Search

The immune system is integral to cardiovascular health and disease. Targeting inflammation ameliorates adverse cardiovascular outcomes. Atherosclerosis, a major underlying cause of cardiovascular disease (CVD), is conceptualised as a lipid-driven inflammation where macrophages play a non-redundant role. However, evidence emerging so far from single cell atlases suggests a dichotomy between lipid associated and inflammatory macrophage states. Here, we present an inclusive reference atlas of human intraplaque immune cell communities. Combining scRNASeq of human surgical carotid endarterectomies in a discovery cohort with bulk RNASeq and immunohistochemistry in a validation cohort (the Carotid Plaque Imaging Project-CPIP), we reveal the existence of PLIN2 hi /TREM1 hi macrophages as a toll-like receptor-dependent inflammatory lipid-associated macrophage state linked to cerebrovascular events. Our study shifts the current paradigm of lipid-driven inflammation by providing biological evidence for a pathogenic macrophage transition to an inflammatory lipid-associated phenotype and for its targeting as a new treatment strategy for CVD., Competing Interests: Competing Interests Statement All authors report no conflict of interest. A.E has received consultancy fees from and/or served on the advisory boards of Novo Nordisk, Sanofi and Amgen but this has not had any relationship with the current study or affected the design/outcome of the study. The remaining authors have no conflicts of interest.

Background: Women's pregnancy history is associated with incident risk of coronary artery disease with some evidence also suggesting a relevance for prognosis following treatment., Objectives: To study the associations between maternal history of preterm delivery, a history of small for gestational age infant, parity and age at first delivery with clinical restenosis after percutaneous coronary intervention (PCI)., Methods: In this prospective cohort study, we included 6027 women < 65 years undergoing their first PCI 2006-2017, merging clinical register data on PCI procedures in Sweden with comprehensive registry data on deliveries since 1973. We used proportional hazards regression to study the association between aspects of pregnancy history and clinical restenosis in per-segment analyses, and with target lesion revascularisation (TLR) in per-patient analyses. We adjusted models for procedural-related and patient-related predictors of restenosis., Results: During 15 981 segment-years of follow-up, 343 (3.7%) events of clinical restenosis occurred. We found no strong evidence of associations between the studied aspects of pregnancy history and clinical restenosis following PCI. For example, the restenosis HR for a history of preterm delivery in the fully adjusted model was 1.09 (95% CI 0.77 to 1.55) and the TLR HR was 1.18 (95% CI 0.91 to 1.52)., Conclusion: Risk of restenosis following treatment with PCI did not differ by the studied aspects of pregnancy history, including preterm delivery, in young and middle-aged women. Larger studies are needed to obtain more precise estimates., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Background: Arterial hemodynamic parameters derived from reservoir-excess pressure analysis exhibit prognostic utility. Reservoir-excess pressure analysis may provide useful information about an influence of altered hemodynamics on target organ such as the kidneys. We determined whether the parameters derived from the reservoir-excess pressure analysis were associated with the reduction in estimated glomerular filtration rate in 542 older adults (69.4±7.9 years, 194 females) at baseline and after 3 years., Methods: Reservoir-excess pressure parameters, including reservoir pressure integral, excess pressure integral, systolic, and diastolic rate constants, were obtained by radial artery tonometry., Results: After 3 years, and in a group of 94 individuals (72.4±7.6 years, 26 females), there was an estimated glomerular filtration rate reduction of >5% per year (median reduction of 20.5% over 3 years). A multivariable logistic regression analysis revealed that higher baseline reservoir pressure integral was independently associated with a smaller reduction in estimated glomerular filtration rate after accounting for conventional cardiovascular risk factors and study centers (odds ratio: 0.660 [95% CIs, 0.494-0.883]; P =0.005). The association remained unchanged after further adjustments for potential confounders and baseline renal function (odds ratio: 0.528 [95% CIs, 0.351-0.794]; P =0.002). No other reservoir-excess pressure parameters exhibited associations with the reduction in renal function., Conclusions: This study demonstrates that baseline reservoir pressure integral was associated with the decline in renal function in older adults at 3-year follow-up, independently of conventional cardiovascular risk factors. This suggests that reservoir pressure integral may play a role in the functional decline of the kidneys.

