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Atherosclerosis (AS) is the leading cause of coronary heart disease, which is the primary cause of death worldwide. Recent studies have identified disulfidptosis as a new type of cell death that may be involved in onset and development of many diseases. However, the role of disulfidptosis in AS is not clear. In this study, bioinformatics analysis and experiments in vivo and in vitro were performed to evaluate the potential relationship between disulfidptosis and AS. AS-related sequencing data were obtained from the Gene Expression Omnibus (GEO). Bioinformatics techniques were used to evaluate differentially expressed genes (DEGs) associated with disulfidptosis-related AS. Hub genes were screened using least absolute shrinkage and selection operator (LASSO) and random forests (RF) methods. In addition, we established a foam cell model in vitro and an AS mouse model in vivo to verify the expressions of hub genes. In addition, we constructed a diagnostic nomogram with hub genes to predict progression of AS. Finally, the consensus clustering method was used to establish two different subtypes, and associations between subtypes and immunity were explored. As the results, 9 disulfidptosis-related AS DEGs were identified from GSE28829 and GSE43292 datasets. Evaluation of DEGs using LASSO and RF methods resulted in identification of 4 hub genes (CAPZB, DSTN, MYL6, PDLIM1), which were analyzed for diagnostic value using ROC curve analysis and verified in vitro and in vivo. Furthermore, a nomogram including hub genes was established that accurately predicted the occurrence of AS. The consensus clustering algorithm was used to separate patients with early atherosclerotic plaques and patients with advanced atherosclerotic plaques into two disulfidptosis subtypes. Cluster B displayed higher levels of infiltrating immune cells, which indicated that patients in cluster B may have a positive immune response for progression of AS. In summary, disulfidptosis-related genes including CAPZB, DSTN, MYL6, and PDLIM1 may be diagnostic markers and therapeutic targets for AS. In addition, these genes are closely related to immune cells, which may inform immunotherapy for AS. [ABSTRACT FROM AUTHOR]

Recently, cellular senescence-induced unstable carotid plaques have gained increasing attention. In this study, we utilized bioinformatics and machine learning methods to investigate the correlation between cellular senescence and the pathological mechanisms of unstable carotid plaques. Our aim was to elucidate the causes of unstable carotid plaque progression and identify new therapeutic strategies. First, differential expression analysis was performed on the test set GSE43292 to identify differentially expressed genes (DEGs) between the unstable plaque group and the control group. These DEGs were intersected with cellular senescence-associated genes to obtain 40 cellular senescence-associated DEGs. Subsequently, key genes were then identified through weighted gene co-expression network analysis, random forest, Recursive Feature Elimination for Support Vector Machines algorithm and cytoHubba plugin. The intersection yielded 3 CSA-signature genes, which were validated in the external validation set GSE163154. Additionally, we assessed the relationship between these CSA-signature genes and the immune landscape of the unstable plaque group. This study suggests that cellular senescence may play an important role in the progression mechanism of unstable plaques and is closely related to the influence of the immune microenvironment. Our research lays the foundation for studying the progression mechanism of unstable carotid plaques and provides some reference for targeted therapy. [ABSTRACT FROM AUTHOR]

The anatomical features spanning from the aortic arch to the proximal carotid artery and the associated cardiovascular risks might significantly influence the development of right carotid plaque. Our research aimed to compare these anatomical and risk factors between individuals with no carotid plaque and those with moderate right-side carotid plaque within a Korean cohort. We conducted a retrospective, cross-sectional analysis involving 413 participants, categorized into a normal group (n = 339) and a right moderate carotid plaque group (defined as > 50% stenosis based on NASCET criteria) (n = 74). We collected data on cardiovascular risk factors and conducted laboratory tests. A 3D model of the carotid artery was constructed using cranio-cervical computed tomography angiography (CTA) data through semi-automated software. Measurements taken on this 3D model included the common carotid artery (CCA), internal carotid artery (ICA), external carotid artery (ECA), and carotid artery bifurcation (CAB) in terms of maximal vascular diameter, sectional area, angles of carotid bifurcation and ICA, and carotid tortuosity. When compared with the normal group, individuals in the right moderate carotid plaque group exhibited smaller angles at the carotid bifurcation, larger CCA diameter and sectional area (p < 0.01), advanced age, and a higher incidence of hypertension, diabetes, and stroke history (p < 0.05), along with reduced glomerular filtration rate (GFR) (p < 0.001). Multivariate analysis revealed that the sectional area of the bifurcation, calcification of the aortic bulb, and GFR were independently associated with the presence of right moderate carotid plaque (p < 0.01). Statistical analyses disclosed significant differences in both clinical risk factors and geometric changes in the region extending from the aortic arch to the proximal carotid artery among subjects with right moderate carotid plaque when compared to those without. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the relationship between complex aortic plaque (CAP) and short-term as well as long-term outcomes following cardioembolic stroke. CAP is a known risk factor for occurrence and recurrence of ischemic stroke. However, the association of CAP on cardioembolic stroke remains unclear. This was retrospective study using prospective cohort of consecutive patients with cardioembolic stroke who underwent transesophageal echocardiography. The functional outcome was evaluated using the modified Rankin Scale score at 3 months, and long-term outcomes were assessed by recurrence of ischemic stroke and occurrence of major adverse cardiovascular events (MACE). Among 759 patients with cardioembolic stroke, 91 (12.0%) had CAP. Early ischemic stroke recurrence within 3 months was associated with CAP (p = 0.025), whereas CAP was not associated with functional outcome at 3 months (odd ratio 1.01, 95% confidence interval [CI] 0.57–1.84, p = 0.973). During a median follow-up of 3.02 years, CAP was significantly associated with ischemic stroke recurrence (hazard ratio = 2.68, 95% CI 1.48–4.88, p = 0.001) and MACE occurrence (hazard ratio = 1.61, 95% CI 1.03–2.51, p = 0.039). In conclusion, CAP was associated with early ischemic stroke recurrence and poor long-term outcomes in patients with cardioembolic stroke. It might be helpful to consider transesophageal echocardiography for patients with cardioembolic stroke to identify CAP. [ABSTRACT FROM AUTHOR]

Abstract To investigate the significance of atherosclerotic plaque location in hybrid surgery comprising both endovascular recanalization approaches and carotid endarterectomy for symptomatic atherosclerotic non-acute long-segment occlusion of the internal carotid artery (ICA), 162 patients were enrolled, including 120 (74.1%) patients in the proximal plaque group and 42 (25.9%) in the distal plaque group. Surgical recanalization was performed in all patients, with successful recanalization in 119 (99.2%) patients in the proximal and 39 (92.9%) in the distal plaque group. The total successful recanalization rate was 97.5% (158/162) with a failure rate of 2.5% (4/162). Periprocedural complications occurred in 5 (4.2% or 5/120) patients in the proximal plaque group, including neck infection in two (1.7%), recurrent nerve injury in 1 (0.8%), and laryngeal edema in 2 (1.7%), and 2 (4.8%) in the distal plaque group, including femoral puncture infection in 2 (4.8%). No severe complications occurred in either group. Univariate analysis showed plaque location was a significant (P = 0.018) risk factor for successful recanalization, and multivariate analysis indicated that the plaque location remained a significant independent risk factor for recanalization success (P = 0.017). In follow-up 6–48 months after the recanalization surgery, reocclusion occurred in two (2.8%) patients in the proximal plaque group and 4 (13.3%) in the distal plaque group. In conclusion, although hybrid surgery achieves similar outcomes in patients with ICA occlusion caused by either proximal or distal atherosclerotic plaques, plaque location may be a significant risk factor for successful recanalization of symptomatic non-acute long-segment ICA occlusion.

Abstract Experimental evidence indicates that follicle-stimulating hormone (FSH), an essential hormone for reproduction, can act directly on endothelial cells inducing atherosclerosis activation and development. However, it remains unknown whether the FSH-receptor (FSHR) is expressed in human atherosclerosis plaques. To demonstrate the FSHR presence, we used immunohistochemical and immunoelectron microscopy involving a specific monoclonal antibody FSHR1A02 that recognizes an epitope present in the FSHR-ectodomain. In all 55 patients with atherosclerotic plaques located in carotid, coronary, femoral arteries, and iliac aneurysm, FSHR was selectively expressed in arterial endothelium covering atherosclerotic plaques and endothelia lining intraplaque neovessels. Lymphatic neovessels were negative for FSHR. M1-macrophages, foam cells, and giant multinucleated cells were also FSHR-positive. FSHR was not detected in normal internal thoracic artery. Immunoelectron microscopy performed in ApoEKO/hFSHRKI mice with atherosclerotic plaques, after injection in vivo with mouse anti-hFSHR monoclonal antibody FSHR1A02 coupled to colloidal gold, showed FSHR presence on the luminal surface of arterial endothelial cells covering atherosclerotic plaques. Therefore, FSHR can bind, internalize, and deliver into the plaque circulating ligands to FSHR-positive cells. In conclusion, we report FSHR expression in endothelial cells, M1-macrophages, M1-derived foam cells, giant multinucleated macrophages, and osteoclasts associated with human atherosclerotic plaques.

