A narrative review of plaque and brain imaging biomarkers for stroke risk stratification in patients with atherosclerotic carotid artery disease

Objective: In this narrative review, we aim to review imaging biomarkers that carry the potential to non-invasively guide stroke risk stratification for treatment optimization. Background: Carotid atherosclerosis plays a fundamental part in the occurrence of ischemic stroke. International guidelines select the optimal treatment strategy still mainly based on the presence of clinical symptoms and the degree of stenosis for stroke prevention in patients with atherosclerotic carotid plaques. These guidelines, based on randomized controlled trials that were conducted three decades ago, recommend carotid revascularization in symptomatic patients with high degree of stenosis versus a conservative approach for most asymptomatic patients. Due to optimization of best medical therapy and risk factor control, it is suggested that a subgroup of symptomatic patients is at lower risk of stroke and may not benefit from revascularization, whereas a selective subgroup of high-risk asymptomatic patients would benefit from this procedure. Methods: A literature search was performed for articles published up to December 2020 using PubMed, EMBASE and Scopus. Based on the literature found, change in stenosis degree and volume, plaque echolucency, plaque surface, intraplaque haemorrhage, lipid-rich necrotic core, thin fibrous cap, inflammation, neovascularization, microembolic signals, cerebrovascular reserve, intracranial collaterals, silent brain infarcts, diffusion weighted imaging lesions and white matters lesions have the potential to predict stroke risk. Conclusions: The applicability of imaging biomarkers needs to be further improved before the potential synergistic prognostic ability of imaging biomarkers can be verified on top of the clinical biomarkers. In the future, the routine and combined assessment of both plaque and brain imaging biomarkers might help to improve optimization of treatment strategies in individual patients with atherosclerotic carotid artery disease

Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles

Plasma extracellular vesicles (EVs) are lipid membrane vesicles involved in several biological processes including coagulation. Both coagulation and lipid metabolism are strongly associated with cardiovascular events. Lowering very-low- and low-density lipoprotein ((V)LDL) particles via dextran sulphate LDL apheresis also removes coagulation proteins. It remains unknown, however, how coagulation proteins are removed in apheresis. We hypothesize that plasma EVs that contain high levels of coagulation proteins are concomitantly removed with (V)LDL particles by dextran sulphate apheresis. For this, we precipitated (V)LDL particles from human plasma with dextran sulphate and analyzed the abundance of coagulation proteins and EVs in the precipitate. Coagulation pathway proteins, as demonstrated by proteomics and a bead-based immunoassay, were over-represented in the (V)LDL precipitate. In this precipitate, both bilayer EVs and monolayer (V)LDL particles were observed by electron microscopy. Separation of EVs from (V)LDL particles using density gradient centrifugation revealed that almost all coagulation proteins were present in the EVs and not in the (V)LDL particles. These EVs also showed a strong procoagulant activity. Our study suggests that dextran sulphate used in LDL apheresis may remove procoagulant EVs concomitantly with (V)LDL particles, leading to a loss of coagulation proteins from the blood

Inhibition of PFKFB3 Hampers the Progression of Atherosclerosis and Promotes Plaque Stability

