Inflammation In Atherosclerotic Lesions Research Articles

The Role of Bacterial Extracellular Membrane Nanovesicles in Atherosclerosis: Unraveling a Potential Trigger.

In this review, we explore the intriguing and evolving connections between bacterial extracellular membrane nanovesicles (BEMNs) and atherosclerosis development, highlighting the evidence on molecular mechanisms by which BEMNs can promote the athero-inflammatory process that is central to the progression of atherosclerosis. Atherosclerosis is a chronic inflammatory disease primarily driven by metabolic and lifestyle factors; however, some studies have suggested that bacterial infections may contribute to the development of both atherogenesis and inflammation in atherosclerotic lesions. In particular, the participation of BEMNs in atherosclerosis pathogenesis has attracted special attention. We provide some general insights into how the immune system responds to potential threats such as BEMNs during the development of atherosclerosis. A comprehensive understanding of contribution of BEMNs to atherosclerosis pathogenesis may lead to the development of targeted interventions for the prevention and treatment of the disease.

Small molecule-assisted assembly of multifunctional ceria nanozymes for synergistic treatment of atherosclerosis

Considering that intravascular reactive oxygen species (ROS) and inflammation are two characteristic features of the atherosclerotic microenvironment, developing an appropriate strategy to treat atherosclerosis by synergistically regulating ROS and inflammation has attracted widespread attention. Herein, a special molecule, zoledronic acid, containing imidazole and bisphosphonate groups, was selected for the first time to assist the assembly of cerium ions and produce functionalized ceria-zoledronic acid nanocomposites (CZ NCs). It not only serves as a new carrier for different kinds of drugs (e.g. probucol, PB) but also exerts an efficient multienzyme activity to achieve collaborative therapy. More importantly, platelet membrane-coated biomimetic nanoplatform (PCZ@PB NCs) specifically accumulate at inflammatory atherosclerotic lesions, synergistically regulate ROS levels and inflammation, and efficiently inhibit foam cell formation. This novel assembly method can also be applied in the treatment of many other diseases associated with oxidative stress and inflammation.

Treatment of vulnerable plaque using combined high-density lipoprotein and pentostatin- loaded microbubbles- mediated shock wave sonoporation therapy: Evaluation with positron emission tomography imaging and histopathology

Background and Aims : The rupture of vulnerable atherosclerosis and the subsequent formation of thrombi are the main factors responsible for myocardial infarctions. Macrophages are responsible for the prevalence of inflammation in atherosclerotic lesions. In the present study, we developed a shock wave sonoporation therapy system and investigated its effect on advanced atherosclerosis regression using Positron Emission Tomography (PET) imaging.

Radiation-induced C-reactive protein triggers apoptosis of vascular smooth muscle cells through ROS interfering with the STAT3/Ref-1 complex.

Damage to normal tissue can occur over a long period after cancer radiotherapy. Free radical by radiation can initiate or accelerate chronic inflammation, which can lead to atherosclerosis. However, the underlying mechanisms remain unclear. Vascular smooth muscle cells (VSMCs) proliferate in response to JAK/STAT3 signalling. C‐reactive protein (CRP) can induce VSMCs apoptosis via triggering NADPH oxidase (NOX). Apoptotic VSMCs promote instability and inflammation of atherosclerotic lesions. Herein, we identified a VSMCs that switched from proliferation to apoptosis through was enhanced by radiation‐induced CRP. NOX inhibition using lentiviral sh‐p22phox prevented apoptosis upon radiation‐induced CRP. CRP overexpression reduced the amount of STAT3/Ref‐1 complex, decreased JAK/STAT phosphorylation and formed a new complex of Ref‐1/CRP in VSMC. Apoptosis of VSMCs was further increased by CRP co‐overexpressed with Ref‐1. Functional inhibition of NOX or p53 also prevented apoptotic activity of the CRP‐Ref‐1 complex. Immunofluorescence showed co‐localization of CRP, Ref‐1 and p53 with α‐actin‐positive VSMC in human atherosclerotic plaques. In conclusion, radiation‐induced CRP increased the VSMCs apoptosis through Ref‐1, which dissociated the STAT3/Ref‐1 complex, interfered with JAK/STAT3 activity, and interacted with CRP‐Ref‐1, thus resulting in transcription‐independent cell death via p53. Targeting CRP as a vascular side effect of radiotherapy could be exploited to improve curability.