Background and Aims: Extracellular matrix (ECM) remodeling is one of the key components in the formation of vulnerable atherosclerotic plaques and cardiovascular events. We recently showed that the full-length ECM-proteoglycan osteoglycin was associated with plaque vulnerability and future cardiovascular events. In the present study, we aimed to investigate the association of cleaved osteoglycin with plaque phenotype., Methods: Two-hundred human carotid plaques were analyzed by immunohistochemistry. Cleaved osteoglycin and active caspase-3 were assessed by ELISA. ECM components (collagen, elastin and glycosaminoglycans) were assessed by colorimetric assays in plaque tissue homogenates. Matrix metalloproteinases (MMPs) were assessed using Milliplex. MMP-cleavage of osteoglycin and its effect on apoptosis were studied in vitro. Cardiovascular events were recorded during follow-up using national registries., Results: Plaque levels of cleaved osteoglycin were significantly higher in asymptomatic plaques and correlated to α-actin plaque area, collagen, elastin and inversely to lipids, active. caspase-3 and a histological vulnerability index. Cleaved osteoglycin correlated to several MMPs, especially MMP-12, which was also shown to cleave osteoglycin in vitro. In vitro cleavage of osteoglycin was also associated with less smooth muscle cell apoptosis. Patients with high plaque levels of cleaved osteoglycin had a significantly lower risk to suffer from future cardiovascular events., Conclusions: The current study shows that cleaved osteoglycin is associated with a stable plaque phenotype and lower risk for future cardiovascular events. Potentially due to reduced cell apoptosis and ability to retain LDL. These results indicate that targeting the cleavage of osteoglycin may be a potential therapeutic strategy to stabilize plaques., (Copyright © 2022. Published by Elsevier B.V.)

Hepatocyte growth factor (HGF) is released by stressed human vascular cells and promotes vascular cell repair responses in both autocrine and paracrine ways. Subjects with a low capacity to express HGF in response to systemic stress have an increased cardiovascular risk. Human atherosclerotic plaques with a low content of HGF have a more unstable phenotype. The present study shows that subjects with a low ability to express HGF in response to metabolic stress have an increased risk to suffer myocardial infarction and stroke., Competing Interests: The work was supported by the Swedish Society for Medical Research, Emil and Wera Cornell Foundation, Hjelt Foundation, the Swedish Research Council, the Swedish Foundation for International Cooperation in Research and Higher Education, Crafoord Foundation, the Swedish Medical Society, Diabetes Foundation, Swedish Heart and Lung Foundation, Diabetes Research and Wellness Foundation, Albert Påhlssons Foundation, Skåne University Hospital (SUS) Foundations and funds, Lund University Infrastructure grant “Malmö Population-based Cohorts” (STYR 2019/2046), and Lund University Diabetes Center (Swedish Research Council–Strategic Research Area Excellence of Diabetes Research in Sweden (Exodiab) grant number 2009-1039 and the Swedish Foundation for Strategic Research grant number IRC15-006), National Natural Science Foundation of China grant 82120108004 (Dr Liang) and 82100479 (Dr Chen). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2022 The Authors.)

Background: Carotid artery disease is highly prevalent and a main cause of ischemic stroke and vascular dementia. There is a paucity of information on predictors of serious vascular events. Besides percentage diameter stenosis, international guidelines also recommend the evaluation of qualitative characteristics of carotid artery disease as a guide to treatment, but with no agreement on which qualitative features to assess. This inadequate knowledge leads to a poor ability to identify patients at risk, dispersion of medical resources, and unproven use of expensive and resource-consuming techniques, such as magnetic resonance imaging, positron emission tomography, and computed tomography., Objectives: The Carotid Artery Multimodality imaging Prognostic (CAMP) study will: prospectively determine the best predictors of silent and overt ischemic stroke and vascular dementia in patients with asymptomatic subcritical carotid artery disease by identifying the noninvasive diagnostic features of the 'vulnerable carotid plaque'; assess whether 'smart' use of low-cost diagnostic methods such as ultrasound-based evaluations may yield at least the same level of prospective information as more expensive techniques., Study Design: We will compare the prognostic/predictive value of all proposed techniques with regard to silent or clinically manifest ischemic stroke and vascular dementia. The study will include ≥300 patients with asymptomatic, unilateral, intermediate degree (40-60% diameter) common or internal carotid artery stenosis detected at carotid ultrasound, with a 2-year follow-up. The study design has been registered on Clinicaltrial.gov on December 17, 2020 (ID number NCT04679727)., (Copyright © 2022 Italian Federation of Cardiology - I.F.C. All rights reserved.)