Aims: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3-mediated glycolysis is pivotal in driving macrophage- and endothelial cell activation and thereby inflammation. Once activated, these cells play a crucial role in the progression of atherosclerosis. Here, we analyzed the expression of PFKFB3 in human atherosclerotic lesions and investigated the therapeutic potential of pharmacological inhibition of PFKFB3 in experimental atherosclerosis by using the glycolytic inhibitor PFK158. Methods and Results: PFKFB3 expression was higher in vulnerable human atheromatous carotid plaques when compared to stable fibrous plaques and predominantly expressed in plaque macrophages and endothelial cells. Analysis of advanced plaques of human coronary arteries revealed a positive correlation of PFKFB3 expression with necrotic core area. To further investigate the role of PFKFB3 in atherosclerotic disease progression, we treated 6–8 weeks old male Ldlr–/– mice. These mice were fed a high cholesterol diet for 13 weeks, of which they were treated for 5 weeks with the glycolytic inhibitor PFK158 to block PFKFB3 activity. The incidence of fibrous cap atheroma (advanced plaques) was reduced in PFK158-treated mice. Plaque phenotype altered markedly as both necrotic core area and intraplaque apoptosis decreased. This coincided with thickening of the fibrous cap and increased plaque stability after PFK158 treatment. Concomitantly, we observed a decrease in glycolysis in peripheral blood mononuclear cells compared to the untreated group, which alludes that changes in the intracellular metabolism of monocyte and macrophages is advantageous for plaque stabilization. Conclusion: High PFKFB3 expression is associated with vulnerable atheromatous human carotid and coronary plaques. In mice, high PFKFB3 expression is also associated with a vulnerable plaque phenotype, whereas inhibition of PFKFB3 activity leads to plaque stabilization. This data implies that inhibition of inducible glycolysis may reduce inflammation, which has the ability to subsequently attenuate atherogenesis

The age- and sex-specific composition of atherosclerotic plaques in vascular surgery patients

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

Carotid atherosclerotic plaque matrix metalloproteinase-12-positive macrophage subpopulation predicts adverse outcome after endarterectomy

BACKGROUND: Matrix metalloproteinase-12 (MMP-12) promotes atherosclerosis in animal models. MMP-12 is expressed in only a subset of foam-cell macrophages (FCMs) in human plaques. We investigated whether the prevalence of this MMP-12–expressing subpopulation is a prognostic indicator of adverse outcome in patients after carotid endarterectomy (CEA). METHODS AND RESULTS: Serial sections of culprit lesions from 236 patients who underwent CEA and had undergone 3 years of clinical follow-up were stained immunocytochemically for MMP-12 and for CD68, and the MMP-12/CD68 ratio was used to quantify the MMP-12–expressing subpopulation. A high MMP-12/CD68 ratio correlated with a high content of lipid and total macrophages and a low content of vascular smooth muscle cells, as well as with MMP-8 (R=0.211, P=0.001), MMP-9 (R=0.251, P<0.001), and cleaved caspase-3 (R=0.142, P=0.036) activity measured in a neighboring segment. Dual immunohistochemical examination confirmed the location of MMP-12 in a subpopulation of MMP-8– and MMP-9–positive FCMs, whereas all apoptotic FCMs were MMP-12 positive. Patients who yielded plaques within the highest quartile compared with the lowest quartile of MMP-12/CD68 ratio had a 2.4-fold (hazard ratio, 2.4; 95% CI, 1.1- to 5.1-fold; adjusted P=0.027) increased risk of major adverse cardiovascular event and a 3.4-fold (3.4; 1.2- to 9.6-fold, P=0.024) increased risk for stroke. CONCLUSIONS: The prevalence of an MMP-12–positive subset of FCMs is a prognostic marker for adverse clinical outcome after CEA

Delayed Development of Aneurysmal Dilatations in Patients with Extracranial Carotid Artery Dissections

Peer reviewe

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 C-reactive protein is associated with a pro-inflammatory and adverse plaque phenotype

