such as lipid core, calcifications, collagen content, SMC content, and macrophage burden. These parameters were evaluated using a semi-quantitative scoring method, and the resulting data was dichotomized as predefined criteria into categories of no/minor or moderate/heavy staining. In addition, the presence or absence of IPH was also scored. The prevalence of IPH and pre-procedural neurological symptoms were 62.4% and 87.1%, respectively. The amount of glycophorin staining was significantly higher in samples from men compared to samples of women (median 7.15 (IQR:3.37, 13.41) versus median 4.06 (IQR:1.98, 8.32), p < 0.001). Glycophorin C was associated with IPH adjusted for clinical confounders (OR 1.90; 95% CI 1.63, 2.21; p = < 0.001). Glycophorin C was significantly associated with ipsilateral pre-procedural neurological symptoms (OR:1.27, 95%CI:1.06-1.41, p = 0.005). Sex-stratified analysis, showed that this was also the case for men (OR 1.37; 95%CI 1.12, 1.69; p = 0.003), but not for women (OR 1.15; 95%CI 0.77, 1.73; p = 0.27). Glycophorin C was associated with classical features of a vulnerable plaque, such as a larger lipid core, a higher macrophage burden, less calcifications, a lower collagen and SMC content. There were marked sex differences, in men, glycophorin C was associated with calcifications and collagen while these associations were not found in women. To conclude, the accumulation of erythrocytes in atherosclerotic plaque quantified and visualized by glycophorin C was independently associated with the presence of IPH, preprocedural symptoms in men, and with a more vulnerable plaque composition in both men and women. These results strengthen the notion that the accumulation of erythrocytes quantified by glycophorin C can be used as a marker for plaque vulnerability.

X chromosome inactivation skewing is common in advanced carotid atherosclerotic lesions in females and predicts secondary peripheral artery events.

BACKGROUND AND AIM: Sex differences in atherosclerosis have been described with female plaques being mostly perceived as stable and fibrous. Sex-specific mechanisms such as mosaic loss of the Y chromosome in men have been linked to cardiovascular health. In women, X-linked mechanisms such as X chromosome inactivation (XCI) skewing is common in several tissues. Yet, information on the role of XCI in female atherosclerotic plaques is lacking. Here, we investigated the presence of XCI skewing in advanced atherosclerotic lesions and its association with cardiovascular risk factors, histological plaque data, and clinical data. METHODS: XCI skewing was quantified in 154 atherosclerotic plaque and 55 blood DNA samples of women included in the Athero-Express study. The skewing status was determined performing the HUMARA assay. Then, we studied the relationship of XCI skewing in female plaque and cardiovascular risk factors using regression models. In addition, we studied if plaque XCI predicted plaque composition, and adverse events during 3-years follow-up using Cox proportional hazard models. RESULTS: XCI skewing was detected in 76 of 154 (49.4%) plaques and in 27 of 55 (67%) blood samples. None of the clinical risk factors were associated with plaque skewing. Plaque skewing was more often detected in plaques with a plaque hemorrhage (OR [95% CI]: 1.44 [1.06-1.98], P = 0.02). Moreover, skewed plaques were not associated with a higher incidence of composite and major events but were specifically associated with peripheral artery events during a 3-year follow-up period in a multivariate model (HR [95%CI]: 1.46 [1.09-1.97]; P = 0.007). CONCLUSIONS: XCI skewing is common in carotid plaques of females and is predictive for the occurrence of peripheral artery events within 3 years after carotid endarterectomy.