Aims: Inflammation is a key factor in atherosclerosis. The transcription factor interferon regulatory factor-5 (IRF5) drives macrophages towards a pro-inflammatory state. We investigated the role of IRF5 in human atherosclerosis and plaque stability., Methods and Results: Bulk RNA sequencing from the Carotid Plaque Imaging Project biobank were used to mine associations between major macrophage associated genes and transcription factors and human symptomatic carotid disease. Immunohistochemistry, proximity extension assays, and Helios cytometry by time of flight (CyTOF) were used for validation. The effect of IRF5 deficiency on carotid plaque phenotype and rupture in ApoE-/- mice was studied in an inducible model of plaque rupture. Interferon regulatory factor-5 and ITGAX/CD11c were identified as the macrophage associated genes with the strongest associations with symptomatic carotid disease. Expression of IRF5 and ITGAX/CD11c correlated with the vulnerability index, pro-inflammatory plaque cytokine levels, necrotic core area, and with each other. Macrophages were the predominant CD11c-expressing immune cells in the plaque by CyTOF and immunohistochemistry. Interferon regulatory factor-5 immunopositive areas were predominantly found within CD11c+ areas with a predilection for the shoulder region, the area of the human plaque most prone to rupture. Accordingly, an inducible plaque rupture model of ApoE-/-Irf5-/- mice had significantly lower frequencies of carotid plaque ruptures, smaller necrotic cores, and less CD11c+ macrophages than their IRF5-competent counterparts., Conclusion: Using complementary evidence from data from human carotid endarterectomies and a murine model of inducible rupture of carotid artery plaque in IRF5-deficient mice, we demonstrate a mechanistic link between the pro-inflammatory transcription factor IRF5, macrophage phenotype, plaque inflammation, and its vulnerability to rupture., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Cardiology.)

Histology is a long standing and well-established gold standard for pathological characterizations. In recent years however, synchrotron radiation-based micro-computed tomography (SRμCT) has become a tool for extending the imaging of two-dimensional thin sections into three-dimensional imaging of tissue blocks, enabling so-called virtual histology with arbitrary clipping planes, volumetric rendering and automatic segmentation. In this study, we present a thorough characterization of human carotid plaques after endarterectomy of patients with stroke or transient ischemic attack (TIA), investigating several different pathologic structures using both SRμCT and histology. Phase-contrast SRμCT was performed with two different magnifications (voxel sizes 6.5 μm and 0.65 μm, respectively), and histology was performed with multiple different stainings (Alpha-actin, Glycophorin A, von Kossa, Movat, CD68). The 0.65 μm high-resolution SRμCT was performed on selected areas with plaque typical relevant morphology, identified on the 6.5 μm low-resolution SRμCT. The tomography datasets were reconstructed with additional 3D volume rendering and compared to histology. In total, nine different regions with typical pathologic structures were identified and imaged with high-resolution SRμCT. The results show many characteristics typical for advanced atherosclerotic plaques, clinically relevant, namely ruptures with thrombosis, neo-vascularization, inflammatory infiltrates in shoulder regions, lipid rich necrotic cores (LRNC), thin fibrous cap, calcifications, lumen irregularities, and changes in vessel wall structures such as the internal elastic membrane. This method's non-destructive nature renders details of micro-structures with an excellent visual likeness to histology, with the additional strength of multiplanar and 3D visualization and the possibility of multiple re-scans., Competing Interests: The authors have declared that no competing interests exist.

Recent studies highlight the importance of lipotoxic damage in aortic cells as the major pathogenetic contributor to atherosclerotic disease. Since the STE20-type kinase STK25 has been shown to exacerbate ectopic lipid storage and associated cell injury in several metabolic organs, we here investigate its role in the main cell types of vasculature. We depleted STK25 by small interfering RNA in human aortic endothelial and smooth muscle cells exposed to oleic acid and oxidized LDL. In both cell types, the silencing of STK25 reduces lipid accumulation and suppresses activation of inflammatory and fibrotic pathways as well as lowering oxidative and endoplasmic reticulum stress. Notably, in smooth muscle cells, STK25 inactivation hinders the shift from a contractile to a synthetic phenotype. Together, we provide several lines of evidence that antagonizing STK25 signaling in human aortic endothelial and smooth muscle cells is atheroprotective, highlighting this kinase as a new potential therapeutic target for atherosclerotic disease., (© 2022. The Author(s).)