The effects of low doses of ionizing radiation on atherosclerosis remain uncertain, particularly as regards the generation of pro- or anti-inflammatory responses, and the time scale at which such effects can occur following irradiation. To explore these phenomena, we exposed atheroprone ApoE(−/−) mice to a single dose of 0, 0.05, 0.5 or 1 Gy of 137Cs (γ) administered at a 10.35 mGy min−1 dose rate and evaluated short-term (1–10 days) and long-term consequences (100 days). Bone marrow-derived macrophages were derived from mice 1 day after exposure. Irradiation was associated with a significant skewing of M0 and M2 polarized macrophages towards the M2 phenotype, as demonstrated by an increased mRNA expression of Retnla, Arg1, and Chil3 in cells from mice exposed to 0.5 or 1 Gy compared with non-irradiated animals. Minimal effects were noted in M1 cells or M1 marker mRNA. Concurrently, we observed a reduced secretion of IL-1β but enhanced IL-10 release from M0 and M2 macrophages. Effects of irradiation on circulating monocytes were most marked at day 10 post-exposure, when the 1 Gy dose was associated with enhanced numbers of both Ly6CHigh and Ly6Low cells. By day 100, levels of circulating monocytes in irradiated and non-irradiated mice were equivalent, but anti-inflammatory Ly6CLow monocytes were significantly increased in the spleen of mice exposed to 0.05 or 1 Gy. Long term exposures did not affect atherosclerotic plaque size or lipid content, as determined by Oil red O staining, whatever the dose applied. Similarly, irradiation did not affect atherosclerotic plaque collagen or smooth muscle cell content. However, we found that lesion CD68+ cell content tended to decrease with rising doses of radioactivity exposure, culminating in a significant reduction of plaque macrophage content at 1 Gy. Taken together, our results show that short- and long-term exposures to low to moderate doses of ionizing radiation drive an anti-inflammatory response, skewing bone marrow-derived macrophages towards an IL-10-secreting M2 phenotype and decreasing plaque macrophage content. These results suggest a low-grade athero-protective effect of low and moderate doses of ionizing radiation. [ABSTRACT FROM AUTHOR]

To investigate the significance of atherosclerotic plaque location in hybrid surgery comprising both endovascular recanalization approaches and carotid endarterectomy for symptomatic atherosclerotic non-acute long-segment occlusion of the internal carotid artery (ICA), 162 patients were enrolled, including 120 (74.1%) patients in the proximal plaque group and 42 (25.9%) in the distal plaque group. Surgical recanalization was performed in all patients, with successful recanalization in 119 (99.2%) patients in the proximal and 39 (92.9%) in the distal plaque group. The total successful recanalization rate was 97.5% (158/162) with a failure rate of 2.5% (4/162). Periprocedural complications occurred in 5 (4.2% or 5/120) patients in the proximal plaque group, including neck infection in two (1.7%), recurrent nerve injury in 1 (0.8%), and laryngeal edema in 2 (1.7%), and 2 (4.8%) in the distal plaque group, including femoral puncture infection in 2 (4.8%). No severe complications occurred in either group. Univariate analysis showed plaque location was a significant (P = 0.018) risk factor for successful recanalization, and multivariate analysis indicated that the plaque location remained a significant independent risk factor for recanalization success (P = 0.017). In follow-up 6–48 months after the recanalization surgery, reocclusion occurred in two (2.8%) patients in the proximal plaque group and 4 (13.3%) in the distal plaque group. In conclusion, although hybrid surgery achieves similar outcomes in patients with ICA occlusion caused by either proximal or distal atherosclerotic plaques, plaque location may be a significant risk factor for successful recanalization of symptomatic non-acute long-segment ICA occlusion. [ABSTRACT FROM AUTHOR]

Elevated remnant cholesterol (RC) is considered a risk factor for atherosclerotic cardiovascular disease, but the evidence on this association applies to the Chinese population with hypertension is limited. We aimed to explore the association between RC levels and carotid plaque in old adults with hypertension. 8523 hypertensive patients aged ≥ 60 years with serum lipids and carotid ultrasonography data were included in this community-based screening. Fasting RC was calculated as total cholesterol minus high-density lipoprotein cholesterol minus low-density lipoprotein cholesterol (LDLC). The associations of RC levels with carotid plaque risk were evaluated using Logistic regression and restricted cubic spline models. Carotid plaque was screened in 4821 (56.56%) subjects. After multivariable-adjusted, RC was significantly related to carotid plaque [Odd ratio (OR)] = 1.043 per 0.1 mmol/L increase, 95% confidence interval (CI): 1.030–1.056). The highest versus the lowest quartile of RC was 1.928 (1.673–2.223) for carotid plaque. A nonlinear association was found between serum RC levels and the risk of carotid plaque (P for nonlinearity < 0.001). Moreover, an RC > 0.78 mmol/L differentiated patients at a higher risk of carotid plaque compared to those at lower concentrations, regardless of whether LDLC was on target at 2.59 mmol/L. In old adults with hypertension, elevated RC was positively associated with carotid plaque, independent of LDLC and other conventional risk factors. [ABSTRACT FROM AUTHOR]

Patients with peripheral artery disease (PAD) have a higher risk of cardiovascular events. We examined the histology of atheromatous plaques in the femoral artery and investigated their association with subsequent cardiovascular events in patients with PAD. Patients who underwent femoral artery endarterectomy between March 2010 and January 2021 were included. We analyzed the expression of myeloperoxidase (MPO), citrullinated histone, and programmed cell death ligand 1 (PD-L1) in femoral artery plaques by immunohistochemistry. Data on the subsequent occurrence of major adverse cardiovascular events (MACEs), major adverse limb events (MALEs), and all-cause mortality were retrospectively collected. A total of 37 patients were included. The median age was 71 (range, 42–90) years, and 25 patients (67.6%) were male. During the median follow-up of 24 months, 10 patients experienced MACEs and 16 patients had MALEs. Patients with MACEs had a higher number of MPO-stained cells (p = 0.044) and lower PD-L1 staining intensity (p = 0.021) in atheromatous plaques compared with those of patients with a stable prognosis. When the patients were grouped according to the immunologic score based on the MPO-stained cell number and PD-L1 staining intensity, those with a higher score had a significantly higher cumulative risk of MACEs (p = 0.014). The immunologic profile of excised peripheral artery plaques may be associated with future cardiovascular events in patients with PAD. [ABSTRACT FROM AUTHOR]

In the study, a biomimetic platform for anti-inflammatory-based treatment of atherosclerotic plaque was developed. Gliclazide (GL) as an anti-inflammasome agent was encapsulated in PLGA nanoparticles (NP), which were coated by monocyte membrane using an extrusion procedure. The size and zeta potential of the nanoghost (NG) changed to 292 and – 10 nm from 189.5 to −34.1 in the core NP. In addition, the actual size of 62.5 nm with a coating layer of 5 nm was measured using TEM. The NG was also showed a sustained release profile with the drug loading content of about 4.7%. Beside to attenuated TNFα, decrease in gene expression levels of NLRP3, MyD88, NOS, IL-1β, IL-18 and caspases 1/3/8/9 in LPS-primed monocytes exposed to NG strongly indicated remarkable inflammation control. After systemic toxicity evaluation and pharmacokinetic analysis of NP and NG, intravenous NG treatment of rabbits with experimentally induced atherosclerosis revealed remarkably less plaque lesions, foam cells, lipid-laden macrophages, and pathological issues in tunica media of aorta sections. Higher expression of CD163 than CD68 in aorta of NG-treated rabbits strongly reveals higher M2/M1 macrophage polarization. The bio/hemocompatible, biomimetic and anti-inflammatory NG can be considered as a potential platform for immunotherapy of particularly atherosclerosis in the field of personalized medicine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a chronic lipid-driven inflammatory response of the innate and adaptive immune systems, and it is responsible for several cardiovascular ischemic events. The present study aimed to determine immune infiltration-related biomarkers in carotid atherosclerotic plaques (CAPs). Gene expression profiles of CAPs were extracted from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between the CAPs and control groups were screened by the "limma" package in R software. Immune cell infiltration between the CAPs and control groups was evaluated by the single sample gene set enrichment analysis. Key infiltrating immune cells in the CAPs group were screened by the Wilcoxon test and least absolute shrinkage and selection operator regression. The weighted gene co-expression network analysis was used to identify immune cell-related genes. Hub genes were identified by the protein–protein interaction (PPI) network. Receiver operating characteristic curve analysis was performed to assess the gene's ability to differentiate between the CAPs and control groups. Finally, we constructed a miRNA-gene-transcription factor network of hub genes by using the ENCODE database. Eleven different types of immune infiltration-related cells were identified between the CAPs and control groups. A total of 1,586 differentially expressed immunity-related genes were obtained through intersection between DEGs and immune-related genes. Twenty hub genes were screened through the PPI network. Eventually, 7 genes (BTK, LYN, PTPN11, CD163, CD4, ITGAL, and ITGB7) were identified as the hub genes of CAPs, and these genes may serve as the estimable drug targets for patients with CAPs. [ABSTRACT FROM AUTHOR]