Sex-differences have been observed in the development of atherosclerosis between men and women. Women tend to have more stable and fibrous plaques compared to men. Sex-specific mechanisms such as mosaic loss of the Y chromosome in men, were associated with cardiovascular health. In women, despite X-linked mechanisms like X chromosome inactivation (XCI) skewing was identified in various tissues. However, its relationship with atherosclerosis has not yet been investigated. In our study, we explored if prevalence of XCI skewing in advanced atherosclerotic lesions related to cardiovascular risk factors, histological plaque data, and clinical information. We found that XCI skewing was present in approximately 50% of human plaques, particularly those with plaque hemorrhage. Interestingly, we did not find any notable relationship between plaque skewing and clinical risk factors. However, we found that XCI was more present in women with peripheral artery events during the 3 years period following carotid endarterectomy. In summary, our findings indicate that XCI skewing is commonly observed in carotid plaques among females and may serve as a predictive factor for the occurrence of peripheral artery events within 3 years after carotid endarterectomy.

Characteristics of peripheral blood cells are independently related to major adverse cardiovascular events after carotid endarterectomy.

Background and aims: Patients who underwent carotid endarterectomy (CEA) still have a residual risk of 13% of developing a major adverse cardiovascular event (MACE) within 3 years. Inflammatory processes leading up to MACE are not fully understood. Therefore, we examined blood cell characteristics (BCCs), possibly reflecting inflammatory processes, in relation to MACE to identify BCCs that may contribute to an increased risk. Methods: We analyzed 75 pretreatment BCCs from the Sapphire analyzer, and clinical data from the Athero-Express biobank in relation to MACE after CEA using Random Survival Forests, and a Generalized Additive Survival Model. To understand biological mechanisms, we related the identified variables to intraplaque hemorrhage (IPH). Results: Of 783 patients, 97 (12%) developed MACE within 3 years after CEA. Red blood cell distribution width (RDW) (HR 1.23 [1.02, 1.68], p = 0.022), CV of lymphocyte size (LACV) (HR 0.78 [0.63, 0.99], p = 0.043), neutrophil complexity of the intracellular structure (NIMN) (HR 0.80 [0.64, 0.98], p = 0.033), mean neutrophil size (NAMN) (HR 0.67 [0.55, 0.83], p < 0.001), mean corpuscular volume (MCV) (HR 1.35 [1.09, 1.66], p = 0.005), eGFR (HR 0.65 [0.52, 0.80], p < 0.001); and HDL-cholesterol (HR 0.62 [0.45, 0.85], p = 0.003) were related to MACE. NAMN was related to IPH (OR 0.83 [0.71-0.98], p = 0.02). Conclusions: This is the first study to present a higher RDW and MCV and lower LACV, NIMN and NAMN as biomarkers reflecting inflammatory processes that may contribute to an increased risk of MACE after CEA.

An automatic entropy method to efficiently mask histology whole-slide images.

Tissue segmentation of histology whole-slide images (WSI) remains a critical task in automated digital pathology workflows for both accurate disease diagnosis and deep phenotyping for research purposes. This is especially challenging when the tissue structure of biospecimens is relatively porous and heterogeneous, such as for atherosclerotic plaques. In this study, we developed a unique approach called 'EntropyMasker' based on image entropy to tackle the fore- and background segmentation (masking) task in histology WSI. We evaluated our method on 97 high-resolution WSI of human carotid atherosclerotic plaques in the Athero-Express Biobank Study, constituting hematoxylin and eosin and 8 other staining types. Using multiple benchmarking metrics, we compared our method with four widely used segmentation methods: Otsu's method, Adaptive mean, Adaptive Gaussian and slideMask and observed that our method had the highest sensitivity and Jaccard similarity index. We envision EntropyMasker to fill an important gap in WSI preprocessing, machine learning image analysis pipelines, and enable disease phenotyping beyond the field of atherosclerosis.

Plasma Lipoprotein Lipase Is Associated with Risk of Future Major Adverse Cardiovascular Events in Patients Following Carotid Endarterectomy.