Exploiting Glutamine Consumption in Atherosclerotic Lesions by Positron Emission Tomography Tracer (2S,4R)-4-18F-Fluoroglutamine.

Increased glutamine metabolism by macrophages is associated with development of atherosclerotic lesions. Positron emission tomography/computed tomography (PET/CT) with a glutamine analog (2S,4R)-4-18F-fluoroglutamine (18F-FGln) allows quantification of glutamine consumption in vivo. Here, we investigated uptake of 18F-FGln by atherosclerotic lesions in mice and compared the results with those obtained using the glucose analog 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG). Uptake of 18F-FGln and 18F-FDG by healthy control mice (C57BL/6JRj) and atherosclerotic low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR−/−ApoB100/100) was investigated. The mice were injected intravenously with 18F-FGln or 18F-FDG for in vivo PET/CT imaging. After sacrifice at 70 minutes post-injection, tracer uptake was analyzed by gamma counting of excised tissues and by autoradiography of aorta cryosections, together with histological and immunohistochemical analyses. We found that myocardial uptake of 18F-FGln was low. PET/CT detected lesions in the aortic arch, with a target-to-background ratio (SUVmax, aortic arch/SUVmean, blood) of 1.95 ± 0.42 (mean ± standard deviation). Gamma counting revealed that aortic uptake of 18F-FGln by LDLR−/−ApoB100/100 mice (standardized uptake value [SUV], 0.35 ± 0.06) was significantly higher than that by healthy controls (0.20 ± 0.08, P = 0.03). More detailed analysis by autoradiography revealed that the plaque-to-healthy vessel wall ratio of 18F-FGln (2.90 ± 0.42) was significantly higher than that of 18F-FDG (1.93 ± 0.22, P = 0.004). Immunohistochemical staining confirmed that 18F-FGln uptake in plaques co-localized with glutamine transporter SLC7A7-positive macrophages. Collectively these data show that the 18F-FGln PET tracer detects inflamed atherosclerotic lesions. Thus, exploiting glutamine consumption using 18F-FGln PET may have translational relevance for studying atherosclerotic inflammation.

Ultraselective Carbon Nanotubes for Photoacoustic Imaging of Inflamed Atherosclerotic Plaques (Adv. Funct. Mater. 37/2021)

Ultraselective Carbon Nanotubes Vulnerable atherosclerotic plaques can result in heart attacks. Inflammation, particularly inflammatory monocytes/macrophages, characterizes such plaques. In article number 2101005, Eliver E. B. Ghosn, Bryan Ronain Smith, and co-workers present single-walled carbon nanotubes' unprecedented selectivity to inflammatory monocytes/macrophages that infiltrate plaques, allowing specific detection of inflamed plaques using photoacoustic imaging (laser in, ultrasound out). This highly selective strategy, confirmed by high-dimensional flow cytometry, can accurately identify and diagnose inflamed atherosclerotic lesions.

Exploiting glutamine consumption in inflamed atherosclerotic lesions by positron emission tomography tracer (2S, 4R)-4-[18F]Fluoroglutamine

Background and Aims: Glutamine metabolism of macrophages has been shown to be associated with the development of atherosclerotic lesions1. Positron emission tomography/computed tomography (PET/CT) with glutamine analog (2S, 4R)-4-[18F]fluoroglutamine ([18F]FGln) allows quantification of glutamine consumption in vivo2. Here, we investigated the uptake of [18F]FGln in inflamed atherosclerotic lesions in mice, and compared the results with glucose analog 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG).

Ultra-selective carbon nanotubes for photoacoustic imaging of inflamed atherosclerotic plaques.

