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Background and aims: Aging is the primary risk factor for cardiovascular disease (CVD), but the mechanisms underlying age-linked atherosclerosis remain unclear. We previously observed that long-lived vascular matrix proteins can acquire 'gain-of-function' isoDGR motifs that might play a role in atherosclerotic pathology. Methods: IsoDGR-specific mAb were generated and used for ELISA-based measurement of motif levels in plasma samples from patients with coronary artery diseases (CAD) and non-CAD controls. Functional consequences of isoDGR accumulation in age-damaged fibronectin were determined by bioassay for capacity to activate monocytes, macrophages, and endothelial cells (signalling activity, pro-inflammatory cytokine expression, and recruitment/adhesion potential). Mice deficient in the isoDGR repair enzyme PCMT1 were used to assess motif distribution and macrophage localisation in vivo. Results: IsoDGR-modified fibronectin and fibrinogen levels in patient plasma were significantly enhanced in CAD and further associated with smoking status. Functional assays demonstrated that isoDGR-modified fibronectin activated both monocytes and macrophages via integrin receptor ‘outside in’ signalling, triggering an ERK:AP-1 cascade and expression of pro-inflammatory cytokines MCP-1 and TNFα to drive additional recruitment of circulating leukocytes. IsoDGR-modified fibronectin also induced endothelial cell expression of integrin β1 to further enhance cellular adhesion and matrix deposition. Analysis of murine aortic tissues confirmed accumulation of isoDGR-modified proteins co-localised with CD68+ macrophages in vivo. Conclusions: Age-damaged fibronectin features isoDGR motifs that increase binding to integrins on the surface of monocytes, macrophages, and endothelial cells. Subsequent activation of ‘outside-in’ signalling elicits a range of potent cytokines and chemokines that drive additional leukocyte recruitment to the developing atherosclerotic matrix. Ministry of Education (MOE) National Medical Research Council (NMRC) This work was in part supported by grants from the National Medical Research Council of Singapore (NMRC–OF–IRG-0003-2016) and Ministry of Education of Singapore (MOE2018-T1-001-078).

Background and aims Electronic cigarette (EC) use is popular among youth, touted as a safer alternative to smoking and promoted as a tool to aid in smoking cessation. EC cardiovascular safety however is not well established. The aim of this study was to examine cardiovascular consequences of EC use by evaluating their effect on the entire atherosclerotic cascade in young adults using noninvasive combined positron emission tomography (PET)/magnetic resonance imaging (MR) and comparing EC use with age matched smokers of traditional cigarettes and nonsmoking controls. Methods Carotid PET/MR was applied to look at vascular inflammation (18-fluorodeoxyglucose (FDG)-PET) and plaque burden (multi-contrast MR of vessel wall) from 60 18-30 year-old subjects (20 electronic cigarette users, 20 traditional smokers and 20 nonsmokers). Results Groups were reasonably well balanced in terms of age, gender, demographics, cardiovascular risk and most biomarkers. There were no differences in vascular inflammation as measured by 18-FDG-PET target to background ratios (TBR) between EC users, traditional cigarette smokers and nonsmokers. However, measures of carotid plaque burden − wall area, normalized wall index, and wall thickness − measured from MR were significantly higher in both traditional smokers and EC users than in nonsmokers. Conclusions Young adult EC users, smokers and nonsmokers in our study did not exhibit vascular inflammation as defined by 18-F-FDG-PET TBR max, but smokers and EC users had significantly more carotid plaque burden compared to matched nonsmokers. Results could indicate that vaping does not cause an increase in vascular inflammation as measured by FDG-PET.

Inflammation is associated with atherosclerotic plaque development and precipitation of myocardial infarction and stroke, and anti-inflammatory therapy may reduce disease severity. Costimulatory molecules are key regulators of immune cell activity and inflammation, and are associated with disease development in atherosclerosis. Accumulating evidence indicates that a costimulatory molecule of the Tumor Necrosis Factor Receptor superfamily, the checkpoint regulator CD137, promotes atherosclerosis and vascular inflammation in experimental models. In light of the burgeoning consideration of CD137-targeted therapy in the clinic, it will be important to better understand costimulator immunobiology in development of cardiovascular disease. Here, we review available data on the costimulator CD137 and its potential role in atherosclerosis.