BACKGROUND AND AIMS: Systemic low-grade inflammation, measured by plasma high-sensitivity C-reactive protein (hsCRP) levels, is an important risk factor for atherosclerotic cardiovascular disease (ASCVD). To date, however, it is unknown whether plasma hsCRP is associated with adverse histological plaque features. METHODS: Plaques were derived during carotid endarterectomy. Patients with hsCRP levels ≥2 mg/L were evaluated for pro-inflammatory and adverse plaque characteristics, as well as future ASCVD events, and compared with patients with low hsCRP levels. Logistic and linear regression analyses in addition to subdistribution hazard ratios were conducted, adjusted for cardiovascular risk factors. RESULTS: A total of 1096 patients were included, of which 494 (46.2 %) had hsCRP levels ≥2 mg/L. Elevated hsCRP levels 2 mg/L were independently associated with levels of plaque interleukin 6, beta coefficient of 109.8 (95 % confidence interval (CI): 33.4, 186.5; p = 0.005) pg/L, interleukin 8 levels, 194.8 (110.4, 378.2; p = 0.03) pg/L and adiponectin plaque levels, -16.8 (-30.1, -3.6; p = 0.01) μg/L, compared with plaques from patients with low hsCRP levels. Histological analysis revealed increased vessel density in high hsCRP patients, odds ratio (OR) of 1.57 (1.20, 2.09; p = 0.001), larger lipid core, 1.35 (1.02, 1.73; p = 0.04), and increased macrophage content, 1.32 (1.02, 1.73; p = 0.04). Over a 3-year follow-up period, hsCRP levels ≥2 mg/L were associated with a hazard ratio of 1.81 (1.03, 3.16; p = 0.04) for coronary artery disease event risk. CONCLUSIONS: The distinct inflammatory and histological features observed in carotid plaques among individuals with hsCRP levels ≥2 mg/L underscore the utility of plasma hsCRP as a potent identifier for patients harboring high-risk plaques

Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes

Secreted by activated cells or passively released by damaged cells, extracellular HMGB1 is a prototypical damage-associated molecular pattern (DAMP) inflammatory mediator. During the course of developing extracorporeal approaches to treating injury and infection, we inadvertently discovered that haptoglobin, the acute phase protein that binds extracellular hemoglobin and targets cellular uptake through CD163, also binds HMGB1. Haptoglobin-HMGB1 complexes elicit the production of antiinflammatory enzymes (heme oxygenase-1) and cytokines (e.g., IL-10) in WT but not in CD163-deficient macrophages. Genetic disruption of haptoglobin or CD163 expression significantly enhances mortality rates in standardized models of intra-abdominal sepsis in mice. Administration of haptoglobin to WT and to haptoglobin gene-deficient animals confers significant protection. These findings reveal a mechanism for haptoglobin modulation of the inflammatory action of HMGB1, with significant implications for developing experimental strategies targeting HMGB1-dependent inflammatory diseases

Damage-Associated Molecular Patterns in Myocardial Infarction and Heart Transplantation: The Road to Translational Success

In the setting of myocardial infarction (MI), ischemia reperfusion injury (IRI) occurs due to occlusion (ischemia) and subsequent re-establishment of blood flow (reperfusion) of a coronary artery. A similar phenomenon is observed in heart transplantation (HTx) when, after cold storage, the donor heart is connected to the recipient's circulation. Although reperfusion is essential for the survival of cardiomyocytes, it paradoxically leads to additional myocardial damage in experimental MI and HTx models. Damage (or danger)-associated molecular patterns (DAMPs) are endogenous molecules released after cellular damage or stress such as myocardial IRI. DAMPs activate pattern recognition receptors (PRRs), and set in motion a complex signaling cascade resulting in the release of cytokines and a profound inflammatory reaction. This inflammatory response is thought to function as a double-edged sword. Although it enables removal of cell debris and promotes wound healing, DAMP mediated signalling can also exacerbate the inflammatory state in a disproportional matter, thereby leading to additional tissue damage. Upon MI, this leads to expansion of the infarcted area and deterioration of cardiac function in preclinical models. Eventually this culminates in adverse myocardial remodeling; a process that leads to increased myocardial fibrosis, gradual further loss of cardiomyocytes, left ventricular dilation and heart failure. Upon HTx, DAMPs aggravate ischemic damage, which results in more pronounced reperfusion injury that impacts cardiac function and increases the occurrence of primary graft dysfunction and graft rejection via cytokine release, cardiac edema, enhanced myocardial/endothelial damage and allograft fibrosis. Therapies targeting DAMPs or PRRs have predominantly been investigated in experimental models and are potentially cardioprotective. To date, however, none of these interventions have reached the clinical arena. In this review we summarize the current evidence of involvement of DAMPs and PRRs in the inflammatory response after MI and HTx. Furthermore, we will discuss various current therapeutic approaches targeting this complex interplay and provide possible reasons why clinical translation still fails