Disruption of vulnerable atherosclerotic plaques often leads to myocardial infarction and stroke, the leading causes of morbidity and mortality in the United States. A diagnostic method that detects high-risk atherosclerotic plaques at early stages could prevent these sequelae. The abundance of immune cells in the arterial wall, especially inflammatory Ly-6Chi monocytes and foamy macrophages, is indicative of plaque‎ inflammation, and may be associated with plaque vulnerability. Hence, we sought to develop a new method that specifically targets these immune cells to offer clinically-relevant diagnostic information about cardiovascular disease. We combine ultra-selective nanoparticle targeting of Ly-6Chi monocytes and foamy macrophages with clinically-viable photoacoustic imaging (PAI) in order to precisely and specifically image inflamed plaques ex vivo in a mouse model that mimics human vulnerable plaques histopathologically. Within the plaques, high-dimensional single-cell flow cytometry (13-parameter) showed that our nanoparticles were almost-exclusively taken up by the Ly-6Chi monocytes and foamy macrophages that heavily infiltrate plaques. PAI identified inflamed atherosclerotic plaques that display ~6-fold greater signal compared to controls (P<0.001) six hours after intravenous injection of ultra-selective carbon nanotubes, with in vivo corroboration via optical imaging. Our highly selective strategy may provide a targeted, non-invasive imaging strategy to accurately identify and diagnose inflamed atherosclerotic lesions.

Evaluation of glucagon-like peptide-1 receptor expression in nondiabetic and diabetic atherosclerotic mice using PET tracer 68Ga-NODAGA-exendin-4.

Cardiovascular effects of glucagon-like peptide-1 receptor (GLP-1R) agonist therapies are potentially mediated by anti-inflammatory effects on atherosclerosis. Our study demonstrates that 68Ga-NODAGA-exendin-4, a radioligand specifically targeting GLP-1R, detects GLP-1R expression in inflamed atherosclerotic lesions in nondiabetic and diabetic hypercholesterolemic mice. Immunofluorescence staining suggests that GLP-1R is primarily localized in M2 macrophages in lesions. This study describes a new potential tool that may have translational relevance for studies of pharmacological modification of GLP-1R signaling in atherosclerosis.

Mitochondrial DAMPs and altered mitochondrial dynamics in OxLDL burden in atherosclerosis

Atherosclerosis results in life-threatening cardiovascular pathologies, including ischemic heart disease, stroke, myocardial infarction, and peripheral arterial disease. The role of increased serum low-density lipoprotein (LDL) and resultant accumulation of oxidized-LDL (oxLDL) in atheroma formation is well established. Recent findings elucidate the significance of mitochondrial damage-associated molecular patterns (mtDAMPs) in triggering sterile inflammation in concert with oxLDL. The mtDAMPs including mitochondrial DNA (mtDNA), cytochrome C, cardiolipin, heat shock protein 60 (HSP60), mitochondrial transcription factor A (TFAM), and N-formyl peptides, are expected to possess proatherogenic roles. However, limited data are available in the literature. The mtDAMPs initiate sterile inflammation in atherosclerotic lesions via numerous signaling pathways, most of which converge to the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. Priming the activation of the NLRP3 inflammasome, mtDAMPs promote secretion of proinflammatory cytokines, including interleukin-1β (IL-1β), implicated in atherosclerotic lesions through vascular smooth muscle and fibroblast proliferation, arterial wall thickening, and plaque formation. In this article we critically reviewed and discussed the central role of the NLRP3 inflammasome in mtDAMP-induced sterile inflammation in atherosclerosis with specific components including caspase-1, pregnane X receptor (PXR), adenosine monophosphate activated protein kinase (AMPK), protein phosphatase 2A (PP2A), thioredoxin-interacting protein (TXNIP), and downstream cytokines including IL-1β and IL-18 as potential mediators of atherosclerosis. Better understanding of the proinflammatory effects of mtDAMPs and its pathological association with oxLDL possess immense translational significance for novel therapeutic intervention.

Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity and overexpression of CARD8 mRNA was previously identified in atherosclerosis. However, very little is known about the regulation of CARD8 in endothelial cells and atherosclerosis. The aim of this study was to investigate CARD8 in the regulation of cytokine and chemokine expression in endothelial cells. Sections of human atherosclerotic lesions and non-atherosclerotic arteries were immunostained for CARD8 protein. Expression of CARD8 was correlated to mediators of inflammation in atherosclerotic lesions using Biobank of Karolinska Endarterectomies microarray data. The CARD8 mRNA was knocked-down in human umbilical vein endothelial cells (HUVECs) in vitro, followed by quantitative RT-PCR analysis and OLINK Proteomics. Endothelial and smooth muscle cells in arterial tissue expressed CARD8 and CARD8 correlated with vWF, CD163 and the expression of inflammatory genes, such as CXCL1, CXCL6 and PDGF-A in plaque. Knock-down of CARD8 in HUVECs significantly altered proteins involved in inflammatory response, such as CXCL1, CXCL6, PDGF-A, MCP-1 and IL-6. The present study suggest that CARD8 regulate the expression of cytokines and chemokines in endothelial cells and atherosclerotic lesions, suggesting that CARD8 plays a significant role in endothelial activation.

Inflammation of carotid plaques and risk of cerebrovascular events.

Carotid atherosclerotic plaques represent a risk for ischemic stroke. The data indicate that the risk for distal embolization from atherosclerotic lesions in internal carotid arteries is not related only to the degree of stenosis but also to the composition of plaques. The stability of atherosclerotic plaque depends on the thickness of the fibrous cap and plaque hemorrhage. Recent research indicated that the inflammatory activity of atherosclerotic lesions is pivotal in the progression of atherosclerotic plaques. It also promotes the development of unstable atherosclerotic lesions and is related to thromboembolic cerebrovascular complications. Inflammation destabilizes atherosclerotic plaques through the degradation of their fibrotic structure. Inflammation of atherosclerotic plaques was confirmed by histopathologic findings and levels of circulating inflammatory markers which were correlated to the intensity of the inflammation in atherosclerotic lesions. Recently, new techniques like fluorodeoxyglucose positron emission tomography (18-FDG PET) were developed for the identification of inflammation of atherosclerotic lesions in the vessel wall in vivo. Systemic inflammatory markers, particularly interleukins, tumor necrosis factor-alpha and metalloproteinases were shown to be related to the intensity of the inflammatory process in atherosclerotic lesions and the cerebrovascular events. Identification of inflamed atherosclerotic plaques may help to identify unstable atherosclerotic lesions and subjects at high risk for cerebrovascular incidents who need intensive preventive measures including anti-inflammatory medication.

Association of glucose uptake of visceral fat and acute myocardial infarction: a pilot 18F-FDG PET/CT study

BackgroundInflamed visceral adipose tissue (VAT) facilitates chronic inflammation in atherosclerotic lesions thereby leading to increased risk of coronary artery disease (CAD). In this study, we evaluated the glucose uptake of VAT and the carotid artery with 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET/CT) and their association with CAD, including acute myocardial infarction (AMI).MethodsA total of 90 participants were enrolled (32 with AMI, 33 with chronic stable angina; CSA, and 25 control participants) and undertook 18F-FDG PET/CT. VAT glucose uptake was measured by using maximum standardized uptake value (SUVmax) of VAT region. The target-to-background ratio (TBR) of carotid artery was defined as the SUVmax of carotid artery divided by the SUVmax of jugular vein. The SUVmax of spleen, bone-marrow (BM), and high-sensitivity C-reactive protein (hsCRP) were used for the assessment of systemic inflammatory activity.ResultsVAT SUVmax was highest in participants with AMI, intermediate in participants with CSA, and lowest in control participants. Carotid artery TBR and systemic inflammatory surrogate markers including spleen SUVmax, BM SUVmax, and hsCRP were also higher in the AMI group than in the CSA or control group. Furthermore, VAT SUVmax showed significant positive correlation with carotid artery TBR, spleen SUVmax, BM SUVmax, and hsCRP. In multivariate linear regression and logistic regression analyses, VAT SUVmax was independently associated with carotid artery TBR and AMI.ConclusionsGlucose uptake of VAT assessed by 18F-FDG PET/CT was associated with the severity of CAD and synchronized with the carotid artery inflammation in participants with CAD.

Molecular Imaging of Inflammation in a Mouse Model of Atherosclerosis Using a Zirconium-89-Labeled Probe.