Background The cardiovagal function can be assessed by quantification of respiratory sinus arrhythmia (RSA) during a deep breathing test. However, population studies of RSA and coronary atherosclerosis are lacking. This population-based study examined the relationship between RSA during deep breathing and coronary atherosclerosis, assessed by coronary artery calcium score (CACS). Methods and Results SCAPIS (Swedish Cardiopulmonary Bioimage Study) randomly invited men and women aged 50 to 64 years from the general population. CACS was obtained from computed tomography scanning, and deep breathing tests were performed in 4654 individuals. Expiration-inspiration differences (E-Is) of heart rates were calculated, and reduced RSA was defined as E-I in the lowest decile of the population. The relationship between reduced RSA and CACS (CACS≥100 or CACS≥300) was calculated using multivariable-adjusted logistic regression. The proportion of CACS≥100 was 24% in the lowest decile of E-I and 12% in individuals with E-I above the lowest decile ( P <0.001), and the proportion of CACS≥300 was 12% and 4.8%, respectively ( P <0.001). The adjusted odds ratio (OR) for CACS≥100 was 1.42 (95% CI, 1.10-1.84) and the adjusted OR for CACS≥300 was 1.62 (95% CI, 1.15-2.28), when comparing the lowest E-I decile with deciles 2 to 10. Adjusted ORs per 1 SD lower E-I were 1.17 ( P =0.001) for CACS≥100 and 1.28 ( P =0.001) for CACS≥300. Conclusions Low RSA during deep breathing is associated with increased coronary atherosclerosis as assessed by CACS, independently of traditional cardiovascular risk factors. Cardiovagal dysfunction could be a prevalent and modifiable risk factor for coronary atherosclerosis in the general population.

Background and Aims: The severity of the atherosclerotic burden is hardly quantifiable in subjects at high cardiovascular (CV) risk under intensive pharmacological therapy. Several molecules have been proposed as circulating biomarkers of atherosclerosis, but none has emerged as clinically meaningful., Methods: Circulating proteins involved in inflammation, plaque remodeling, smooth muscle cell migration, apoptosis and endothelial activity were measured by Proximity Extension Assay in the SUMMIT study cohort (n = 1500), including patients with type 2 diabetes (66%) and established CV disease (50%), who underwent ultrasound assessment of carotid atherosclerosis with total plaque area quantification., Results: In patients with evidence of carotid artery atherosclerosis (n = 1174), seven biomarkers were identified as the more closely related to atherosclerosis extension. Compared with a multivariable model including major traditional CV risk factors, the percentage gain of explained variability in total plaque area was the greatest (33%) after inclusion of CD40 receptor (CD40R) ligand, followed by PDGF (30%), CD40R (26%), EGF (22%), CXCL1 (15%), HBEGF and MMP-17 (both 11%). The relationship of total plaque area with CD40R, PDGF was hyperbolic. In the whole study cohort, including subjects without carotid plaques, CD40R was the strongest predictor of the presence and extension of carotid atherosclerosis. Subjects in the third CD40R tertile had a more than two-fold greater atherosclerotic burden compared with lower CD40R tertiles, despite an only marginally higher load of CV risk factors., Conclusions: CD40R stands among an extended set of plausible atherosclerosis-related biomarkers as the most powerful predictor of carotid atherosclerosis burden in a high CV risk cohort., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Rationale: Vascular calcification is a prominent feature of late-stage diabetes, renal and cardiovascular disease (CVD), and has been linked to adverse events. Recent studies in patients reported that plasma levels of osteomodulin (OMD), a proteoglycan involved in bone mineralisation, associate with diabetes and CVD. We hypothesised that OMD could be implicated in these diseases via vascular calcification as a common underlying factor and aimed to investigate its role in this context., Methods and Results: In patients with chronic kidney disease, plasma OMD levels correlated with markers of inflammation and bone turnover, with the protein present in calcified arterial media. Plasma OMD also associated with cardiac calcification and the protein was detected in calcified valve leaflets by immunohistochemistry. In patients with carotid atherosclerosis, circulating OMD was increased in association with plaque calcification as assessed by computed tomography. Transcriptomic and proteomic data showed that OMD was upregulated in atherosclerotic compared to control arteries, particularly in calcified plaques, where OMD expression correlated positively with markers of smooth muscle cells (SMCs), osteoblasts and glycoproteins. Immunostaining confirmed that OMD was abundantly present in calcified plaques, localised to extracellular matrix and regions rich in α-SMA + cells. In vivo, OMD was enriched in SMCs around calcified nodules in aortic media of nephrectomised rats and in plaques from ApoE -/- mice on warfarin. In vitro experiments revealed that OMD mRNA was upregulated in SMCs stimulated with IFNγ, BMP2, TGFβ1, phosphate and β-glycerophosphate, and by administration of recombinant human OMD protein (rhOMD). Mechanistically, addition of rhOMD repressed the calcification process of SMCs treated with phosphate by maintaining their contractile phenotype along with enriched matrix organisation, thereby attenuating SMC osteoblastic transformation. Mechanistically, the role of OMD is exerted likely through its link with SMAD3 and TGFB1 signalling, and interplay with BMP2 in vascular tissues., Conclusion: We report a consistent association of both circulating and tissue OMD levels with cardiovascular calcification, highlighting the potential of OMD as a clinical biomarker. OMD was localised in medial and intimal α-SMA + regions of calcified cardiovascular tissues, induced by pro-inflammatory and pro-osteogenic stimuli, while the presence of OMD in extracellular environment attenuated SMC calcification., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

