Therapeutic S100A8/A9 blockade inhibits myocardial and systemic inflammation and mitigates sepsis-induced myocardial dysfunction.

BACKGROUND AND AIMS: The triggering factors of sepsis-induced myocardial dysfunction (SIMD) are poorly understood and are not addressed by current treatments. S100A8/A9 is a pro-inflammatory alarmin abundantly secreted by activated neutrophils during infection and inflammation. We investigated the efficacy of S100A8/A9 blockade as a potential new treatment in SIMD. METHODS: The relationship between plasma S100A8/A9 and cardiac dysfunction was assessed in a cohort of 62 patients with severe sepsis admitted to the intensive care unit of Linköping University Hospital, Sweden. We used S100A8/A9 blockade with the small-molecule inhibitor ABR-238901 and S100A9-/- mice for therapeutic and mechanistic studies on endotoxemia-induced cardiac dysfunction in mice. RESULTS: In sepsis patients, elevated plasma S100A8/A9 was associated with left-ventricular (LV) systolic dysfunction and increased SOFA score. In wild-type mice, 5 mg/kg of bacterial lipopolysaccharide (LPS) induced rapid plasma S100A8/A9 increase and acute LV dysfunction. Two ABR-238901 doses (30 mg/kg) administered intraperitoneally with a 6 h interval, starting directly after LPS or at a later time-point when LV dysfunction is fully established, efficiently prevented and reversed the phenotype, respectively. In contrast, dexamethasone did not improve cardiac function compared to PBS-treated endotoxemic controls. S100A8/A9 inhibition potently reduced systemic levels of inflammatory mediators, prevented upregulation of inflammatory genes and restored mitochondrial function in the myocardium. The S100A9-/- mice were protected against LPS-induced LV dysfunction to an extent comparable with pharmacologic S100A8/A9 blockade. The ABR-238901 treatment did not induce an additional improvement of LV function in the S100A9-/- mice, confirming target specificity. CONCLUSION: Elevated S100A8/A9 is associated with the development of LV dysfunction in severe sepsis patients and in a mouse model of endotoxemia. Pharmacological blockade of S100A8/A9 with ABR-238901 has potent anti-inflammatory effects, mitigates myocardial dysfunction and might represent a novel therapeutic strategy for patients with severe sepsis.

High levels of autoantibodies against apoB100 p210 are associated with lower incidence of atrial fibrillation in women.

BACKGROUND AND OBJECTIVES: Atrial fibrillation (AF) is associated with inflammation, both systemically and in the atrial tissue. Oxidized low-density lipoprotein (LDL) is increased in patients with AF and is suggested to be one of the molecules that drives inflammation. Autoantibodies against oxidized LDL and apolipoprotein B100, the protein component of LDL, are linked to atherosclerotic disease. However, whether these autoantibodies are associated with occurrence of AF is not known. We investigated autoantibodies against oxidized apolipoprotein B100 peptides and incidence of AF in a large population-based cohort. METHODS: IgM and IgG against native and aldehyde-modified apoB100 peptides 210 (p210) and 45 were analyzed by enzyme-linked immunosorbent assay (ELISA) in 5169 individuals from the Malmö Diet and Cancer cohort. RESULTS: Seven hundred sixty-nine incident AF cases were recorded during a follow-up of 21.3 years. Individuals with high levels of IgM against native p210 at baseline had a lower risk of developing AF; however, the association did not remain after adjustment for age and sex. Women had higher levels of IgM against native p210 than men (0.70 ± 0.22 AU vs. 0.63 ± 0.21 AU, p < 0.001). The association of IgM against native p210 and AF was significantly different between sexes (p for interaction = 0.024), where females with high IgM against p210 had a lower risk for incidence of AF (hazard ratio [95% confidence interval] 4th versus 1st quartile: 0.67 [0.49-0.91]; p = 0.01) after adjusting for risk factors and comorbidities. CONCLUSION: These findings support an association of humoral autoimmunity with AF.

Circulating Monocyte Chemoattractant Protein-1 and Risk of Stroke: Meta-Analysis of Population-Based Studies Involving 17 180 Individuals.