To evaluate the association of intracranial non-stenotic atherosclerotic plaque with cerebral small vessel disease (CSVD) imaging markers in a CSVD population using 3.0 T high-resolution magnetic resonance imaging (HRMRI), which was validated in embolic stroke of undetermined source (ESUS) cohort. We retrospectively recruited consecutive patients who were diagnosed with CSVD or ESUS from January 2015 to December 2019. All patients underwent intracranial HRMRI to assess intracranial non-stenotic atherosclerotic plaques. Baseline and imaging data were collected and were measured among all patients. Among 153 patients with CSVD, there were 59 with intracranial atherosclerotic plaque (IAP) and 94 with non-IAP, including 36 with intracranial atherosclerotic complicated plaque (IACP). Among 227 ESUS patients, there were 155 with IAP and 72 with non-IAP, including 127 with IACP. In the CSVD population, we found that: (1) CSVD burden was associated with IAP (p = 0.036) and IACP (p = 0.008); (2) IAP was associated with white matter hyperintensity (51% vs. 34%; P = 0.039), and IACP was associated with lacunes (69% vs. 35%; P = 0.009) and enlarge perivascular space (69% vs. 39%; P = 0.022). A similar association of CSVD imaging markers with IAP or IACP was found in the ESUS population. Furthermore, the association of unilateral IAP or IACP with CSVD imaging markers of ipsilateral hemisphere was identified in the two cohorts. This is the first report that intracranial non-stenotic atherosclerotic plaque, especially complicated plaque, is closely associated with CSVD imaging markers, which provide further evidence for the association of large artery atherosclerosis with CSVD. [ABSTRACT FROM AUTHOR]

Antigen-naive IgM-producing B cells are atheroprotective, whereas mature B cells producing class-switched antibodies promote atherosclerosis. Activation-induced cytidine deaminase (AID), which mediates class switch recombination (CSR), would thus be expected to foster atherosclerosis. Yet, AID also plays a major role in the establishment of B cell tolerance. We sought to define whether AID affects atherosclerotic plaque formation. We generated Ldlr-/- chimeras transplanted with bone marrow from Aicda-/- or wild-type (WT) mice, fed a HFD for 14 weeks. Decreased B cell maturation in Ldlr-/-Aicda-/- mice was demonstrated by 50% reduction in splenic and aortic BAFFR expression, a key signaling component of B2 cell maturation. This was associated with increased plasma IgM in Ldlr–/-Aicda-/- compared with Ldlr-/-WT animals. Importantly, Ldlr-/-Aicda-/- mice had reduced atherosclerotic lesion area (0.20 ± 0.03mm2) compared with Ldlr-/-WT (0.30 ± 0.04mm2, P < 0.05), although no differences in plaque composition were noted between groups. In addition, immunofluorescence analysis revealed increased splenic B and T cell areas independent of cell number. AID depletion directly inhibits atherosclerotic plaque formation. [ABSTRACT FROM AUTHOR]

Extra- and intracranial carotid plaque calcification might have plaque-stabilizing effects, yet information on changes in plaque calcification remains scarce. We evaluated changes in carotid plaque calcification over 2 years follow-up in patients with symptomatic carotid artery disease. This study is based on the PARISK-study, a multicenter cohort study, with TIA/minor stroke patients with ipsilateral mild-to-moderate carotid artery stenosis (< 70%). We included 79 patients (25% female, mean age 66 years) who underwent CTA imaging with 2 year interval. We assessed the volume of extra- and intracranial carotid artery calcification (ECAC and ICAC) and calculated the difference between baseline and follow-up ECAC and ICAC volume. We performed multivariable regression analyses to investigate the association between change of ECAC or ICAC with cardiovascular determinants. ECAC. We found increase (46.2%) and decrease (34%) in ECAC volume during 2 year follow-up, both significantly correlation with baseline ECAC volume (OR = 0.72, 95% CI 0.58–0.90 respectively OR = 2.24, 95% CI 1.60–3.13).We found significant correlation for change in ECAC volume with diabetes (β = 0.46, 95% CI 0.03–0.89) and baseline ECAC volume (β = 0.81, 95% CI 0.73–0.88). ICAC. We found increase (45.0%) and decrease (25.0%) in ICAC volume. The ICAC decrease was significantly correlated with baseline ICAC volume (OR = 2.17, 95% CI 1.48–3.16), age (OR = 2.00, 95% CI 1.19–3.38) and use of antihypertensive drugs (OR = 3.79, 95% CI 1.20–11.96]).The overall change of ICAC volume was also significantly correlated with diabetes (β = 0.92, 95% CI 1.59–7.02), use of oral hypoglycemic drugs (β = 0.86, 95% CI 0.12–1.59) and baseline ICAC volume (β = 0.71, 95% CI 0.55–0.87). We provide novel insights into the dynamics of carotid plaque calcification in symptomatic stroke patients. [ABSTRACT FROM AUTHOR]

Beverages play a substantial role meeting water, calorie, and nutrient requirements; however, they are presented as being major contributors to the current obesity epidemic. Although, the relationship between beverage consumption and metabolic risk factors for cardiovascular disease (CVD) in adults has been frequently studied, its association with subclinical atherosclerosis is of increased interest. We studied the association of beverage consumption with the presence of peripheral subclinical atherosclerosis among Spanish workers. We performed a cross-sectional study of 2089 middle-aged males, with a mean age of 50.9 (SD 3.9), and without CVD, carried out in the Aragon Workers' Health Study (AWHS). A food frequency questionnaire was used to measure beverage consumption of low-fat milk, coffee and tea (unsweetened), whole-fat milk, sugar-sweetened beverages, bottled fruit juice, artificially-sweetened beverages and 100% fruit juice. Atherosclerotic plaques were measured by ultrasound (in carotid arteries, and in femoral arteries). Atherosclerotic plaque was defined as a focal structure protruding ≥ 0.5 mm into the lumen, or reaching a thickness ≥ 50% of the surrounding intima-media thickness. As statistical analysis, we use logistic regression models, simultaneously adjusted for all beverage groups. As results, unsweetened coffee was the beverage most associated with peripheral subclinical atherosclerosis with an odds ratio (OR) of 1.25 (1.10–1.41), and 1.23 (1.09–1.40) 100g/day] for carotid, and femoral territories respectively. Moreover, subclinical atherosclerosis was positively associated with whole-fat milk [OR 1.10 (1.02–1.18) 100 g/day] in the femoral territory. The association was protective for low-fat milk in the carotid territory [OR 0.93 (0.88–0.99) 100g/day]. There was also a protective association with bottled fruit juices in the femoral territory [0.84 (0.74–0.94) 100g/day]. Our results suggest a detrimental association with the consumption of coffee, as well as with whole-fat milk and the presence of subclinical atherosclerosis. Therefore, an element of prudence excluding water and low-fat milk, must be applied when recommending beverage consumption. [ABSTRACT FROM AUTHOR]

The gene expression profile of abdominal aortic aneurysm (AAA) neck is not fully understood. The etiology of AAA is considered to be related to atherosclerosis and the inflammatory response, involving congenital, genetic, metabolic, and other factors. The level of proprotein convertase subtilisin/kexin type 9 (PCSK9) is related to those of cholesterol, oxidized low-density lipoprotein, and triglycerides. PCSK9 inhibitors have significant effects on lowering LDL-cholesterol, reversing atherosclerotic plaques, and reducing the risk of cardiovascular events and have been approved by several lipid-lowering guidelines. This work was aimed to investigate the potential role of PCSK9 in the neck of AAA. We extracted the expression dataset (GSE47472) containing 14 AAA patients and 8 donors and single-cell RNAseq (scRNA-seq) data (GSE164678) of CaCl2-induced (AAA) samples from the Gene Expression Omnibus dataset. Through bioinformatics methods, we found that PCSK9 was up-regulated in the proximal neck of human AAA. In AAA, PCSK9 was mainly expressed in fibroblasts. Additionally, immune check-point PDCD1LG2 was also expressed higher in AAA neck than donor, while CTLA4, PDCD1, and SIGLEC15 were down-regulated in AAA neck. The expression of PCSK was correlated with PDCD1LG2, LAG3, and CTLA4 in AAA neck. Additionally, some ferroptosis-related genes were also down-regulated in AAA neck. PCSK9 was also correlated with ferroptosis-related genes in AAA neck. In conclusion, PCSK9 was highly expressed in AAA neck, and may exert its role through interacting with immune check-points and ferroptosis-related genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To improve the assessment of carotid plaque vulnerability, a comprehensive characterization of their composition is paramount. Multispectral photoacoustic imaging (MSPAI) can provide plaque composition based on their absorption spectra. However, although various spectral unmixing methods have been developed to characterize different tissue constituents, plaque analysis remains a challenge since its composition is highly complex and diverse. In this study, we employed an adapted piecewise convex multiple-model endmember detection method to identify carotid plaque constituents. Additionally, we explore the selection of the imaging wavelengths in linear models by conditioning the coefficient matrix and its synergy with our unmixing approach. We verified our method using plaque mimicking phantoms and performed ex-vivo MSPAI on carotid endarterectomy samples in a spectral range from 500 to 1300 nm to identify the main spectral features of plaque materials for vulnerability assessment. After imaging, the samples were processed for histological analysis to validate the photoacoustic decomposition. Results show that our approach can perform spectral unmixing and classification of highly heterogeneous biological samples without requiring an extensive fluence correction, enabling the identification of relevant components to assess plaque vulnerability. [ABSTRACT FROM AUTHOR]