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 and Aims: After aortic coarctation (CoA) repair, patients still suffer from cardiovascular complications. The aim of this study was to measure cardiovascular markers, intima-media thickness (IMT) and plaques in controls and patients with CoA., Methods: Sixty-four patients with CoA (66% male, mean age 48 ± 15 years) and controls (54% men, mean age 47 ± 16 years) underwent ultrasound of their arteries. A multiplex platform to analyze circulating blood levels biomarkers reflecting inflammation, tissue remodeling and repair was used., Results: In men following CoA repair, a significantly increased carotid bulb IMT was observed in comparison to the control group (1.05 [0.72-1.24] vs. 0.67 [0.59-0.95] mm; p = 0.003). Median common carotid artery (CCA) IMT was increased in men compared to controls (0.82 [0.61-0.97] mm vs. 0.58 [0.53-0.76] mm, p < 0.003) and in women compared to controls (0.83 [0.70-0.92] vs. 0.60 [0.55-0.69], p < 0.004). CoA demonstrated an independent association with IMT in both men and women. Men with CoA were also more likely to have a plaque in their carotid arteries (p = 0.010). In women with CoA, we observed significantly lower levels of stem cell factor (SCF, p = 0.004) while in men with CoA we observed significantly lower levels of matrix metalloproteinase-3 (MMP-3, p = 0.048), tumor necrosis factor receptor 1 (TNF-R1, p = 0.032), tumor necrosis factor receptor superfamily member 10B (TRAIL-R2, p = 0.019) and monocyte chemotactic protein 1 (MCP-1, p = 0.015)., Conclusions: This is the first study to show that despite successful CoA repair, patients have more carotid atherosclerosis than can be explained by changes in tissue remodeling and repair., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background The balance between stabilizing and destabilizing atherosclerotic plaque components is used in experimental studies and in imaging studies to identify rupture prone plaques. However, we lack the evidence that this balance predicts future cardiovascular events. Here we explore whether a calculated histological ratio, referred to as vulnerability index (VI), can predict patients at higher risk to suffer from future cardiovascular events. Methods and Results Carotid plaques and clinical information from 194 patients were studied. Tissue sections were used for histological analysis to calculate the VI (CD68 [cluster of differentiation 68], alpha-actin, Oil red O, Movat pentachrome, and glycophorin A). Postoperative cardiovascular events were identified through the Swedish National Inpatient Health Register (2005-2013). During the follow-up (60 months) 45 postoperative cardiovascular events were registered. Patients with a plaque VI in the fourth quartile compared with the first to third quartiles had significantly higher risk to suffer from a future cardiovascular event ( P =0.0002). The VI was an independent predictor and none of the 5 histological variables analyzed separately predicted events. In the 13 patients who underwent bilateral carotid endarterectomy, the VI of the right plaque correlated with the VI of the left plaque and vice versa ( r =0.7, P =0.01). Conclusions Our findings demonstrate that subjects with a high plaque VI have an increased risk of future cardiovascular events, independently of symptoms and other known cardiovascular risk factors . This strongly supports that techniques which image such plaques can facilitate risk stratification for subjects in need of more intense treatment.

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4 + T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c + dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.

Background and Aims: Histopathology is the gold standard for analysis of atherosclerotic plaques but has drawbacks due to the destructive nature of the method. Ex vivo MRI is a non-destructive method to image whole plaques. Our aim was to use quantitative high field ex vivo MRI to classify plaque components, with histology as gold standard., Methods: Surgically resected carotid plaques from 12 patients with recent TIA or stroke were imaged at 11.7 T MRI. Quantitative T1/T2* mapping sequences and qualitative T1/T2* gradient echo sequences with voxel size of 30 × 30 × 60 μm 3 were obtained prior to histological preparation, sectioning and staining for lipids, inflammation, hemorrhage, and fibrous tissue. Regions of interest (ROI) were selected based on the histological staining at multiple levels matched between histology and MRI. The MRI parameters of each ROI were then analyzed with quadratic discriminant analysis (QDA) for classification., Results: A total of 965 ROIs, at 70 levels matched between histology and MRI, were registered based on histological staining. In the nine plaques where three or more plaque components were possible to co-localize with MRI, the mean degree of misclassification by QDA was 16.5 %. One of the plaques contained mostly fibrous tissue and lipids and had no misclassifications, and two plaques mostly contained fibrous tissue. QDA generally showed good classification for fibrous tissue and lipids, whereas plaques with hemorrhage and inflammation had more misclassifications., Conclusion: 11.7 T ex vivo high field MRI shows good visual agreement with histology in carotid plaques. T1/T2* maps analyzed with QDA is a promising non-destructive method to classify plaque components, but with a higher degree of misclassifications in plaques with hemorrhage or inflammation., Competing Interests: The authors report no declarations of interest., (© 2021 The Author(s).)