Background and aims The CD40−CD40 Ligand (CD40L) system has an important role in vascular inflammation. For this reason, we assessed the association of soluble CD40L with cardiovascular and all-cause mortality in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. Methods Plasma levels of sCD40L were determined in 2759 persons using an enzyme immunoassay. Cox proportional hazard regressions were performed to evaluate the association between plasma concentration of sCD40 ligand and short-term (12 months) and long-term (10 years) mortality. Subpopulation analyses were conducted in seven different risk groups. Cox regression models were adjusted for traditional risk factors. Results The present study did not reveal significant association between sCD40L plasma levels and all-cause mortality, as well as cardiovascular mortality at one-year follow-up. In selected subgroups only, significant association between elevated sCD40L plasma levels and short-term all-cause and cardiovascular mortality could be observed. With regard to long-term all-cause and cardiovascular mortality analyses, no significant correlation with increased plasma levels of sCD40L could be detected, neither overall nor in any subgroup. Conclusions Soluble sCD40L is not associated with cardiovascular and all-cause mortality in this large cohort. Only in selected patient subgroups elevated levels of sCD40L correlate with short-term mortality but this correlation disappears in long-term analysis.

Background and aims Young cigarette smokers may already present with early signs of vascular inflammation and damage; biglycan (BGN) has been shown to play a critical role in the initiation and progression of vascular lesions, also in young smokers. We investigated whether after smoke cessation, monocyte BGN expression is reduced; moreover, we evaluated any improvement of pro-atherogenic profile and arterial stiffness (AS), and their relationship with BGN in abstinent smokers. Methods Two-hundred-fifty-one young people who had decided to quit smoking were enrolled; of these, 71 had completed the 12-month observation period maintaining smoking abstinence. At enrollment and 12 months later, we evaluated anthropometrics, laboratory profile, carotid-femoral pulse wave velocity (cf-PWV), carotid intima-media thickness (cIMT), BGN expression. Results After 12-month smoke abstinence, we found a significant decrease in inflammatory markers (Hs-CRP: −23.3%; fibrinogen: −11.8%; IL-6: −9.2%), and increased HDL-C levels (+9.3%); blood pressure values were also slightly reduced. cf-PWV (−8.9%) appeared to be improved; cIMT remained unchanged. BGN expression appeared to be reduced (−42.8% relative reduction). BGN reduction appeared to be associated with fibrinogen reduction, and smoking burden. Reduced cf-PWV appeared to be dependent on change in fibrinogen, SBP, IL-6, and BGN by multiple regression analysis. Conclusions After the first year of smoke abstinence, the levels of IL-6, CRP, fibrinogen, HDL-C, and BGN expression, as well cf-PWV, are significantly improved as compared to baseline. This is the first evidence that removing exposure to a well-known cardiovascular risk factor, such as cigarette smoking, leads to significant reduction of BGN expression.

Arachidonate 5-lipoxygenase (5-LO) is a key enzyme in the synthesis of leukotrienes. VIA-2291 is a potent 5-LO inhibitor, which has been shown to reduce hsCRP and noncalcified coronary plaque volume following an acute coronary syndrome (ACS). We aim to evaluate the effect of VIA-2291 on vascular inflammation compared to placebo using FDG-PET.A Phase II, randomized, double-blind, parallel-group study was conducted in 52 patients with recent ACS assigned 1:1 to either 100 mg VIA-2291 or placebo for 24 weeks. The primary outcome was the effect of VIA-2291 relative to placebo on arterial inflammation detected by (18)fluorodeoxyglucose positron emission tomography (FDG-PET) within the index vessel after 24 weeks of daily treatment, compared to baseline.VIA-2291 was relatively well tolerated and was associated with a significant inhibition of the potent chemo-attractant LTB4, with a mean inhibition of activity of 92.8% (p0.0001) at 6 weeks in the VIA-2291 group, without further significant change in inhibition at 24 weeks. However, for VIA-2291 was not associated with significant difference in inflammation (target-to-background ratio) compared to placebo at 24 weeks or 6 weeks of treatment. Further, VIA-2291 was not associated with a significant reduction in hsCRP from baseline after either 6 or 24 weeks of treatment.VIA-2291 is well-tolerated and effectively reduces leukotriene production. However, inhibition of 5-LO with VIA-2291 is not associated with significant reductions in vascular inflammation (by FDG-PET) or in blood inflammatory markers. Accordingly, this study does not provide evidence to support a significant anti-inflammatory effect of VIA-2291 in patients with recent ACS.

