Increased aldehyde-modification of collagen type IV in symptomatic plaques - A possible cause of endothelial dysfunction.

Subendothelial LDL-adhesion and its subsequent oxidation are considered as key events in the development of atherosclerotic lesions. During oxidation of LDL, reactive aldehydes such as malondialdehyde (MDA) are formed, which modify apolipoprotein B100. However, the possibility that these reactive aldehydes could leak out of the LDL-particle and modify surrounding extracellular matrix proteins has been largely unexplored. We have investigated if aldehyde-modification of collagen type IV, one of the major basement membrane components, in plaques is associated with cardiovascular events

Circulating cytokines reflect the expression of pro-inflammatory cytokines in atherosclerotic plaques.

Inflammation is a key factor in the development of plaque rupture and acute cardiovascular events. Although imaging techniques can be used to identify vulnerable atherosclerotic plaques, we are lacking non-invasive methods, such as plasma markers of plaque inflammation that could help to identify presence of vulnerable plaques. The aim of the present study was to investigate whether increased plasma levels of pro-inflammatory cytokines reflects inflammatory activity within atherosclerotic plaques

Circulating soluble IL-6 receptor associates with plaque inflammation but not with atherosclerosis severity and cardiovascular risk

Background: The residual cardiovascular risk in subjects receiving guideline-recommended therapy is related to persistent vascular inflammation and IL-6 represents a target for its treatment. IL-6 binds to receptors on leukocytes and hepatocytes and/or by forming complexes with soluble IL-6 receptors (sIL-6R) binding to gp130 which is present on all cells. Here we aimed to estimate the associations of these two pathways with risk of cardiovascular disease (CVD).Methods: IL-6 and sIL-6R were analyzed using the proximity extension assay. Baseline plasma samples were obtained from participants in the prospective Malmö Diet and Cancer (MDC) study (n = 4661), the SUMMIT VIP study (n = 1438) and the Carotid Plaque Imaging Project (CPIP, n = 285). Incident clinical events were obtained through national registers. Plaques removed at surgery were analyzed by immunohistochemistry and biochemical methods.Results: During 23.1 ± 7.0 years follow-up, 575 subjects in the MDC cohort suffered a first myocardial infarction. Subjects in the highest tertile of IL-6 had an increased risk compared to the lowest tertile (HR and 95% CI 2.60 [2.08-3.25]). High plasma IL-6 was also associated with more atherosclerosis, increased arterial stiffness, and impaired endothelial function in SUMMIT VIP, but IL-6 was only weakly associated with plaque inflammation in CPIP. sIL-6R showed no independent association with risk of myocardial infarction, atherosclerosis severity or vascular function, but was associated with plaque inflammation.Conclusions: Our findings show that sIL-6R is a poor marker of CVD risk and associated vascular changes. However, the observation that sIL-6R reflects plaque inflammation highlights the complexity of the role of IL-6 in CVD.</p

Soluble CD40 levels in plasma are associated with cardiovascular disease and in carotid plaques with a vulnerable phenotype

Background and Purpose CD40 and CD40 ligand (CD40L) are costimulatory molecules of the tumor necrosis factor receptor superfamily and well known for their involvement in inflammatory diseases: atherosclerotic mouse models with disrupted CD40 signalling develop lesions of reduced size with a more stable plaque profile. This study investigated the potential of plasma and intraplaque levels of CD40 and CD40L as markers for cardiovascular disease (CVD) in humans and their association with plaque stability. Methods Soluble CD40 and CD40L (sCD40L) were measured in plasma in 1,437 subjects from The SUrrogate markers for Micro-and Macro-vascular hard endpoints for Innovative diabetes Tools (SUMMIT) cohort. Intra-plaque levels of sCD40 and sCD40L were measured in atherosclerotic plaque homogenates from 199 subjects of the Carotid Plaque Imaging Project (CPIP) cohort. Results Both plasma sCD40 and sCD40L levels were elevated in individuals with prevalent stroke, while sCD40 levels also were higher in individuals with a prior acute myocardial infarction. Plasma levels of sCD40 correlated with carotid intima-media thickness and total carotid plaque area and were associated with risk of cardiovascular events over a 3-year follow-up period. Intra-plaque levels of sCD40 and sCD40L were associated with plaque components characteristic for plaque vulnerability and extracellular matrix remodelling. Conclusions Higher plasma sCD40 and sCD40L levels are associated with prevalent CVD. Plasma sCD40 levels also correlate with the severity of carotid atherosclerosis and predict future cardiovascular events, while intra-plaque levels correlate with a vulnerable plaque phenotype. Our findings thus demonstrate that elevated levels of sCD40 and sCD40L are markers of CVD.</p

Plasma levels of PRO-C3, a type III collagen synthesis marker, are associated with arterial stiffness and increased risk of cardiovascular death