Background: Type 2 diabetes (T2D) patients are at a greater risk of cardiovascular events due to aggravated atherosclerosis. Oxidized LDL (oxLDL) has been shown to be increased in T2D plaques and suggested to contribute to plaque ruptures. Despite intensified statin treatment during the last decade the higher risk for events remains. Here, we explored if intensified statin treatment was associated with reduced oxLDL in T2D plaques and if oxLDL predicts cardiovascular events, to elucidate whether further plaque oxLDL reduction would be a promising therapeutic target., Methods: Carotid plaque OxLDL levels and plasma lipoproteins were assessed in 200 patients. Plaque oxLDL was located by immunohistochemistry. Plaque cytokines, cells and scavenger receptor gene expression were quantified by Luminex, immunohistochemistry and RNA sequencing, respectively. Clinical information and events during follow-up were obtained from national registers., Results: Plaque oxLDL levels correlated with markers of inflammatory activity, endothelial activation and plasma LDL cholesterol (r = 0.22-0.32 and p ≤ 0.01 for all). T2D individuals exhibited lower plaque levels of oxLDL, sLOX-1(a marker of endothelial activation) and plasma LDL cholesterol (p = 0.001, p = 0.006 and p = 0.009). No increased gene expression of scavenger receptors was identified in T2D plaques. The lower oxLDL content in T2D plaques was associated with a greater statin usage (p = 0.026). Supporting this, a linear regression model showed that statin treatment was the factor with the strongest association to plaque oxLDL and plasma LDL cholesterol (p < 0.001 for both). However, patients with T2D more frequently suffered from symptoms and yet plaque levels of oxLDL did not predict cardiovascular events in T2D (findings are summarized in Fig. 1a)., Conclusions: This study points out the importance of statin treatment in affecting plaque biology in T2D. It also implies that other biological components, beyond oxLDL, need to be identified and targeted to further reduce the risk of events among T2D patients receiving statin treatment.

Placental growth factor (PlGF) is a mitogen for endothelial cells, but it can also act as a proinflammatory cytokine. Because it promotes early stages of plaque formation in experimental models of atherosclerosis and was implicated in epidemiological associations with risk of cardiovascular disease (CVD), PlGF has been attributed a pro-atherogenic role. Here, we investigated whether PlGF has a protective role in CVD and whether elevated PlGF reflects activation of repair processes in response to vascular stress. In a population cohort of 4742 individuals with 20 years of follow-up, high baseline plasma PlGF was associated with increased risk of cardiovascular death, myocardial infarction, and stroke, but these associations were lost or weakened when adjusting for cardiovascular risk factors known to cause vascular stress. Exposure of cultured endothelial cells to high glucose, oxidized low-density lipoprotein (LDL) or an inducer of apoptosis enhanced the release of PlGF. Smooth muscle cells and endothelial cells treated with PlGF small interference RNA demonstrated that autocrine PlGF stimulation plays an important role in vascular repair responses. High expression of PlGF in human carotid plaques removed at surgery was associated with a more stable plaque phenotype and a lower risk of future cardiovascular events. When adjusting associations of PlGF with cardiovascular risk in the population cohort for plasma soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2, a biomarker of cellular stress, a high PlGF/TRAIL receptor-2 ratio was associated with a lower risk. Our findings provide evidence for a protective role of PlGF in CVD., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