Age and inflammation are risk factors for cardiovascular disease but the impact of inflammation on cardiovascular risk across the lifespan is not understood. We investigated whether an inflammatory burden is modulated by age in healthy subjects.Caucasian and African-American families were recruited from the general population (age range: 6-74 years, n = 267). Systemic inflammation was assessed by C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen, haptoglobin and α-acid glycoprotein, and vascular inflammation was assessed by pentraxin-3 (PTX-3), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM). To collectively assess systemic or vascular factors across the age spectrum, a composite z-score for each marker category was calculated.There was a contrasting pattern in systemic versus vascular inflammatory burden over age with an increase in systemic but a decrease in vascular markers in both ethnic groups. The results remained unchanged after adjustments for the covariates and covariance. When looking at individual markers to examine which markers are most contributing factors to the composite scores, CRP and SAA were significantly and positively associated with age, while PTX-3 and sVCAM were significantly and negatively associated with age in both ethnic groups.The composite z-score for systemic inflammation increased with age, while the composite z-score for vascular inflammation declined with age, irrespective of ethnicity. The findings illustrate a regulatory relationship between age and inflammation, and suggest that a perceived elevation of vascular markers among the very young may be an indication of physiological changes rather than reflecting a disease process.

Objectives:\ud The toll-like receptors (TLRs), including TLR4, have been shown to play a crucial role in vascular inflammatory diseases, such as atherosclerosis and aneurysm. The main goal of this study was to determine the potential of IAXO-102 (Innaxon, Tewkesbury), a novel small molecule TLR4 antagonist, to modulate non-hematopoietic TLR4 proinflammatory signalling and inhibit experimental abdominal aortic aneurysm (AAA) development.\ud \ud Methods:\ud Human umbilical vein endothelial cells (HUVEC) and Angiotensin II-induced experimental AAA development were our in vitro and in vivo models respectively. Western blotting, antibody array and ELISA approaches were used to explore the effect of IAXO-102 on TLR4 functional activity on two levels: modulation of TLR4-induced mitogen activated protein kinases (MAPK) and p65 NF-kB phosphorylation and expression of TLR4 dependent proinflammatory proteins.\ud \ud Results:\ud Following activation of TLR4, in vitro/in vivo data revealed that IAXO-102 inhibited MAPK and p65 NF-kB phosphorylation associated with down regulation of the expression of TLR4 and TLR4 dependent proinflammatory proteins. Furthermore, IAXO-102 decreased Angiotensin II-induced aortic expansion, rupture and incidence of AAA.\ud \ud Conclusions:\ud These results demonstrate the ability of IAXO-102 to negatively regulate TLR4 signalling and to inhibit experimental AAA development, suggesting the potential therapeutic use of this TLR4 antagonist for pharmacological intervention of AAA.

Objective : Cigarette smokers present early signs of vascular damage and systemic inflammation. Biglycan (BGN), an ubiquitous component of extracellular matrix orchestrating several physiological functions, has recently been indicated as a major source of low-density lipoprotein retention in the normal arterial intima-media layer. We evaluated whether BGN-mRNA expression was enhanced in peripheral monocytes of smokers with no additional cardiovascular risk factors (CVRFs), and if it was associated with altered carotid arterial stiffness (AS) or intima media thickness (cIMT). We also evaluated plasma markers of systemic and vascular inflammation, and correlation with BGN-mRNA. Methods : Two-hundred-fifty-one young smokers were enrolled, with no additional CVRFs, and 60 controls. Plasma lipids, fibrinogen, C-reactive protein (CRP), interleukin-6 (IL-6), AS and cIMT were assessed. A smoke exposure index (SEIx) was calculated. Results : Fibrinogen, CRP, AS indices, cIMT, and BGN-mRNA were higher in smokers compared to controls; HDL-C levels were lower, no difference was detected in IL-6 levels. After stratification of smokers in quartiles based on SEIx values, smokers in the highest quartiles presented highest fibrinogen, CRP, AS, cIMT, BGN, and also IL-6 values, and lowest HDL-C. Conclusion : BGN-mRNA was enhanced in young smokers, compared to controls, and appears associated to a proatherogenic profile, characterized by increased fibrinogen, CRP, and IL-6, lower HDL-C, altered AS and cIMT values, particularly in those with higher SEIx: the more cigarettes smoked over years, the more marked the alterations. Although we cannot state whether BGN have a direct causal role in inducing, maintaining and developing vascular damage, including intima-media wall thickening and arterial stiffening, our data could suggest that it may represent a link between proatherogenic status induced by cigarette smoking, and the development and progression of vascular damage.