Metabolic dysfunction, partly driven by altered liver function, predisposes to coronary artery disease (CAD), but the role of liver in vulnerable atherosclerotic plaque development remains unclear. Here we produced hepatocyte-like cells (HLCs) from 27 induced pluripotent stem cell (iPSC) lines derived from 15 study subjects with stable CAD (n = 5), acute CAD (n = 5) or healthy controls (n = 5). We performed a miRNA microarray screening throughout the differentiation, as well as compared iPSC-HLCs miRNA profiles of the patient groups to identify miRNAs involved in the development of CAD. MicroRNA profile changed during differentiation and started to resemble that of the primary human hepatocytes. In the microarray, 35 and 87 miRNAs were statistically significantly deregulated in the acute and stable CAD patients, respectively, compared to controls. Down-regulation of miR-149-5p, -92a-3p and -221-3p, and up-regulation of miR-122-5p was verified in the stable CAD patients when compared to other groups. The predicted targets of deregulated miRNAs were enriched in pathways connected to insulin signalling, inflammation and lipid metabolism. The iPSC-HLCs derived from stable CAD patients with extensive lesions had a distinct genetic miRNA profile possibly linked to metabolic dysfunction, potentially explaining the susceptibility to developing CAD. The iPSC-HLCs from acute CAD patients with only the acute rupture in otherwise healthy coronaries did not present a distinct miRNA profile, suggesting that hepatic miRNAs do not explain susceptibility to plaque rupture. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EV) are a novel biomarker source for diagnosis and prognosis of cardiovascular disease. A protein comparison of plasma EVs in relation to blood plasma and atherosclerotic plaque has not been performed but would provide insight into the origin and content of biomarker sources and their association with atherosclerotic progression. Using samples of 88 carotid endarterectomy patients in the Athero-Express, 92 proteins (Olink Cardiovascular III panel) were measured in citrate plasma, plasma derived LDL-EVs and atherosclerotic plaque. Proteins were correlated between sources and were related to pre-operative stroke and 3-year major adverse cardiovascular events (MACE). Plasma and EV proteins correlated moderately on average, but with substantial variability. Both showed little correlation with plaque, suggesting that these circulating biomarkers may not originate from the latter. Plaque (n = 17) contained most differentially-expressed proteins in patients with stroke, opposed to EVs (n = 6) and plasma (n = 5). In contrast, EVs contained most differentially-expressed proteins for MACE (n = 21) compared to plasma (n = 9) and plaque (n = 1). EVs appear to provide additional information about severity and progression of systemic atherosclerosis than can be obtained from plasma or atherosclerotic plaque. [ABSTRACT FROM AUTHOR]

Thin-cap fibroatheroma (TCFA) and plaque rupture have been recognized as the most frequent risk factor for thrombosis and acute coronary syndrome. Intravascular optical coherence tomography (IVOCT) can identify TCFA and assess cap thickness, which provides an opportunity to assess plaque vulnerability. We developed an automated method that can detect lipidous plaque and assess fibrous cap thickness in IVOCT images. This study analyzed a total of 4360 IVOCT image frames of 77 lesions among 41 patients. Expert cardiologists manually labeled lipidous plaque based on established criteria. To improve segmentation performance, preprocessing included lumen segmentation, pixel-shifting, and noise filtering on the raw polar (r, θ) IVOCT images. We used the DeepLab-v3 plus deep learning model to classify lipidous plaque pixels. After lipid detection, we automatically detected the outer border of the fibrous cap using a special dynamic programming algorithm and assessed the cap thickness. Our method provided excellent discriminability of lipid plaque with a sensitivity of 85.8% and A-line Dice coefficient of 0.837. By comparing lipid angle measurements between two analysts following editing of our automated software, we found good agreement by Bland–Altman analysis (difference 6.7° ± 17°; mean ~ 196°). Our method accurately detected the fibrous cap from the detected lipid plaque. Automated analysis required a significant modification for only 5.5% frames. Furthermore, our method showed a good agreement of fibrous cap thickness between two analysts with Bland–Altman analysis (4.2 ± 14.6 µm; mean ~ 175 µm), indicating little bias between users and good reproducibility of the measurement. We developed a fully automated method for fibrous cap quantification in IVOCT images, resulting in good agreement with determinations by analysts. The method has great potential to enable highly automated, repeatable, and comprehensive evaluations of TCFAs. [ABSTRACT FROM AUTHOR]

Cardiovascular disease is the leading cause of death worldwide. New therapeutic strategies are aimed to modulate the athero-inflammatory process that partially orchestrates underlying vascular damage. Peripheral blood circulating cells include different immune cells with a central role in the development of the atherogenic inflammatory response. The anti-aging protein α-Klotho has been related to protective effects against CVD. KL is expressed in monocytes, macrophages, and lymphocytes where it exerts anti-inflammatory effects. In this work, we analyse the relationships of the levels of inflammatory markers with the expression of the KL gene in PBCCs and with the serum levels of soluble KL in atherosclerotic vascular disease. For this, we conducted a cross-sectional single-center case–control study including a study group of 76 CVD patients and a control group of 16 cadaveric organ donors without medical antecedent or study indicating CVD. Vascular artery fragments and whole blood and serum samples were obtained during elective or organ retrieval surgery. Serum levels of sKL, TNFα and IL10, and gene expression levels of KL, TNF, IL10, NFKB1, DNMT1, and DNMT3A in PBCCs were measured. In these cells, we also determined KL promoter methylation percentage. Histological and immunohistochemical analyses were employed to visualize atherosclerotic lesions and to measure IL10 and TNFα levels in vascular fragments. Patients with CVD presented higher values of proinflammatory markers both at systemic and in the vasculature and in the PBCCs, compared to the control group. In PBCCs, CVD patients also presented lower gene expression levels of KL gene (56.4% difference, P < 0.001), higher gene expression levels of DNMT1 and DNMT3A (P < 0.0001, for both) and a higher methylation status of in the promoter region of KL (34.1 ± 4.1% vs. 14.6 ± 3.4%, P < 0.01). In PBCCs and vasculature, KL gene expression correlated inversely with pro-inflammatory markers and directly with anti-inflammatory markers. sKL serum levels presented similar associations with the expression levels of pro- and anti-inflammatory markers in PBCCs. The differences in KL expression levels in PBCCs and in serum sKL levels with respect to control group was even greater in those CVD patients with macroscopically observable atheromatous plaques. We conclude that promoter methylation-mediated downregulation of KL gene expression in PBCCs is associated with the pro-inflammatory status in atherosclerotic vascular disease. [ABSTRACT FROM AUTHOR]

Some studies suggest that being an apolipoprotein e4 (APOE e4) carrier increases the risk of atherosclerosis, and others suggest that cardiorespiratory fitness (CRF) could play a key role in atherosclerotic prevention. Our aim was to analyze the association of APOE e4 with carotid atherosclerosis and the association of CRF with atherosclerosis in APOE e4 carriers. A cross-sectional analysis based on a subsample of 90 participants in the Aragon Workers' Health Study was carried out. Ultrasonography was used to assess the presence of plaques in carotid territory; the submaximal Chester Step Test was used to assess CRF; and behavioral, demographic, anthropometric, and clinical data were obtained by trained personnel during annual medical examinations. APOE e4e4 participants were categorized into Low-CRF (VO2max < 35 mL/kg/min) and High-CRF (VO2max ≥ 35 mL/kg/min) groups. After adjusting for several confounders, compared with APOE e3e3, those participants genotyped as APOE e3e4 and APOE e4e4 showed an OR = 1.60 (95% CI 0.45, 5.71) and OR = 4.29 (95% CI 1.16, 15.91), respectively, for carotid atherosclerosis. Compared to Low-CRF APOE e4e4 carriers, the odds of carotid plaque detection were 0.09 (95% CI 0.008, 0.98) times lower among High-CRF APOE e4e4 carriers. The APOE e4e4 genotype was associated with increased carotid atherosclerosis. However, CRF is a modifiable factor that may be targeted by APOE e4e4 to decrease the elevation of atherosclerotic risk due to this genetic condition. [ABSTRACT FROM AUTHOR]