BackgroundBeyond clinical atherosclerosis imaging of vessel stenosis and plaque morphology, early detection of inflamed atherosclerotic lesions by molecular imaging could improve risk assessment and clinical management in high-risk patients. To identify inflamed atherosclerotic lesions by molecular imaging in vivo, we studied the specificity of our radiotracer based on maleylated (Mal) human serum albumin (HSA), which targets key features of unstable atherosclerotic lesions.Materials and MethodsMal-HSA was radiolabeled with a positron-emitting metal ion, zirconium-89 (89Zr4+). The targeting potential of this probe was compared with unspecific 89Zr-HSA and 18F-FDG in an experimental model of atherosclerosis (Apoe–/– mice, n=22), and compared with wild-type (WT) mice (C57BL/6J, n=21) as controls.ResultsPET/MRI, gamma counter measurements, and autoradiography showed the accumulation of 89Zr-Mal-HSA in the atherosclerotic lesions of Apoe–/– mice. The maximum standardized uptake values (SUVmax) for 89Zr-Mal-HSA at 16 and 20 weeks were 26% and 20% higher (P<0.05) in Apoe–/– mice than in control WT mice, whereas no difference in SUVmax was observed for 18F-FDG in the same animals. 89Zr-Mal-HSA uptake in the aorta, as evaluated by a gamma counter 48 h postinjection, was 32% higher (P<0.01) for Apoe–/– mice than in WT mice, and the aorta-to-blood ratio was 8-fold higher (P<0.001) for 89Zr-Mal-HSA compared with unspecific 89Zr-HSA. HSA-based probes were mainly distributed to the liver, spleen, kidneys, bone, and lymph nodes. The phosphor imaging autoradiography (PI-ARG) results corroborated the PET and gamma counter measurements, showing higher accumulation of 89Zr-Mal-HSA in the aortas of Apoe–/– mice than in WT mice (9.4±1.4 vs 0.8±0.3%; P<0.001).Conclusion89Zr radiolabeling of Mal-HSA probes resulted in detectable activity in atherosclerotic lesions in aortas of Apoe–/– mice, as demonstrated by quantitative in vivo PET/MRI. 89Zr-Mal-HSA appears to be a promising diagnostic tool for the early identification of macrophage-rich areas of inflammation in atherosclerosis.

Bone Marrow Endothelial Cells Regulate Myelopoiesis in Diabetes Mellitus.

Diabetes mellitus is a prevalent public health problem that affects about one-third of the US population and leads to serious vascular complications with increased risk for coronary artery disease. How bone marrow hematopoiesis contributes to diabetes mellitus complications is incompletely understood. We investigated the role of bone marrow endothelial cells in diabetic regulation of inflammatory myeloid cell production. In 3 types of mouse diabetes mellitus, including streptozotocin, high-fat diet, and genetic induction using leptin-receptor-deficient db/db mice, we assayed leukocytes, hematopoietic stem and progenitor cells (HSPC). In addition, we investigated bone marrow endothelial cells with flow cytometry and expression profiling. In diabetes mellitus, we observed enhanced proliferation of HSPC leading to augmented circulating myeloid cell numbers. Analysis of bone marrow niche cells revealed that endothelial cells in diabetic mice expressed less Cxcl12, a retention factor promoting HSPC quiescence. Transcriptome-wide analysis of bone marrow endothelial cells demonstrated enrichment of genes involved in epithelial growth factor receptor (Egfr) signaling in mice with diet-induced diabetes mellitus. To explore whether endothelial Egfr plays a functional role in myelopoiesis, we generated mice with endothelial-specific deletion of Egfr (Cdh5CreEgfrfl/fl). We found enhanced HSPC proliferation and increased myeloid cell production in Cdh5CreEgfrfl/fl mice compared with wild-type mice with diabetes mellitus. Disrupted Egfr signaling in endothelial cells decreased their expression of the HSPC retention factor angiopoietin-1. We tested the functional relevance of these findings for wound healing and atherosclerosis, both implicated in complications of diabetes mellitus. Inflammatory myeloid cells accumulated more in skin wounds of diabetic Cdh5CreEgfrfl/fl mice, significantly delaying wound closure. Atherosclerosis was accelerated in Cdh5CreEgfrfl/fl mice, leading to larger and more inflamed atherosclerotic lesions in the aorta. In diabetes mellitus, bone marrow endothelial cells participate in the dysregulation of bone marrow hematopoiesis. Diabetes mellitus reduces endothelial production of Cxcl12, a quiescence-promoting niche factor that reduces stem cell proliferation. We describe a previously unknown counterregulatory pathway, in which protective endothelial Egfr signaling curbs HSPC proliferation and myeloid cell production.