The underlying role of inflammation in atherosclerosis has been characterized. However, current treatment of coronary artery disease (CAD) predominantly consists of targeted reductions in serum lipoprotein levels rather than combating the deleterious effects of acute and chronic inflammation. Vascular inflammation acts by a number of different molecular and cellular pathways to contribute to atherogenesis. Over the last decades, both basic studies and clinical trials have provided evidence for the potential benefits of treatment of inflammation in CAD. During this period, development of pharmacotherapies directed towards inflammation in atherosclerosis has accelerated quickly. This review will highlight specific therapies targeting interleukin-1β (IL-1β), P-selectin and 5-lipoxygenase (5-LO). It will also aim to examine the anti-inflammatory effects of serpin administration, colchicine and intravenous HDL-directed treatment of CAD. We summarize the mechanistic rationale and evidence for these novel anti-inflammatory treatments at both the experimental and clinical levels.

The rapidly evolving field of molecular imaging promises important advances in the diagnosis, characterization and pharmacological treatment of vascular disease. Magnetic resonance imaging (MRI) provides a modality that is well suited to vascular imaging as it can provide anatomical, structural and functional data on the arterial wall. Its capabilities are further enhanced by the use of a range of increasingly sophisticated contrast agents that target specific molecules, cells and biological processes. This article will discuss one such approach, using microparticles of iron oxide (MPIO).MPIO have been shown to create highly conspicuous contrast effects on T2*-weighted MR images. We have developed a range of novel ligand-conjugated MPIO for molecular MRI of endothelial adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and P-selectin expressed in vascular inflammation, as well as activated platelet thrombosis. This review discusses the application of ligand-targeted MPIO for in vivo molecular MRI in a diverse range of vascular disease models including acute vascular inflammation, atherosclerosis, thrombosis, ischemia-reperfusion injury and ischemic stroke. The exceptionally conspicuous contrast effects of ligand-conjugated MPIO provide a versatile and sensitive tool for quantitative vascular molecular imaging that could refine diagnosis and measure response to treatment. The potential for clinical translation of this new class of molecular contrast agent for clinical imaging of vascular syndromes is discussed.

Objective Atherosclerosis is associated with Alzheimer's disease (AD) in humans, but the nature of this link is still elusive. Aim of this study was to investigate aortic atherosclerosis development in a mouse model with central nervous system (CNS) restricted β-amyloid precursor protein (APP) overexpression. Methods and results APP23 mice, overexpressing the Swedish mutated human APP selectively in the brain, were crossed with mice lacking apolipoprotein E (ApoE KO). Nine weeks old mice were fed a western type diet for eight weeks, then atherosclerotic lesions, aortic wall and cortical tissues gene expression and β-amyloid (Aβ) deposition were evaluated. Compared with ApoE KO, APP23/ApoE KO mice developed larger aortic atherosclerotic lesions and showed significantly increased expression of MCP-1, IL-6, ICAM-1 and MTPase 6, a marker of oxidative stress in the vascular wall. Of note brain limited APP synthesis was associated with an increased microglia and brain endothelial cells activation, in spite of the absence of β-amyloid deposits in the brain or alteration in the levels of oxidized metabolites of cholesterol such as 4-cholesten-3-one. Conclusion Our study suggests that the vascular pro-inflammatory effects of CNS-localised APP overexpression lead to atherogenesis before parenchymal Aβ deposition and neuronal dysfunction.