Mast cells have been associated with the progression and destabilization of advanced atherosclerotic plaques. Reducing intraplaque mast cell accumulation upon atherosclerosis progression could be a potent therapeutic strategy to limit plaque destabilization. Leukotriene B4 (LTB4) has been reported to induce mast cell chemotaxis in vitro. Here, we examined whether antagonism of the LTB4-receptor BLT1 could inhibit mast cell accumulation in advanced atherosclerosis. Expression of genes involved in LTB4 biosynthesis was determined by single-cell RNA sequencing of human atherosclerotic plaques. Subsequently, Western-type diet fed LDLr−/− mice with pre-existing atherosclerosis were treated with the BLT1-antagonist CP105,696 or vehicle control three times per week by oral gavage. In the spleen, a significant reduction in CD11b+ myeloid cells was observed, including Ly6Clo and Ly6Chi monocytes as well as dendritic cells. However, atherosclerotic plaque size, collagen and macrophage content in the aortic root remained unaltered upon treatment. Finally, BLT1 antagonism did not affect mast cell numbers in the aortic root. Here, we show that human intraplaque leukocytes may be a source of locally produced LTB4. However, BLT1-antagonism during atherosclerosis progression does not affect either local mast cell accumulation or plaque size, suggesting that other mechanisms participate in mast cell accumulation during atherosclerosis progression. [ABSTRACT FROM AUTHOR]

M1 macrophages secrete a large number of proinflammatory factors and promote the expansion of atherosclerotic plaques and processes. Salvianolic acid B (Sal B) exerts anti-inflammatory, antitumor and other effects, but no study has addressed whether Sal B can regulate the polarization of macrophages to exert these anti-atherosclerotic effects. Therefore, we investigated the inhibition of Sal B in M1 macrophage polarization and the underlying mechanism. The effects of different treatments on cell viability, gene expression and secretion of related proteins, phenotypic markers and cytokines were detected by MTT and western blot assays, RT‒qPCR and ELISAs. Cell viability was not significantly changed when the concentration of Sal B was less than 200 μM, and Lipopolysaccharide (LPS) (100 ng/mL) + interferon-γ (IFN-γ) (2.5 ng/mL) successfully induced M1 polarization. RT‒qPCR and ELISAs indicated that Sal B can downregulate M1 marker (Inducible Nitric Oxide Synthase (iNOS), Tumor Necrosis Factor-α (TNF-α), and Interleukin-6 (IL-6)) and upregulate M2 marker (Arginase-1 (Arg-1) and Interleukin-10 (IL-10)) expression. Western blotting was performed to measure the expression of Nuclear Factor-κB (NF-κB), p-Akt, p-mTOR, LC3-II, Beclin-1, and p62, and the results suggested that Sal B inhibits the M1 polarization of RAW264.7 macrophages by promoting autophagy via the NF-κB signalling pathway. The study indicated that Sal B inhibits M1 macrophage polarization by inhibiting NF-κB signalling pathway activation and downregulating Akt/mTOR activation to promote autophagy. [ABSTRACT FROM AUTHOR]

The mechanism underlying the association between elevated red cell distribution width (RDW) and poor prognosis in variety of diseases is unknown although many researchers consider RDW a marker of inflammation. We hypothesized that RDW directly affects intravascular hemodynamics, interactions between circulating cells and vessel wall, inducing local changes predisposing to atherothrombosis. We applied different human and animal models to verify our hypothesis. Carotid plaques harvested from patients with high RDW had increased expression of genes and proteins associated with accelerated atherosclerosis as compared to subjects with low RDW. In microfluidic channels samples of blood from high RDW subjects showed flow pattern facilitating direct interaction with vessel wall. Flow pattern was also dependent on RDW value in mouse carotid arteries analyzed with Magnetic Resonance Imaging. In different mouse models of elevated RDW accelerated development of atherosclerotic lesions in aortas was observed. Therefore, comprehensive biological, fluid physics and optics studies showed that variation of red blood cells size measured by RDW results in increased interactions between vascular wall and circulating morphotic elements which contribute to vascular pathology. [ABSTRACT FROM AUTHOR]

Atherosclerotic plaque rupture is the etiology of ischemic stroke and myocardial infarction. The molecular mechanisms responsible for rupture remain unclear, in part, due to the lack of data from plaques at the time of rupture. Ribosome-depleted total RNA was sequenced from carotid plaques obtained from patients undergoing carotid endarterectomy with high-grade stenosis and either (1) a carotid-related ischemic cerebrovascular event within the previous 5 days ('recently ruptured,' n = 6) or (2) an absence of a cerebrovascular event ('asymptomatic,' n = 5). Principal component analysis confirmed plaque rupture was responsible for the greatest percentage of the variability between samples (23.2%), and recently ruptured plaques were enriched for transcripts associated with inflammation and extracellular matrix degradation. Hierarchical clustering achieved differentiation of the asymptomatic from the recently ruptured plaques. This analysis also found co-expression of transcripts for immunoglobulins and B lymphocyte function, matrix metalloproteinases, and interferon response genes. Examination of the differentially expressed genes supported the importance of inflammation and inhibition of proliferation and migration coupled with an increase in apoptosis. Thus, the transcriptome of recently ruptured plaques is enriched with transcripts associated with inflammation and fibrous cap thinning and support further examination of the role of B lymphocytes and interferons in atherosclerotic plaque rupture. [ABSTRACT FROM AUTHOR]

The development of atherosclerotic plaques is the result of a chronic inflammatory response coordinated by stromal and immune cellular components of the vascular wall. While endothelial cells and leukocytes are well-recognised mediators of inflammation in atherosclerosis, the role of smooth muscle cells (SMCs) remains incompletely understood. Here we aimed to address the role of canonical NF-κB signalling in SMCs in the development of atherosclerosis. We investigated the role of NF-κB signalling in SMCs in atherosclerosis by employing SMC-specific ablation of NEMO, an IKK complex subunit that is essential for canonical NF-κB activation, in ApoE−/− mice. We show that SMC-specific ablation of NEMO (NEMOSMCiKO) inhibited high fat diet induced atherosclerosis in ApoE−/− mice. NEMOSMCiKO/ApoE−/− mice developed less and smaller atherosclerotic plaques, which contained fewer macrophages, decreased numbers of apoptotic cells and smaller necrotic areas and showed reduced inflammation compared to the plaques of ApoE−/− mice. In addition, the plaques of NEMOSMCiKO/ApoE−/− mice showed higher expression of α-SMA and lower expression of the transcriptional factor KLF4 compared to those of ApoE−/− mice. Consistently, in vitro, NEMO-deficient SMCs exhibited reduced proliferation and migration, as well as decreased KLF4 expression and lower production of IL-6 and MCP-1 upon inflammatory stimulus (TNF or LPS) compared to NEMO-expressing SMCs. In conclusion, NEMO-dependent activation of NF-κB signalling in SMCs critically contributes to the pathogenesis of atherosclerosis by regulating SMC proliferation, migration and phenotype switching in response to inflammatory stimuli. [ABSTRACT FROM AUTHOR]

The regression, or resolution, of inflammation in atherosclerotic plaques is impaired in diabetes. However, the factors mediating this effect remain incomplete. We identified protein arginine methyltransferase 2 (PRMT2) as a protein whose expression in macrophages is reduced in hyperglycemia and diabetes. PRMT2 catalyzes arginine methylation to target proteins to modulate gene expression. Because PRMT2 expression is reduced in cells in hyperglycemia, we wanted to determine whether PRMT2 plays a causal role in the impairment of atherosclerosis regression in diabetes. We, therefore, examined the consequence of deleting PRMT2 in myeloid cells during the regression of atherosclerosis in normal and diabetic mice. Remarkably, we found significant impairment of atherosclerosis regression under normoglycemic conditions in mice lacking PRMT2 (Prmt2−/−) in myeloid cells that mimic the decrease in regression of atherosclerosis in WT mice under diabetic conditions. This was associated with increased plaque macrophage retention, as well as increased apoptosis and necrosis. PRMT2-deficient plaque CD68+ cells under normoglycemic conditions showed increased expression of genes involved in cytokine signaling and inflammation compared to WT cells. Consistently, Prmt2−/− bone marrow-derived macrophages (BMDMs) showed an increased response of proinflammatory genes to LPS and a decreased response of inflammation resolving genes to IL-4. This increased response to LPS in Prmt2−/− BMDMs occurs via enhanced NF-kappa B activity. Thus, the loss of PRMT2 is causally linked to impaired atherosclerosis regression via a heightened inflammatory response in macrophages. That PRMT2 expression was lower in myeloid cells in plaques from human subjects with diabetes supports the relevance of our findings to human atherosclerosis. [ABSTRACT FROM AUTHOR]

Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in atherosclerotic plaques and implicated in the development of cardiovascular diseases. Peripheral arterial disease (PAD) is an atherosclerotic disease that often results in major cardiovascular events. This study aimed to prospectively examine the potential of urine NGAL (uNGAL) in predicting worsening PAD status and major adverse limb events (MALE). Baseline urine NGAL (uNGAL) and urine creatinine (uCr) concentrations were measured in PAD (n = 121) and non-PAD (n = 77) patients. Levels of uNGAL were normalized for urine creatinine (uNGAL/uCr). Outcomes included worsening PAD status, which was defined as a drop in ankle brachial index (ABI) > 0.15, and major adverse limb events (MALE), which was defined as a need for surgical revascularization or amputations. PAD patients had 2.30-fold higher levels of uNGAL/uCr [median (IQR) 31.8 (17.0–62.5) μg/g] in comparison to non-PAD patients [median (IQR) 73.3 (37.5–154.7) μg/g] (P = 0.011). Multivariate cox analysis showed that uNGAL/uCr levels were independently associated with predicting worsening PAD status and MALE outcomes. Cumulative survival analysis, over follow up period, demonstrated a direct correlation between elevated uNGAL/uCr levels and PAD disease progression and MALE outcomes. These data demonstrate an association between elevated uNGAL/uCr levels and worsening PAD disease status and MALE outcomes, indicating its potential for risk-stratification of PAD patients. [ABSTRACT FROM AUTHOR]

A significant amount of vascular thrombotic events are associated with rupture of the fibrous cap that overlie atherosclerotic plaques. Cap rupture is however difficult to predict due to the heterogenous composition of the plaque, unknown material properties, and the stochastic nature of the event. Here, we aim to create tissue engineered human fibrous cap models with a variable but controllable collagen composition, suitable for mechanical testing, to scrutinize the reciprocal relationships between composition and mechanical properties. Myofibroblasts were cultured in 1 × 1.5 cm-sized fibrin-based constrained gels for 21 days according to established (dynamic) culture protocols (i.e. static, intermittent or continuous loading) to vary collagen composition (e.g. amount, type and organization). At day 7, a soft 2 mm ∅ fibrin inclusion was introduced in the centre of each tissue to mimic the soft lipid core, simulating the heterogeneity of a plaque. Results demonstrate reproducible collagenous tissues, that mimic the bulk mechanical properties of human caps and vary in collagen composition due to the presence of a successfully integrated soft inclusion and the culture protocol applied. The models can be deployed to assess tissue mechanics, evolution and failure of fibrous caps or complex heterogeneous tissues in general. [ABSTRACT FROM AUTHOR]

The Apolipoprotein E (APOE) gene polymorphism (rs429358 and rs7412) shows a well-established association with lipid profiles, but its effect on cardiovascular disease is still conflicting. Therefore, we examined the association of different APOE alleles with common carotid artery intima-media thickness (CCA-IMT), carotid plaques, incident myocardial infarction (MI) and stroke. We analyzed data from 3327 participants aged 20–79 years of the population-based Study of Health in Pomerania (SHIP) from Northeast Germany with a median follow-up time of 14.5 years. Linear, logistic, and Cox-regression models were used to assess the associations of the APOE polymorphism with CCA-IMT, carotid plaques, incident MI and stroke, respectively. In our study, the APOE E2 allele was associated with lower CCA-IMT at baseline compared to E3 homozygotes (β: − 0.02 [95% CI − 0.04, − 0.004]). Over the follow-up, 244 MI events and 218 stroke events were observed. APOE E2 and E4 allele were not associated with incident MI (E2 HR: 1.06 [95% CI 0.68, 1.66]; E4 HR: 1.03 [95% CI 0.73, 1.45]) and incident stroke (E2 HR: 0.79 [95% CI 0.48, 1.30]; E4 HR: 0.96 [95% CI 0.66, 1.38]) in any of the models adjusting for potential confounders. However, the positive association between CCA-IMT and incident MI was more pronounced in E2 carriers than E3 homozygotes. Thus, our study suggests that while APOE E2 allele may predispose individuals to lower CCA-IMT, E2 carriers may be more prone to MI than E3 homozygotes as the CCA-IMT increases. APOE E4 allele had no effect on CCA-IMT, plaques, MI or stroke. [ABSTRACT FROM AUTHOR]

Non-apoptotic regulated cell death (ferroptosis and necroptosis) leads to the release of damage-associated molecular patterns (DAMPs), which initiate and perpetuate a non-infectious inflammatory response. We hypothesize that DAMPs and non-apoptotic regulated cell death are critical players of atherosclerotic plaque progression with inadequate response to lipid-lowering treatment. We aimed to uncover the silent mechanisms that govern the existing residual risk of cardiovascular-related mortality in experimental atherosclerosis. Proteomic and genomic approaches were applied on the ascending aorta of hyperlipidemic rabbits and controls with and without lipid-lowering treatment. The hyperlipidemic animals, which presented numerous heterogeneous atherosclerotic lesions, exhibited high concentrations of serum lipids and increased lipid peroxidation oxidative stress markers. The analyses revealed the significant upregulation of DAMPs and proteins implicated in ferroptosis and necroptosis by hyperlipidemia. Some of them did not respond to lipid-lowering treatment. Dysregulation of five proteins involved in non-apoptotic regulated cell death proteins (VDAC1, VDAC3, FTL, TF and PCBP1) and nine associated DAMPs (HSP90AA1, HSP90AB1, ANXA1, LGALS3, HSP90B1, S100A11, FN, CALR, H3-3A) was not corrected by the treatment. These proteins could play a key role in the atherosclerotic silent evolution and may possess an unexplored therapeutic potential. Mass spectrometry data are available via ProteomeXchange with identifier PXD026379. [ABSTRACT FROM AUTHOR]

Extracellular adenosine-5′-triphosphate (ATP) acts as an import signaling molecule mediating inflammation via purinergic P2 receptors. ATP binds to the purinergic receptor P2X4 and promotes inflammation via increased expression of pro-inflammatory cytokines. Because of the central role of inflammation, we assumed a functional contribution of the ATP-P2X4-axis in atherosclerosis. Expression of P2X4 was increased in atherosclerotic aortic arches from low-density lipoprotein receptor-deficient mice being fed a high cholesterol diet as assessed by real-time polymerase chain reaction and immunohistochemistry. To investigate the functional role of P2X4 in atherosclerosis, P2X4-deficient mice were crossed with low-density lipoprotein receptor-deficient mice and fed high cholesterol diet. After 16 weeks, P2X4-deficient mice developed smaller atherosclerotic lesions compared to P2X4-competent mice. Furthermore, intravital microscopy showed reduced ATP-induced leukocyte rolling at the vessel wall in P2X4-deficient mice. Mechanistically, we found a reduced RNA expression of CC chemokine ligand 2 (CCL-2), C-X-C motif chemokine-1 (CXCL-1), C-X-C motif chemokine-2 (CXCL-2), Interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) as well as a decreased nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)-inflammasome priming in atherosclerotic plaques from P2X4-deficient mice. Moreover, bone marrow derived macrophages isolated from P2X4-deficient mice revealed a reduced ATP-mediated release of CCL-2, CC chemokine ligand 5 (CCL-5), Interleukin-1β (IL-1β) and IL-6. Additionally, P2X4-deficient mice shared a lower proportion of pro-inflammatory Ly6Chigh monocytes and a higher proportion of anti-inflammatory Ly6Clow monocytes, and expressend less endothelial VCAM-1. Finally, increased P2X4 expression in human atherosclerotic lesions from carotid endarterectomy was found, indicating the importance of potential implementations of this study's findings for human atherosclerosis. Collectively, P2X4 deficiency reduced experimental atherosclerosis, plaque inflammation and inflammasome priming, pointing to P2X4 as a potential therapeutic target in the fight against atherosclerosis. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction (ED) is a hallmark of atherosclerosis and is influenced by well-defined risk factors, including hypoxia, dyslipidemia, inflammation, and oscillatory flow. However, the individual and combined contributions to the molecular underpinnings of ED remain elusive. We used global gene expression in human coronary artery endothelial cells to identify gene pathways and cellular processes in response to chemical hypoxia, oxidized lipids, IL-1β induced inflammation, oscillatory flow, and these combined stimuli. We found that clustering of the surrogate risk factors differed from the sum of the individual insults that gave rise to emergent phenotypes such as cell proliferation. We validated these observations in samples of human coronary artery atherosclerotic plaques analyzed using single-cell RNA sequencing. Our findings suggest a hierarchical interaction between surrogates of CV risk factors and the advent of emergent phenotypes in response to combined stimulation in endothelial cells that may influence ED. [ABSTRACT FROM AUTHOR]