INTRODUCTION: Carotid plaque intraplaque haemorrhage (IPH) is associated with future cardiovascular events. It was hypothesised that plasma proteins associated with carotid plaque IPH are also likely to be associated with major adverse cardiovascular events (MACE) after carotid endarterectomy (CEA). METHODS: In pre-operative blood samples from patients undergoing CEA within the Athero-Express biobank, proteins involved in cardiovascular disease were measured using three OLINK proteomics immunoassays. The association between proteins and IPH was analysed using logistic regression analyses. Subsequently, the association between the IPH associated plasma proteins and the three year post-operative risk of MACE (including stroke, myocardial infarction, or cardiovascular death) was analysed. RESULTS: Within the three year follow up, 130 patients (18.9%) of 688 symptomatic and asymptomatic patients undergoing CEA developed MACE. Six of 276 plasma proteins were found to be significantly associated with IPH, from which only lipoprotein lipase (LPL) was associated with the post-operative risk of MACE undergoing CEA. Within the 30 day peri-operative period, high plasma LPL was independently associated with an increased risk of MACE (adjusted hazard ratio [HR] per standard deviation [SD] 1.60, 1.10 - 2.30), p = .014). From 30 days to three years, however, high LPL was associated with a lower risk of MACE (adjusted HR per SD 0.80, 0.65 - 0.99, p= .036). CONCLUSION: High LPL concentrations were found to be associated with a higher risk of MACE in the first 30 post-operative days but with a lower risk MACE between 30 days and three years, meaning that LPL has different hazards at different time points.

Plasma Extracellular Vesicle Serpin G1 and CD14 Levels are Associated with Major Adverse Cardiovascular Events and Major Adverse Limb Events in Patients Undergoing Femoral Endarterectomy.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Serpin G1 and CD14 plasma EV protein levels are associated with future MACE and MALE in patients with severe PAD.

Editor's Choice - Risk of Bleeding Complications With Different Peri-Operative Antithrombotic Regimens During Carotid Endarterectomy: a National Registry Analysis.

OBJECTIVE: Antithrombotic therapy is one of the cornerstones of the prevention of (recurrent) ocular or cerebral ischaemic events in patients with carotid artery stenosis. Randomised controlled trials on antithrombotic therapy for patients with minor ischaemic stroke and transient ischaemic attack (TIA) have recommended dual antiplatelet therapy (DAPT) in the three weeks following the index event. However, these trials excluded patients undergoing carotid revascularisation. To date, the optimal antithrombotic therapy during the peri-operative period of carotid endarterectomy (CEA) remains unclear. METHODS: Symptomatic and asymptomatic patients with carotid artery stenosis undergoing primary CEA from the Dutch Audit for Carotid Interventions registry between June 2013 and December 2020 were eligible for inclusion. The primary outcome was defined as post-operative cervical bleeding needing re-intervention or intracranial haemorrhage during the first 30 days following CEA. The secondary outcomes were ischaemic stroke or TIA or all cause mortality during the first 30 days following CEA. Descriptive statistics and multiple logistic regressions analyses were applied, with acetylsalicylic acid (ASA) as the reference value. RESULTS: A total of 12 317 patients were included. In the peri-operative phase, 31.0% of patients were treated with ASA, 32.4% with clopidogrel, 11.1% with ASA plus clopidogrel, 10.4% with ASA plus dipyridamole, 10.3% with vitamin K antagonist, and 4.8% with direct acting oral anticoagulants therapy. After multiple logistic regression analysis, no association was seen with the primary outcome in ASA plus clopidogrel (odds ratio [OR] 0.81; confidence interval [CI] 0.58 - 1.13; p = .23), and ASA plus dipyridamole (OR 0.69; CI 0.47 - 1.00; p = .059). Both the DAPT therapies were not associated with the secondary outcome. CONCLUSION: The effectiveness and safety of DAPT did not differ from single antiplatelet therapy (SAPT) in patients undergoing CEA and further evaluation is needed in prospective studies. Considering additional data from the literature and guideline recommendations, DAPT should be started immediately after stroke until 30 days after CEA followed by SAPT, due to a possible reduction in the risk of recurrence.

Transcriptomic-based clustering of human atherosclerotic plaques identifies subgroups with different underlying biology and clinical presentation.

