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Background: Atherosclerotic plaque ruptures, triggered by blood flow–associated biomechanical forces, cause most myocardial infarctions and strokes. Objectives: This study aims to investigate the exact location and underlying mechanisms of atherosclerotic plaque ruptures, identifying therapeutic targets against cardiovascular events. Methods: Histology, electron microscopy, bulk and spatial RNA sequencing on human carotid plaques were studied in proximal, most stenotic, and distal regions along the longitudinal blood flow direction. Genome-wide association studies were used to examine heritability enrichment and causal relationships of atherosclerosis and stroke. Associations between top differentially expressed genes (DEGs) and preoperative and postoperative cardiovascular events were examined in a validation cohort. Results: In human carotid atherosclerotic plaques, ruptures predominantly occurred in the proximal and most stenotic regions but not in the distal region. Histologic and electron microscopic examination showed that proximal and most stenotic regions exhibited features of plaque vulnerability and thrombosis. RNA sequencing identified DEGs distinguishing the proximal and most stenotic regions from the distal region which were deemed as most relevant to atherosclerosis-associated diseases as shown by heritability enrichment analyses. The identified pathways associated with the proximal rupture-prone regions were validated by spatial transcriptomics, firstly in human atherosclerosis. Of the 3 top DEGs, matrix metallopeptidase 9 emerged particularly because Mendelian randomization suggested that its high circulating levels were causally associated with atherosclerosis risk. Conclusions: Our findings show plaque site–specific transcriptional signatures associated with proximal rupture-prone regions of carotid atherosclerotic plaques. This led to the geographical mapping of novel therapeutic targets, such as matrix metallopeptidase 9, against plaque rupture.

There is emerging evidence of an association between epigenetic modifications, glycemic control and atherosclerosis risk. In this study, we mapped genome-wide epigenetic changes in patients with type 2 diabetes (T2D) and advanced atherosclerotic disease. We performed chromatin immunoprecipitation sequencing (ChIP-seq) using a histone 3 lysine 9 acetylation (H3K9ac) mark in peripheral blood mononuclear cells from patients with atherosclerosis with T2D (n = 8) or without T2D (ND, n = 10). We mapped epigenome changes and identified 23,394 and 13,133 peaks in ND and T2D individuals, respectively. Out of all the peaks, 753 domains near the transcription start site (TSS) were unique to T2D. We found that T2D in atherosclerosis leads to an H3K9ac increase in 118, and loss in 63 genomic regions. Furthermore, we discovered an association between the genomic locations of significant H3K9ac changes with genetic variants identified in previous T2D GWAS. The transcription factor 7-like 2 (TCF7L2) rs7903146, together with several human leukocyte antigen (HLA) variants, were among the domains with the most dramatic changes of H3K9ac enrichments. Pathway analysis revealed multiple activated pathways involved in immunity, including type 1 diabetes. Our results present novel evidence on the interaction between genetics and epigenetics, as well as epigenetic changes related to immunity in patients with T2D and advanced atherosclerotic disease.

Background: Chronic Kidney Disease (CKD) is associated with an increased risk for cardiovascular events such as stroke. However, it is still unclear if decreased kidney function is associated with a vulnerable atherosclerotic plaque phenotype. To explore if renal function was associated with carotid plaque vulnerability we analyzed carotid plaques obtained at surgery from the Carotid Plaque Imaging Project (CPIP). Methods: Patients were enrolled through the CPIP cohort. The indication for surgery was plaques with stenosis >70%, associated with ipsilateral symptoms or plaques with stenosis >80% not associated with symptoms. Transversal sections from the most stenotic plaque region were analyzed for connective tissue, calcium, lipids, macrophages, intraplaque hemorrhage, and smooth muscle cells. Homogenates were analyzed for collagen and elastin. Results: Carotid endarterectomy specimens from 379 patients were obtained. The median GFR was 73 ml/min/1.73 m 2 . Plaque characteristics showed no significant association with eGFR, neither when eGFR was divided in CKD groups nor when eGFR was handled as a continuous variable and adjusting for other known risk factors (ie, age, diabetes, hypertension, and smoking). Conclusions: The higher risk of cardiovascular disease such as stroke in CKD is not associated with increased plaque vulnerability and other factors have to be sought.