P26 Soluble programed cell death ligand-1 is associated with acute coronary syndrome

Abstract Background Immune checkpoint by programmed cell death (PD)-1 and its ligand (PD-L1) play crucial role in T cell tolerance toward vascular wall antigens. PD-L1 is widely expressed on a number of cells including immune cells and vascular endothelium. It was reported that increased expression of PD-L1 in dendritic cells implicates upregulated inflammation in atherosclerotic lesions that is associated with plaque instability. Although plaque rupture in coronary atherosclerosis is an important pathogenesis of acute coronary syndrome (ACS), the association between PD-L1 and ACS is still unknown. Purpose We hypothesize that circulating PD-L1 might be associated with ACS, reflecting endothelial damage and coronary plaque rupture. To elucidate this hypothesis, we compared serum levels of soluble PD-L1 (sPD-L1) in stable coronary artery disease (CAD) patients with those in ACS patients. Methods Serum levels of sPD-L1 were measured by using commercially available ELISA kit (Human PD-L1/B7-H1 DuoSet, R&amp;D Systems) in consecutive patients with CAD admitted to our University Hospital from February 2016 to March 2017. Patients with any malignant disease or severe inflammatory disease were excluded from this study. Serum levels of sPD-L1 and clinical backgrounds were compared between stable-CAD and ACS patients. Results In total, 269 patients with CAD were enrolled (28 cases [10.4 %] with ACS and 241 cases [89.6 %] with stable-CAD). PD-L1 had no correlation to C-reactive protein, cardiac troponin, and classical atherosclerotic risks such as age, body mass index, estimated glomerular filtration rate, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol, and hemoglobin A1c. Although age, sex, history of smoking, and the prevalences of hypertension, diabetes mellitus and dyslipidemia were comparable between both groups, the level of LDL-C was significantly higher in patients with ACS compared with those with stable-CAD (94.0 [77.0–112.0] mg/dL vs. 78.5 [65.0–97.0] mg/dL, P = 0.008). Also serum level of sPD-L1 was significantly increased in patients with ACS compared with those with stable-CAD (106.1 [60.9–157.7] pg/mL vs. 64.8 [30.9–102.5] pg/mL, P = 0.003). Univariate logistic regression analysis identified that serum levels of both sPD-L1 and LDL-C were independently associated with ACS. Moreover, multivariable logistic regression analysis with factors from univariate analysis identified that serum level of sPD-L1 was significantly and independently associated with ACS (odds ratio: 1.006, 95 % confidence interval: 1.001–1.012, P = 0.03). Conclusions This is the first study to elucidate that the increased serum levels of sPD-L1 was associated with ACS. This study suggests that sPD-L1 could be a risk marker and therapeutic target for ACS.

P5507Growth differentiation factor-15 and mortality in suspected or known coronary heart disease patients with chronic kidney disease: a subanalysis of the ANOX study