It has been hypothesised that oral bacteria can migrate, through the blood, from the mouth to the arterial plaques, thus exacerbating atherosclerosis. This study compared bacteria present in the peripheral blood of individuals with and without coronary artery disease (CAD). RNA sequences obtained from blood were downloaded from GEO (GSE58150). Eight patients with coronary artery calcification (CAC) scoring > 500 and eight healthy individuals were analysed. After conducting quality control, the sequences were aligned to the hg38 reference genome using Hisat2. Bacterial taxa were analysed by inputting the unmapped sequences into Kraken. Ecological indices were calculated using Vegan. The package DESeq2 was used to compare the counts of bacteria per standard rank between groups. A total of 51 species were found only in patients with CAD and 41 were exclusively present in healthy individuals. The counts of one phylum, one class, three orders, two families and one genus were significantly different between the analysed groups (p < 0.00032, FDR < 10%), including the orders Cardiobacteriales, Corynebacteriales and Fusobacteriales. Twenty-three bacterial species belonging to the subgingival plaque bacterial complexes were also identified in the blood of individuals from both the groups; Fusobacterium nucleatum was significantly less frequent in patients with CAD (p = 0.0012, FDR = 4.8%). Furthermore, the frequency of another 11 bacteria differed significantly among patients with CAD than that among healthy individuals (p < 0.0030, FDR < 10%). These bacteria have not been previously reported in patients with atherosclerosis and periodontitis. The presence of members of the subgingival plaque bacterial complexes in the blood of patients with CAC supports the hypothesis that the periodontopathogens can be disseminated through the blood flow to other body parts where they may enhance inflammatory processes that can lead to the development or exacerbation of atherosclerosis. [ABSTRACT FROM AUTHOR]

Nephrosclerosis patients are at an exceptionally high cardiovascular (CV) risk. We aimed to determine whether genetic variability represented by 38 tag-SNPs in genes of the cyclooxygenase pathway (PTGS1, PTGS2, PTGES, PTGES2 and PTGES3) leading to prostaglandin E2 (PGE2) synthesis, modified CV traits and events in 493 nephrosclerosis patients. Additionally, we genotyped 716 controls to identify nephrosclerosis risk associations. The addition of three variants, namely PTGS2 rs4648268, PTGES3 rs2958155 and PTGES3 rs11300958, to a predictive model for CV events containing classic risk factors in nephrosclerosis patients, significantly enhanced its statistical power (AUC value increased from 78.6 to 87.4%, p = 0.0003). Such increase remained significant after correcting for multiple testing. In addition, two tag-SNPs (rs11790782 and rs2241270) in PTGES were linked to higher systolic and diastolic pressure [carriers vs. non-carriers = 5.23 (1.87–9.93), p = 0.03 and 5.9 (1.87–9.93), p = 0.004]. PTGS1(COX1) rs10306194 was associated with higher common carotid intima media thickness (ccIMT) progression [OR 1.90 (1.07–3.36), p = 0.029], presence of carotid plaque [OR 1.79 (1.06–3.01), p = 0.026] and atherosclerosis severity (p = 0.041). These associations, however, did not survive Bonferroni correction of the data. Our findings highlight the importance of the route leading to PGE2 synthesis in the CV risk experienced by nephrosclerosis patients and add to the growing body of evidence pointing out the PGE2 synthesis/activity axis as a promising therapeutic target in this field. [ABSTRACT FROM AUTHOR]

Wall shear stress (WSS), the frictional force of the blood on the vessel wall, plays a crucial role in atherosclerotic plaque development. Low WSS has been associated with plaque growth, however previous research used different approaches to define low WSS to investigate its effect on plaque progression. In this study, we used four methodologies to allocate low, mid and high WSS in one dataset of human coronary arteries and investigated the predictive power of low WSS for plaque progression. Coronary reconstructions were based on multimodality imaging, using intravascular ultrasound and CT-imaging. Vessel-specific flow was measured using Doppler wire and computational fluid dynamics was performed to calculate WSS. The absolute WSS range varied greatly between the coronary arteries. On the population level, the established pattern of most plaque progression at low WSS was apparent in all methodologies defining the WSS categories. However, for the individual patient, when using measured flow to determine WSS, the absolute WSS values range so widely, that the use of absolute thresholds to determine low WSS was not appropriate to identify regions at high risk for plaque progression. [ABSTRACT FROM AUTHOR]

Atherosclerosis and its consequences cause considerable morbidity and mortality world-wide. We have previously shown that expression of the DNA glycosylase NEIL3 is regulated in human atherosclerotic plaques, and that NEIL3-deficiency enhances atherogenesis in Apoe−/− mice. Herein, we identified a time point prior to quantifiable differences in atherosclerosis between Apoe−/−Neil3−/− mice and Apoe−/− mice. Mice at this age were selected to explore the metabolic and pathophysiological processes preceding extensive atherogenesis in NEIL3-deficient mice. Untargeted metabolomic analysis of young Apoe−/−Neil3−/− mice revealed significant metabolic disturbances as compared to mice expressing NEIL3, particularly in metabolites dependent on the gut microbiota. 16S rRNA gene sequencing of fecal bacterial DNA indeed confirmed that the NEIL3-deficient mice had altered gut microbiota, as well as increased circulating levels of the bacterially derived molecule LPS. The mice were challenged with a FITC-conjugated dextran to explore gut permeability, which was significantly increased in the NEIL3-deficient mice. Further, immunohistochemistry showed increased levels of the proliferation marker Ki67 in the colonic epithelium of NEIL3-deficient mice, suggesting increased proliferation of intestinal cells and gut leakage. We suggest that these metabolic alterations serve as drivers of atherosclerosis in NEIL3-deficient mice. [ABSTRACT FROM AUTHOR]

Aortic wall 18F-fluorodeoxyglucose (FDG)-uptake does not allow differentiation of aortitis from atheroma, which is problematic in clinical practice for diagnosing large vessel vasculitis giant-cell arteritis (GCA) in elderly patients. The purpose of this study was to compare the FDG uptake characteristics of GCA aortitis and aortic atheroma using positron emission tomography/FDG computed tomography (FDG-PET/CT). This study compared FDG aortic uptake between patients with GCA aortitis and patients with aortic atheroma; previously defined by contrast enhanced CT. Visual grading according to standardized FDG-PET/CT interpretation criteria and semi-quantitative analyses (maximum standardized uptake value (SUVmax), delta SUV (∆SUV), target to background ratios (TBR)) of FDG aortic uptake were conducted. The aorta was divided into 5 segments for analysis. 29 GCA aortitis and 66 aortic atheroma patients were included. A grade 3 FDG uptake of the aortic wall was identified for 23 (79.3%) GCA aortitis patients and none in the atheroma patient group (p < 0.0001); grade 2 FDG uptake was as common in both populations. Of the 29 aortitis patients, FDG uptake of all 5 aortic segments was positive for 21 of them (72.4%, p < 0.0001). FDG uptake of the supra-aortic trunk was identified for 24 aortitis (82.8%) and no atheromatous cases (p < 0.0001). All semi-quantitative analyses of FDG aortic wall uptake (SUVmax, ∆SUV and TBRs) were significantly higher in the aortitis group. ∆SUV was the feature with the largest differential between aortitis and aortic atheroma. In this study, GCA aortitis could be distinguished from an aortic atheroma by the presence of an aortic wall FDG uptake grade 3, an FDG uptake of the 5 aortic segments, and FDG uptake of the peripheral arteries. [ABSTRACT FROM AUTHOR]

Patient-specific phenotyping of coronary atherosclerosis would facilitate personalized risk assessment and preventive treatment. We explored whether unsupervised cluster analysis can categorize patients with coronary atherosclerosis according to their plaque composition, and determined how these differing plaque composition profiles impact plaque progression. Patients with coronary atherosclerotic plaque (n = 947; median age, 62 years; 59% male) were enrolled from a prospective multi-national registry of consecutive patients who underwent serial coronary computed tomography angiography (median inter-scan duration, 3.3 years). K-means clustering applied to the percent volume of each plaque component and identified 4 clusters of patients with distinct plaque composition. Cluster 1 (n = 52), which comprised mainly fibro-fatty plaque with a significant necrotic core (median, 55.7% and 16.0% of the total plaque volume, respectively), showed the least total plaque volume (PV) progression (+ 23.3 mm3), with necrotic core and fibro-fatty PV regression (− 5.7 mm3 and − 5.6 mm3, respectively). Cluster 2 (n = 219), which contained largely fibro-fatty (39.2%) and fibrous plaque (46.8%), showed fibro-fatty PV regression (− 2.4 mm3). Cluster 3 (n = 376), which comprised mostly fibrous (62.7%) and calcified plaque (23.6%), showed increasingly prominent calcified PV progression (+ 21.4 mm3). Cluster 4 (n = 300), which comprised mostly calcified plaque (58.7%), demonstrated the greatest total PV increase (+ 50.7mm3), predominantly increasing in calcified PV (+ 35.9 mm3). Multivariable analysis showed higher risk for plaque progression in Clusters 3 and 4, and higher risk for adverse cardiac events in Clusters 2, 3, and 4 compared to that in Cluster 1. Unsupervised clustering algorithms may uniquely characterize patient phenotypes with varied atherosclerotic plaque profiles, yielding distinct patterns of progressive disease and outcome. [ABSTRACT FROM AUTHOR]