Background and aims: The N-terminal propeptide of type III collagen (PRO-C3) assay measures a pro-peptide released during type III collagen synthesis, an important feature of arterial stiffening and atherogenesis. There is a clinical need for improved non-invasive, cheap and easily accessible methods for evaluating individuals at risk of cardiovascular disease (CVD). In this study, we investigate the potential of using circulating levels of PRO-C3 to mark the degree of vascular stenosis and risk of cardiovascular events. Methods: Baseline plasma levels of PRO-C3 were measured by ELISA in subjects belonging to the SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools (SUMMIT) cohort (N = 1354). Associations between PRO-C3 levels with vascular characteristics, namely stiffness and stenosis, and risk of future cardiovascular events were explored. Subjects were followed up after a median of 35 months (interquartile range 34–36 months), with recorded outcomes cardiovascular death and all-cause mortality. Results: We found a correlation between PRO-C3 levels and pulse wave velocity (rho 0.13, p = 0.000009), a measurement of arterial stiffness. Higher PRO-C3 levels were also associated with elevated blood pressure (rho 0.07, p = 0.014), as well as risk of cardiovascular mortality over a three-year follow-up period (OR 1.56, confidence interval 1.008–2.43, p = 0.046). Conclusions: Elevated circulating PRO-C3 levels are associated with arterial stiffness and future cardiovascular death, in the SUMMIT cohort, suggesting a potential value of PRO-C3 as a novel marker for declining vascular health.</p

Epigenome-Wide Histone Acetylation Changes in Peripheral Blood Mononuclear Cells in Patients with Type 2 Diabetes and Atherosclerotic Disease

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.Peer reviewe

The Novel Collagen Matrikine, Endotrophin, is Associated with Mortality and Cardiovascular Events in Patients with Atherosclerosis

Background: Rupture of atherosclerotic plaques is the major cause of acute cardiovascular events. The biomarker PRO-C6 measuring Endotrophin, a matrikine of collagen type VI, may provide valuable information detecting subjects in need of intensified strategies for secondary prevention. Objective: In this study, we evaluate endotrophin in human atherosclerotic plaques and circulating levels of PRO-C6 in patients with atherosclerosis, to determine the predictive potential of the biomarker. Methods: Sections from the stenotic human carotid plaques were stained with the PRO-C6 antibody. PRO-C6 was measured in serum of patients enrolled in the Carotid Plaque Imagining Project (CPIP) (discovery cohort, n = 577) and the innovative medicines initiative surrogate markers for micro- and macrovascular hard end-points for innovative diabetes tools (IMI-SUMMIT, validation cohort, n = 1,378). Median follow-up was 43 months. Kaplan–Meier curves and log-rank tests were performed in the discovery cohort. Cox proportional hazard regression analysis (HR with 95% CI) was used in the discovery cohort and binary logistic regression (OR with 95% CI) in the validation cohort. Results: PRO-C6 was localized in the core and shoulder of the atherosclerotic plaque. In the discovery cohort, PRO-C6 independently predicted future cardiovascular events (HR 1.089 [95% CI 1.019 −1.164], p = 0.01), cardiovascular death (HR 1.118 [95% CI 1.008 −1.241], p = 0.04) and all-cause death (HR 1.087 [95% CI 1.008 −1.172], p = 0.03). In the validation cohort, PRO-C6 predicted future cardiovascular events (OR 1.063 [95% CI 1.011 −1.117], p = 0.017). Conclusion: PRO-C6 is present in the atherosclerotic plaque and associated with future cardiovascular events, cardiovascular death and all-cause mortality in two large prospective cohorts

Detecting the Vulnerable Plaque

Myocardial infarction and stroke, mainly caused by a rupture of an atherosclerotic plaque, are two of the most common causes of mortality and morbidity in the world. Previous studies have pointed out inflammation, driven by inflammatory lipids, as the keystone in the formation of the rupture prone vulnerable plaque. The risk for a plaque rupture is greatly enhanced in the diabetic population and increased inflammation has been suggested as the contributing factor. However, the mechanism remains unknown. One of the current greatest challenges in the atherosclerotic field is to identify subjects with plaques that are vulnerable before the occurrence of a plaque rupture. In this thesis we provide evidence that the enzyme lipoprotein associated phospholipase A2 (LpPLA2) and the lipids lysophosphatidylcholines and sphingolipids (ceramides, lactosylceramides, glucosylceramides) contribute to the inflammatory process and that they are associated with the vulnerable plaque. This suggests that the synthesis of these lipids may be possible therapeutic targets. We also show that the inflammatory process in the plaque is reflected in the peripheral blood, as measured by cytokines, whereas no association was found for soluble urokinase-type plasminogen activator receptor. The inflammatory cytokines MIP-1β, TNF-α and fractalkine predict the inflammatory plaque with high sensitivity and specificity, proposing that these inflammatory cytokines could be used as plasma markers for plaque inflammation. Finally, we provide novel data regarding the biology of the diabetic plaque. According to our data the inflammatory activity is not increased in the diabetic plaque tissue. Diabetic plaques contained less stabilizing extracellular matrix proteins (collagen and elastin). Additionally the plaque levels of the inflammation regulating cytokine IL-10 and the growth factor platelet derived growth factor as well as the smooth muscle cell stimulating matrix metalloproteinase 2 were significantly decreased in diabetic plaque tissue. These findings were independent of the presence of symptoms. In summary, this thesis shows that lipids as LysoPCs and glycosphingolipids are important inducers of plaque inflammation and may be possible therapeutic targets. In the general population the inflammatory activity is an important factor for the formation of a vulnerable plaque and this inflammatory process is reflected in blood, hinting a role for fractalkine, MIP-1β and TNF-α as future biomarkers. In the diabetic population, however, an impaired tissue repair mechanism seems to be the keystone of plaque vulnerability, rather than inflammation, emphasizing the need for new therapeutic approaches for diabetics