Abstract Background Chronic kidney disease (CKD) is an independent risk factor for the development and progression of coronary heart disease (CHD). Growth differentiation factor-15 (GDF-15), a distant member of the transforming growth factor-β cytokine superfamily, plays a role in the initiation of inflammation in atherosclerotic lesions. Elevated GDF-15 was found in various diseases including CKD and stable CHD, and was reported to predict mortality and cardiovascular events in general or established CHD population. However, the prognostic value of GDF-15 in suspected or known CHD patients with CKD is unknown. Methods Serum GDF-15 levels were measured in 999 suspected or known CHD patients with CKD undergoing elective coronary angiography, enrolled in the development of novel biomarkers related to angiogenesis or oxidative stress to predict cardiovascular events (ANOX) study, and followed up for 3 years. The primary outcome was all-cause death. The secondary outcomes were cardiovascular death, and major adverse cardiovascular events (MACE) defined as a composite of cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke. Results During the follow-up, 154 patients died from any cause, 61 died from cardiovascular disease, and 96 developed MACE. After adjustment for established risk factors, GDF-15 levels were significantly associated with all-cause death (hazard ratio [HR] for 1-SD increase, 1.78; 95% confidence interval [CI], 1.57–2.00), cardiovascular death (HR, 1.88; 95% CI, 1.58–2.25), and MACE (HR, 1.54; 95% CI, 1.32–1.79). Even after incorporation of N-terminal pro-brain natriuretic peptide, contemporary sensitive cardiac troponin-I, and high-sensitivity C-reactive protein into a model with established risk factors, the addition of GDF-15 levels further improved the prediction of all-cause death (continuous net reclassification improvement [NRI], 0.401; 95% CI, 0.231–0.571; P&lt;0.001; integrated discrimination improvement [IDI], 0.041; 95% CI, 0.021–0.062; P&lt;0.001) and cardiovascular death (NRI, 0.297; 95% CI, 0.039–0.555; P=0.024; IDI, 0.032; 95% CI, 0.010–0.055; P=0.005), but not that of MACE (NRI, 0.138; 95% CI, v0.070–0.347; P=0.194; IDI, 0.012; 95% CI, −0.000–0.024; P=0.053). Conclusions In suspected or known CHD patients with CKD undergoing elective coronary angiography, elevated GDF-15 levels may predict all-cause and cardiovascular mortality independent of established risk factors and cardiovascular biomarkers. Acknowledgement/Funding The ANOX study is supported by a Grant-in-Aid for Clinical Research from the National Hospital Organization.

P1217Zirconium-89 labelled probe for molecular imaging of inflammation in experimental atherosclerosis

Abstract Background Early detection of inflamed atherosclerotic lesions by molecular imaging might improve risk assessment beyond that of vascular stenosis and plaque morphology imaging, and improve the clinical management of high-risk patients. Purpose To target the key features of unstable atherosclerotic lesions, we studied the feasibility of our radiotracer, based on modified human serum albumin (HSA), to identify inflamed atherosclerotic lesions by in vivo molecular imaging. Methods We applied a maleylated HSA (Mal-HSA) probe, recognised by scavenger receptors on macrophages, in an experimental in vivo imaging study of atherosclerosis. Mal-HSA was coupled with a positron-emittingmetal ion, Zirconium-89 (89Zr). The targeting potential of this probe was evaluated and compared with unspecific 89Zr-HSA and 18F-FDG in a mouse model of atherosclerosis (Apoe−/−, n=22) and compared with wild-type (WT) mice (C57BL/6, n=21) as controls. Radiotracer accumulation in the aortic arch was analysed in vivo by the fusion of positron emission tomography–magnetic resonance imaging (PET-MRI), radiotracer bio-distribution was measured ex vivo by gamma counter, and plaque uptake was evaluated by phosphor imaging (PI) autoradiography (ARG). Results PET-MRI, gamma counter measurements, and PI-ARG showed the accumulation of 89Zr-Mal-HSA in the atherosclerotic lesions of Apoe−/− mice. The maximum standardised uptake value (SUVmax) for 89Zr-Mal-HSA at 16 and 20 weeks were 26% and 20% higher (P&lt;0.05) in Apoe−/− mice than control WT mice, whereas no difference in SUVmax was found for 18F-FDG in the same animals. 89Zr-Mal-HSA uptake in the aorta as evaluated by gamma counter 48 h post-injection was 32% higher (P&lt;0.01) for Apoe−/− mice compared to WT mice, and the aorta-to-blood ratio was 10-fold higher (P&lt;0.001) for 89Zr-Mal-HSA compared with unspecific 89Zr-HSA. HSA probes were mainly distributed to the liver, spleen, kidneys, bone and lymph nodes. The PI-ARG results corroborated the PET and gamma counter measurements, showing higher accumulation of 89Zr-Mal-HSA in the aortas of Apoe−/− mice compared to WT mice; 9.4±1.4 vs 0.8±0.3% (P&lt;0.001). Conclusions The modified HSA-based radiotracer showed in vivo targeting of inflamed atherosclerotic lesions of mouse aorta, which could also be verified ex vivo. 89Zr-Mal-HSA seems to be a promising diagnostic tool for the identification of vascular inflammation. Further methodological studies are needed to verify its applicability for detecting rupture-prone plaques. Acknowledgement/Funding Swedish Research Council (22036); the Swedish HLF (20150423, 20170669); ALF (20150517, 447561, 726481); Söderberg Foundations, VINNOVA and KI