Additional file 1. Supplementary methods and table SI, SII and SIII.

Supplemental material, Supplemented_figure_1 for Carotid Plaque Morphology is Similar in Patients with Reduced and Normal Renal Function by Caroline Heijl, Fredrik Kahn, Andreas Edsfeldt, Christoffer Tengryd, Jan Nilsson and Isabel Goncalves in Clinical Medicine Insights: Cardiology

Supplemental Digital Content is available in the text., Background: Myeloid cells are central to atherosclerotic lesion development and vulnerable plaque formation. Impaired ability of arterial phagocytes to uptake apoptotic cells (efferocytosis) promotes lesion growth and establishment of a necrotic core. The transcription factor interferon regulatory factor (IRF)-5 is an important modulator of myeloid function and programming. We sought to investigate whether IRF5 affects the formation and phenotype of atherosclerotic lesions. Methods: We investigated the role of IRF5 in atherosclerosis in 2 complementary models. First, atherosclerotic lesion development in hyperlipidemic apolipoprotein E-deficient (ApoE-/-) mice and ApoE-/- mice with a genetic deletion of IRF5 (ApoE-/-Irf5-/-) was compared and then lesion development was assessed in a model of shear stress-modulated vulnerable plaque formation. Results: Both lesion and necrotic core size were significantly reduced in ApoE-/-Irf5-/- mice compared with IRF5-competent ApoE-/- mice. Necrotic core size was also reduced in the model of shear stress-modulated vulnerable plaque formation. A significant loss of CD11c+ macrophages was evident in ApoE-/-Irf5-/- mice in the aorta, draining lymph nodes, and bone marrow cell cultures, indicating that IRF5 maintains CD11c+ macrophages in atherosclerosis. Moreover, we revealed that the CD11c gene is a direct target of IRF5 in macrophages. In the absence of IRF5, CD11c- macrophages displayed a significant increase in expression of the efferocytosis-regulating integrin-β3 and its ligand milk fat globule-epidermal growth factor 8 protein and enhanced efferocytosis in vitro and in situ. Conclusions: IRF5 is detrimental in atherosclerosis by promoting the maintenance of proinflammatory CD11c+ macrophages within lesions and controlling the expansion of the necrotic core by impairing efferocytosis.

Background: Activation of the renin-angiotensin-aldosterone-system (RAAS) has been proposed to contribute to development of vascular complications in type 2 diabetes (T2D). The aim of the present study was to determine if plasma renin levels are associated with the severity of vascular changes in subjects with and without T2D.Methods: Renin was analyzed by the Proximity Extension Assay in subjects with (n = 985) and without (n = 515) T2D participating in the SUMMIT (SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools) study and in 205 carotid endarterectomy patients. Vascular changes were assessed by determining ankle-brachial pressure index (ABPI), carotid intima-media thickness (IMT), carotid plaque area, pulse wave velocity (PWV) and the reactivity hyperemia index (RHI).Results: Plasma renin was elevated in subjects with T2D and demonstrated risk factor-independent association with prevalent cardiovascular disease both in subjects with and without T2D. Renin levels increased with age, body mass index, HbA1c and correlated inversely with HDL. Subjects with T2D had more severe carotid disease, increased arterial stiffness, and impaired endothelial function. Risk factor-independent associations between renin and APBI, bulb IMT, carotid plaque area were observed in both T2D and non-T2D subjects. These associations were independent of treatment with RAAS inhibitors. Only weak associations existed between plasma renin and the expression of pro-inflammatory and fibrous components in plaques from 205 endarterectomy patients.Conclusions: Our findings provide clinical evidence for associations between systemic RAAS activation and atherosclerotic burden and suggest that this association is of particular importance in T2D.