Rationale: The alarmin S100A9 has been identified as a potential therapeutic target in myocardial infarction. Short-term S100A9 blockade during the inflammatory phase post-myocardial infarction inhibits systemic and cardiac inflammation and improves cardiac function long term., Objective: To evaluate the impact of S100A9 blockade on postischemic cardiac repair., Methods and Results: We assessed cardiac function, hematopoietic response, and myeloid phagocyte dynamics in WT (wild type) C57BL/6 mice with permanent coronary artery ligation, treated with the specific S100A9 blocker ABR-238901 for 7 or 21 days. In contrast to the beneficial effects of short-term therapy, extended S100A9 blockade led to progressive deterioration of cardiac function and left ventricle dilation. The treatment reduced the proliferation of Lin - Sca-1 + c-Kit + hematopoietic stem and progenitor cells in the bone marrow and the production of proreparatory CD150 + CD48 - CCR2 + hematopoietic stem cells. Monocyte trafficking from the spleen to the myocardium and subsequent phenotype switching to reparatory Ly6C lo MerTK hi macrophages was also impaired, leading to inefficient efferocytosis, accumulation of apoptotic cardiomyocytes, and a larger myocardial scar. The transcription factor Nur77 (Nr4a1 [nuclear receptor subfamily 4 group A member 1]) mediates the transition from inflammatory Ly6C hi monocytes to reparatory Ly6C lo macrophages. S100A9 upregulated the levels and activity of Nur77 in monocytes and macrophages in vitro and in Ly6C hi/int monocytes in vivo, and S100A9 blockade antagonized these effects. Finally, the presence of reparatory macrophages in the myocardium was also impaired in S100A9 -/ - mice with permanent myocardial ischemia, leading to depressed cardiac function long term., Conclusions: We show that S100A9 plays an important role in both the inflammatory and the reparatory immune responses to myocardial infarction. Long-term S100A9 blockade negatively impacts cardiac recovery and counterbalances the beneficial effects of short-term therapy. These results define a therapeutic window targeting the inflammatory phase for optimal effects of S100A9 blockade as potential immunomodulatory treatment in acute myocardial infarction.