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. Here we analyze the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These plaque phenotypes were associated with clinical presentation and showed differences in cellular compositions. Validation in coronary segments showed that the molecular signature of these plaques was linked to coronary ischemia. One of the plaque types with the most severe clinical symptoms pointed to both inflammatory and fibrotic cell lineages. Further, we did a preliminary analysis of potential circulating biomarkers that mark the different plaques phenotypes. In 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 distinct underlying biology of symptomatic lesions.

High lipoprotein(a) is associated with major adverse limb events after femoral artery endarterectomy.

BACKGROUNDS AND AIMS: Elevated lipoprotein(a) (Lp[a]) has been identified as a causal risk factor for cardiovascular disease including peripheral arterial disease (PAD). Although Lp(a) is associated with the diagnosis of PAD, it remains elusive whether there is an association of Lp(a) with cardiovascular and limb events in patients with severe PAD. METHODS: Preoperative plasma Lp(a) levels were measured in 384 consecutive patients that underwent iliofemoral endarterectomy and were included in the Athero-Express biobank. Our primary objective was to assess the association of Lp(a) levels with Major Adverse Limb Events (MALE). Our secondary objective was to relate Lp(a) levels to Major Adverse Cardiovascular Events (MACE) and femoral plaque composition that was acquired from baseline surgery. RESULTS: During a median follow-up time of 5.6 years, a total of 225 MALE were recorded in 132 patients. Multivariable analysis, including history of peripheral intervention, age, diabetes mellitus, end stage renal disease and PAD disease stages, showed that Lp(a) was independently associated with first (HR of 1.36 (95% CI 1.02-1.82) p = .036) and recurrent MALE (HR 1.36 (95% CI 1.10-1.67) p = .004). A total of 99 MACE were recorded but Lp(a) levels were not associated with MACE.sLp(a) levels were significantly associated with a higher presence of smooth muscle cells in the femoral plaque, although this was not associated with MALE or MACE. CONCLUSIONS: Plasma Lp(a) is independently associated with first and consecutive MALE after iliofemoral endarterectomy. Hence, in patients who undergo iliofemoral endarterectomy, Lp(a) could be considered as a biomarker to enhance risk stratification for future MALE.

Mast Cell Distribution in Human Carotid Atherosclerotic Plaque Differs Significantly by Histological Segment.

OBJECTIVE: 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. METHODS: Twenty-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. RESULTS: A 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. CONCLUSION: MC 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.

Monocyte-Chemoattractant Protein-1 Levels in Human Atherosclerotic Lesions Associate With Plaque Vulnerability.

[Figure: see text].

Circulating Tumor DNA Analysis: Clinical Implications for Colorectal Cancer Patients. A Systematic Review.

BACKGROUND: Liquid biopsies could improve diagnosis, prognostication, and monitoring of colorectal cancer (CRC). Mutation, chromosomal copy number alteration, and methylation analysis in circulating tumor DNA (ctDNA) from plasma or serum has gained great interest. However, the literature is inconsistent on preferred candidate markers, hampering a clear direction for further studies and clinical translation. This review assessed the potential of ctDNA analysis for clinical utility. METHODS: A systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines was conducted up to December 3, 2018, followed by methodological quality assessment. Primary endpoints were accuracy for detection, prognostication, and monitoring. RESULTS: Eighty-four studies were included. For CRC detection, sensitivity was 75% using ctDNA mutation analysis and up to 96% using copy number analysis. Septin 9 (SEPT9) hypermethylation analysis showed sensitivities of 100% and specificities of 97%. Regarding prognostication, ctDNA KRAS mutations were associated with oncological outcome and could predict response to anti-epidermal growth factor receptor therapy. For monitoring, sequential ctDNA KRAS mutation analysis showed promise for detection of relapses or therapy resistance. CONCLUSIONS: This comprehensive overview of ctDNA candidate markers demonstrates SEPT9 methylation analysis to be promising for CRC detection, and KRAS mutation analysis could assist in prognostication and monitoring. Prospective evaluation of marker panels in clinical decision making should bring ctDNA analysis into practice.