Regulatory T cells suppress the expression of COX-2 in vulnerable plaque.

COX-2 contributes to local inflammation in atherosclerotic lesions. Regulatory T cells (Tregs) enhance the stability of atherosclerotic plaques. The aim of this study was to detect the potential relationship between Tregs and COX-2 in vulnerable plaques. Thirty ApoE -/- mice were fed a high-fat diet, and a silastic perivascular collar was placed around the right common carotid artery to induce vulnerable plaques. Eight weeks after collar placement, the mice were divided randomly into three groups: control, PBS, and Treg groups. Four weeks later, the right common carotid arteries were collected to detect the expression of COX-2. The results showed that Tregs significantly suppressed the expression of COX-2 in vulnerable plaques. In an in vitro experiment, RAW264.7 cells were divided randomly into three groups, which were precultured without T cells or with CD4 + CD25- T cells or Tregs for 48h with an anti-CD3 antibody; then the cells were stimulated with LPS for 24h. The RAW264.7 cells were harvested for RT-PCR and western blot assays and the results showed that Tregs downregulated COX-2 expression in RAW264.7 cells. Therefore, Tregs inhibited the expression of COX-2 in vulnerable plaques and macrophages, and COX-2 inhibition may be an important effect of Tregs that results in atherosclerotic plaque stabilization.

IL-33/IL-33R in various types of carotid artery atherosclerotic lesions

ObjectiveInflammation plays a crucial role in the progression of atherosclerotic plaques. The aim of the study was to investigate serum levels and expression of Interleukin-33 (IL-33) and ST2 receptor in atherosclerotic plaques and to analyze correlation with the type of the carotid plaques in patients with carotid disease. MethodsThis study included 191 consecutive patients submitted for carotid endarterectomy (CEA). Preoperative serum levels of IL-33 and soluble ST2 (sST2) were measured. Atherosclerotic plaques obtained during surgery were initially histologically classified and immunohistochemical analyzes of IL-33, IL-33R, CD68 and alpha-SMA expression was performed. Ultrasound assessment of the level of carotid stenosis in each patient was performed prior to carotid surgery. Demographic and clinical data such as gender, age, smoking status, blood pressure, glycaemia, hemoglobin and creatinine levels, and comorbidities were collected and the comparisons between variables were statistically evaluated. ResultsSerum levels of IL-33 (35.86 ± 7.93 pg/ml vs.12.29 ± 1.8 pg/ml, p < 0.05) and sST2 (183 ± 8.03 pg/ml vs. 122.31 ± 15.89 pg/ml, p < 0.05) were significantly higher in the group of CEA patients vs. healthy subjects. We demonstrated abundant tissue expression of IL-33 and ST2 in atherosclerotic carotid artery lesions. The levels of IL-33 and IL-33R expression were significantly higher in vulnerable plaques and significantly correlated with the degree of inflammatory cells infiltration in these plaques (R = 0.579, p = 0.049). Immunohistochemical analysis also revealed that cells responsible for IL-33 expression are not only mononuclear cells confined to inflammatory atherosclerotic lesions, but also smooth muscle cells which gained phenotypic characteristics of foam cells and were loaded with lipid droplets. ConclusionThe obtained results confirm the importance of IL-33/ST2 axis in the process of atherosclerosis, and indicate its ambiguous function in immune response, whether as proinflammatory cytokine in advanced atherosclerotic lesions, or as profibrotic, in early lesions.