Background and Aims: The pro-inflammatory alarmin S100A12 (EN-RAGE) and the soluble form of its receptor, the receptor for advanced glycation endproducts (sRAGE), have diverging roles in cardiovascular disease. In experimental studies, S100A12 promoted atherosclerosis while sRAGE treatment was anti-atherogenic and reduced myocardial infarction size by scavenging RAGE ligands. Here, we aimed to explore the links between S100A12, sRAGE, and long-term prognosis after an acute coronary syndrome (ACS)., Methods: We measured S100A12 and sRAGE in 524 patients within 24 h after an ACS, and again 6 weeks later in a subgroup of 114 patients. This subgroup also completed a follow-up echocardiography after 1 year. The median follow-up time for recurrent major adverse cardiovascular events (MACE), defined as recurrent ACS or cardiovascular death, was 25.7 ± 12.6 months., Results: In Cox proportional hazard analyses, baseline S100A12 and sRAGE were positively associated with the risk of MACE, independently of traditional cardiovascular risk factors. The association between sRAGE and MACE remained significant after additional adjustment for troponin T, NT-proBNP and hsCRP [HR 95%CI for highest versus lowest tertile 3.2 (1.5-6.5), p = 0.002]. High sRAGE was also associated with deteriorating left ventricular function and an increased rate of heart failure hospitalization post-discharge. In contrast, patients with increasing sRAGE at 6 weeks compared to baseline had lower incidence of recurrent ACS., Conclusions: Our data suggest that sRAGE has a dual, phase-dependent association with residual cardiovascular risk after ACS. These findings are important for the design and interpretation of future studies on sRAGE as biomarker and potential treatment in ACS patients., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The relationship between dyslipidemia, inflammation and CV organ damage in type 2 diabetes mellitus (T2DM) is complex. Insulin resistance and inflammatory cytokines interleukins (ILs) increase plasma triglycerides (TG). ILs also up-regulate expression of matrix-metalloproteinases (MMPs) that, together with TG, decrease high density lipoprotein cholesterol (HDL) levels. High TG, low HDL, increased ILs and MMPs trigger structural and functional changes in different parts of cardiovascular (CV) system. To understand better the role of lipids and inflammation in CV organ damage, the present study investigated the inter-relationships between lipids, ILs and MMPs, as well as the associations of lipids, ILs and MMPs with various CV measures, both in diabetic and non-diabetic population (nonT2DM)., Methods: In T2DM patients (N = 191) and nonT2DM subjects (N = 94) were assessed carotid intima-media thickness (cIMT) and inter-adventitial diameter (IADiam), carotid wave speed (ccaWS), carotid-femoral pulse wave velocity (cfPWV), left ventricular (LV) mass, LV systolic (s') and early diastolic (e') longitudinal velocities of mitral annulus, together with glycemic control, lipid profile, IL-6, IL-18 and MMP-12., Results: T2DM patients, as compared to nonT2DM subjects, had significantly higher plasma levels of IL-6, IL-18, MMP-12 and lower HDL (P < 0.05-0.0001). They had also higher cIMT, IADiam, ccaWS, cfPWV and LV mass, and lower e' velocity (P < 0.005-0.0001). Both in T2DM patients and nonT2DM subjects, MMP-12 increased with IL-6 (r = 0.43 and 0.39; P < 0.0001) and IL-18 (r = 0.32 and 0.42; P < 0.0001), and HDL decreased with MMP-12 (r = - 0.29 and - 0.42; P < 0.0001). In both populations, MMP-12 was directly associated with IADiam, ccaWS, cfPWV and LV mass (r = 0.42, 0.32, 0.26 and 0.29; P < 0.0001 in T2DM patients, and r = 0.39, 0.28, 0.32 and 0.27; P < 0.01-0.0001 in nonT2DM subjects). In multivariate analysis, MMP-12 remained independently related to IADiam, ccaWS, cfPWV and LV mass in T2DM patients, and to IADiam only in nonT2DM subjects., Conclusions: This cross-sectional study demonstrated a direct association between ILs and MMP-12, as well as an inverse association between MMP-12 and HDL, both in T2DM patients and in nonT2DM subjects. In T2DM patients, who had higher levels of ILs and MMP-12, the latter was independently related to several structural and functional markers of preclinical CV organ damage.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone with extrapancreatic effects beyond glycemic control. Here we demonstrate unexpected effects of GIP signaling in the vasculature. GIP induces the expression of the proatherogenic cytokine osteopontin (OPN) in mouse arteries via local release of endothelin-1 and activation of CREB. Infusion of GIP increases plasma OPN concentrations in healthy individuals. Plasma endothelin-1 and OPN concentrations are positively correlated in patients with critical limb ischemia. Fasting GIP concentrations are higher in individuals with a history of cardiovascular disease (myocardial infarction, stroke) when compared with control subjects. GIP receptor (GIPR) and OPN mRNA levels are higher in carotid endarterectomies from patients with symptoms (stroke, transient ischemic attacks, amaurosis fugax) than in asymptomatic patients, and expression associates with parameters that are characteristic of unstable and inflammatory plaques (increased lipid accumulation, macrophage infiltration, and reduced smooth muscle cell content). While GIPR expression is predominantly endothelial in healthy arteries from humans, mice, rats, and pigs, remarkable upregulation is observed in endothelial and smooth muscle cells upon culture conditions, yielding a "vascular disease-like" phenotype. Moreover, the common variant rs10423928 in the GIPR gene is associated with increased risk of stroke in patients with type 2 diabetes., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Introduction: Elevated levels of markers for thrombin activation are associated with plaque echogenicity and degree of stenosis in patients with carotid artery stenosis. The Activated Protein C-Protein C Inhibitor (APC-PCI) complex reflects activation of the Protein C system and is a measure of thrombin generation. The aim of the present study was to examine APC-PCI complex in patients undergoing thrombendartherectomy for carotid artery stenosis, and to relate the findings to clinical characteristics and plaque morphology as determined by ultrasound., Materials and Methods: Blood was obtained from 125 patients (39 female, median age 71 years) with carotid artery stenosis admitted from September 2005 to May 2007. The APC-PCI complex was measured using a sandwich immunofluorometric method and compared to an age- and sex-matched healthy control-group. Clinical and demographic characteristics, routine laboratory markers and ultrasound characteristics were analysed using univariate and multivariate analysis., Results: APC-PCI complex concentration was significantly increased in patients with carotid artery stenosis (median 0.21 microg/L; 10th to 90th percentile 0.15-0.36) compared to a healthy control-group (0.19 microg/L; 0.11-0.31; P=.009). There was no significant difference in APC-PCI-values between asymptomatic (n=48) and symptomatic (n=77) patients with carotid artery stenosis (0.22 vs. 0.20 microg/L; p=0.626). Patients with minor stroke (n=31) had a higher median APC-PCI-concentration (0.27 microg/L; 0.15-0.63) than patients with amaurosis fugax (0.19 microg/L; 0.15-0.36) or transient ischemic attack (0.21 microg/L; 0.12-0.36) (p=0.016). No association was found between APC-PCI-values and the degrees of carotid artery stenosis or the time from the latest neurological symptoms to blood sampling. Patients with echolucent plaques had significantly lower APC-PCI concentrations (0.20 microg/L; 0.14-0.35 vs. 0.24 microg/L; 0.15-0.60; p=0.043), according to the Gray-Weale classification., Conclusions: Patients with carotid artery disease exhibit increased concentrations of APC-PCI compared to a healthy control-group, particularly those patients with echogenic plaques, who have significantly higher APC-PCI levels than patients with echolucent plaques., (Copyright 2009 Elsevier Ltd. All rights reserved.)