Inflammation inside Atherosclerotic plaques represents a major pathophysiological process driving plaques towards rupture. Pre-clinical studies suggest a relationship between lipid rich necrotic core, intraplaque hemorrhage and inflammation, not previously explored in patients. Therefore, we designed a pilot study to investigate the feasibility of assessing the relationship between these plaque features in a quantitative manner using PET/MRI. In 12 patients with high-grade carotid stenosis the extent of lipid rich necrotic core and intraplaque hemorrhage was quantified from fat and R2* maps acquired with a previously validated 4-point Dixon MRI sequence in a stand-alone MRI. PET/MRI was used to measure 18F-FDG uptake. T1-weighted images from both scanners were used for registration of the quantitative Dixon data with the PET images. The plaques were heterogenous with respect to their volumes and composition. The mean values for the group were as follows: fat fraction (FF) 0.17% (± 0.07), R2* 47.6 s−1 (± 10.9) and target-to-blood pool ratio (TBR) 1.49 (± 0.48). At group level the correlation between TBR and FFmean was − 0.406, p 0.19 and for TBR and R2*mean 0.259, p 0.42. The lack of correlation persisted when analysed on a patient-by-patient basis but the study was not powered to draw definitive conclusions. We show the feasibility of analysing the quantitative relationship between lipid rich necrotic cores, intraplaque haemorrhage and plaque inflammation. The 18F-FDG uptake for most patients was low. This may reflect the biological complexity of the plaques and technical aspects inherent to 18F-FDG measurements. Trial registration: ISRCTN, ISRCTN30673005. Registered 05 January 2021, retrospectively registered. [ABSTRACT FROM AUTHOR]

Atherosclerotic plaques are characterized by an accumulation and subsequent oxidation of LDL, resulting in adaptive immune responses against formed or exposed neoepitopes of the LDL particle. Autoantibodies against native p210, the 3136–3155 amino acid sequence of the LDL protein apolipoprotein B-100 (apoB100) are common in humans and have been associated with less severe atherosclerosis and decreased risk for cardiovascular events in clinical studies. However, whether apoB100 native p210 autoantibodies play a functional role in atherosclerosis is not known. In the present study we immunized apoE-/- mice with p210-PADRE peptide to induce an antibody response against native p210. We also injected mice with murine monoclonal IgG against native p210. Control groups were immunized with PADRE peptide alone or with control murine monoclonal IgG. Immunization with p210-PADRE induced an IgG1 antibody response against p210 that was associated with reduced atherosclerotic plaque formation in the aorta and reduced MDA-LDL content in the lesions. Treatment with monoclonal p210 IgG produced a similar reduction in atherosclerosis as immunization with p210-PADRE. Our findings support an atheroprotective role of antibodies against the apoB100 native p210 and suggest that vaccines that induce the expression of native p210 IgG represent a potential therapeutic strategy for lowering cardiovascular risk. [ABSTRACT FROM AUTHOR]

Since alterations in the intestinal microbiota may induce systemic inflammation and polarization of macrophages to the M1 state, the microbiome role in atherosclerosis, an M1-driven disease, requires evaluation. We aimed to determine if antibiotic (Abx) induced alterations to the intestinal microbiota interferes with atherosclerotic plaque inflammation resolution after lipid-lowering in mice. Hyperlipidemic Apoe−/− mice were fed a western diet to develop aortic atherosclerosis with aortas then transplanted into normolipidemic wild-type (WT) mice to model clinically aggressive lipid management and promote atherosclerosis inflammation resolution. Gut microbial composition pre and post-transplant was altered via an enteral antibiotic or not. Post aortic transplant, after Abx treatment, while plaque size did not differ, compared to Apoe−/− mice, Abx– WT recipient mice had a 32% reduction in CD68-expressing cells (p = 0.02) vs. a non-significant 12% reduction in Abx+ WT mice. A trend toward an M1 plaque CD68-expresing cell phenotype was noted in Abx+ mice. By 16S rRNA sequence analysis, the Abx+ mice had reduced alpha diversity and increased Firmicutes/Bacteroidetes relative abundance ratio with a correlation between gut Firmicutes abundance and plaque CD68-expressing cell content (p < 0.05). These results indicate that in a murine atherosclerotic plaque inflammation resolution model, antibiotic-induced microbiome perturbation may blunt the effectiveness of lipid-lowering to reduce the content of plaque inflammatory CD68-expressing cells. [ABSTRACT FROM AUTHOR]

To assess the influence of lipid-lowering therapy on coronary plaque volume, and to identify the LDL and HDL targets for plaque regression to provide a comprehensive overview. The databases searched (from inception to 15 July 2020) to identify prospective studies investigating the impact of lipid-lowering therapy on coronary plaque volume and including quantitative measurement of plaque volume by intravascular ultrasound after treatment. Thirty-one studies that included 4997 patients were selected in the final analysis. Patients had significantly lower TAV (SMD: 0.123 mm3; 95% CI 0.059, 0.187; P = 0.000) and PAV (SMD: 0.123%; 95% CI 0.035, 0.212; P = 0.006) at follow-up. According to the subgroup analyses, TAV was significantly reduced in the LDL < 80 mg/dL and HDL > 45 mg/dL group (SMD: 0.163 mm3; 95% CI 0.092, 0.234; P = 0.000), and PAV was significantly reduced in the LDL < 90 mg/dL and HDL > 45 mg/dL group (SMD: 0.186%; 95% CI 0.081, 0.291; P = 0.001).Thirty-one studies that included 4997 patients were selected in the final analysis. Patients had significantly lower TAV (SMD: 0.123 mm3; 95% CI 0.059, 0.187; P = 0.000) and PAV (SMD: 0.123%; 95% CI 0.035, 0.212; P = 0.006) at follow-up. According to the subgroup analyses, TAV was significantly reduced in the LDL < 80 mg/dL and HDL > 45 mg/dL group (SMD: 0.163 mm3; 95% CI 0.092, 0.234; P = 0.000), and PAV was significantly reduced in the LDL < 90 mg/dL and HDL > 45 mg/dL group (SMD: 0.186%; 95% CI 0.081, 0.291; P = 0.001). Our meta-analysis suggests that not only should LDL be reduced to a target level of < 80 mg/dL, but HDL should be increased to a target level of > 45 mg/dL to regress coronary plaques. Trial Registration PROSPERO identifier: CRD42019146170. [ABSTRACT FROM AUTHOR]

Endothelial erosion of atherosclerotic plaques is the underlying cause of approximately 30% of acute coronary syndromes (ACS). As the vascular endothelium is profoundly affected by the haemodynamic environment to which it is exposed, we employed computational fluid dynamic (CFD) analysis of the luminal geometry from 17 patients with optical coherence tomography (OCT)-defined plaque erosion, to determine the flow environment permissive for plaque erosion. Our results demonstrate that 15 of the 17 cases analysed occurred on stenotic plaques with median 31% diameter stenosis (interquartile range 28–52%), where all but one of the adherent thrombi located proximal to, or within the region of maximum stenosis. Consequently, all flow metrics related to elevated flow were significantly increased (time averaged wall shear stress, maximum wall shear stress, time averaged wall shear stress gradient) with a reduction in relative residence time, compared to a non-diseased reference segment. We also identified two cases that did not exhibit an elevation of flow, but occurred in a region exposed to elevated oscillatory flow. Our study demonstrates that the majority of OCT-defined erosions occur where the endothelium is exposed to elevated flow, a haemodynamic environment known to evoke a distinctive phenotypic response in endothelial cells. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process. We used a VEGF-A targeted fluorescent antibody (bevacizumab-IRDye800CW [bevacizumab-800CW]) to image and visualize the distribution of VEGF-A in (non-)culprit carotid plaques ex vivo. Freshly endarterectomized human plaques (n = 15) were incubated in bevacizumab-800CW ex vivo. Subsequent NIRF imaging showed a more intense fluorescent signal in the culprit plaques (n = 11) than in the non-culprit plaques (n = 3). A plaque received from an asymptomatic patient showed pathologic features similar to the culprit plaques. Cross-correlation with VEGF-A immunohistochemistry showed co-localization of VEGF-A over-expression in 91% of the fluorescent culprit plaques, while no VEGF-A expression was found in the non-culprit plaques (p < 0.0001). VEGF-A expression was co-localized with CD34, a marker for angiogenesis (p < 0.001). Ex vivo near-infrared fluorescence (NIRF) imaging by incubation with bevacizumab-800CW shows promise for visualizing VEGF-A overexpression in culprit atherosclerotic plaques in vivo. [ABSTRACT FROM AUTHOR]