Rationale: Proinflammatory cytokines have been identified as potential targets for lowering vascular risk. Experimental evidence and Mendelian randomization suggest a role of MCP-1 (monocyte chemoattractant protein-1) in atherosclerosis and stroke. However, data from large-scale observational studies are lacking. Objective: To determine whether circulating levels of MCP-1 are associated with risk of incident stroke in the general population. Methods and Results: We used previously unpublished data on 17 180 stroke-free individuals (mean age, 56.7±8.1 years; 48.8% men) from 6 population-based prospective cohort studies and explored associations between baseline circulating MCP-1 levels and risk of any stroke, ischemic stroke, and hemorrhagic stroke during a mean follow-up interval of 16.3 years (280 522 person-years at risk; 1435 incident stroke events). We applied Cox proportional-hazards models and pooled hazard ratios (HRs) using random-effects meta-analyses. After adjustments for age, sex, race, and vascular risk factors, higher MCP-1 levels were associated with increased risk of any stroke (HR per 1-SD increment in ln-transformed MCP-1, 1.07; 95% CI, 1.01-1.14). Focusing on stroke subtypes, we found a significant association between baseline MCP-1 levels and higher risk of ischemic stroke (HR, 1.11 [1.02-1.21]) but not hemorrhagic stroke (HR, 1.02 [0.82-1.29]). The results followed a dose-response pattern with a higher risk of ischemic stroke among individuals in the upper quartiles of MCP-1 levels as compared with the first quartile (HRs, second quartile: 1.19 [1.00-1.42]; third quartile: 1.35 [1.14-1.59]; fourth quartile: 1.38 [1.07-1.77]). There was no indication for heterogeneity across studies, and in a subsample of 4 studies (12 516 individuals), the risk estimates were stable after additional adjustments for circulating levels of IL (interleukin)-6 and high-sensitivity CRP (C-reactive protein). Conclusions: Higher circulating levels of MCP-1 are associated with increased long-term risk of stroke. Our findings along with genetic and experimental evidence suggest that MCP-1 signaling might represent a therapeutic target to lower stroke risk.Visual Overview: An online visual overview is available for this article.

Low Levels of CD4+CD28null T Cells at Baseline Are Associated With First-Time Coronary Events in a Prospective Population-Based Case-Control Cohort.

OBJECTIVE: CD4+CD28null T cells have been shown to be associated with recurrent coronary events and suggested as potential biomarker and therapeutic target. It is unknown whether CD4+CD28null T cells associate with first-time cardiovascular events. We examined CD4+CD28null T cells in a prospective population-based cohort and in patients with advanced atherosclerosis. Approach and Results: CD4+CD28null T cells were quantified in 272 individuals experiencing a first-time coronary event during up to 17 years of follow-up and 272 age- and sex-matched controls in a case-control study, nested within the population-based Malmö Diet and Cancer study. The highest tertile of CD4+CD28null T cells was associated with a lower incidence of first-time coronary events compared with the lowest tertile (odds ratio, 0.48 [95% CI, 0.29-0.79], P=0.004) when adjusting for Framingham risk factors. This association remained significant for events recorded after >9 years of follow-up, when most coronary events occurred, but not during the first 9 years of follow-up, despite similar odds ratio. Additionally, we analyzed CD4+CD28null T cells in 201 patients with advanced atherosclerosis undergoing carotid endarterectomy. The adjusted hazard ratio for cardiovascular events in patients with advanced atherosclerosis was 2.11 (95% CI, 1.10-4.05, P=0.024), comparing the highest with the lowest CD4+CD28null T-cell tertile. CONCLUSIONS: Our findings reveal complex associations between CD4+CD28null T cells and cardiovascular disease. Although we confirm the reported positive associations with an adverse prognosis in patients with already established disease, the opposite associations with first-time coronary events in the population-based cohort may limit the clinical use of CD4+CD28null T cells.

Lack of Ability to Present Antigens on Major Histocompatibility Complex Class II Molecules Aggravates Atherosclerosis in ApoE-/- Mice.

BACKGROUND: Hypercholesterolemic mice lacking factors required for activation of CD4+ T cells are characterized by reduced development of atherosclerosis. Consequently, it has been assumed that atherosclerosis involves loss of tolerance against modified self-antigens generated in response to hypercholesterolemia and that presentation of such antigens on major histocompatibility complex class II (MHCII) leads to activation of proatherogenic Th1 cells. In this study, we wanted to determine the role of antigen presentation on MHCII in atherosclerosis development. METHODS: Apolipoprotein E (ApoE-/-) mice deficient for MHCII (ApoE-/-MHCII-/-) were used to study the role of MHCII in atherosclerosis development. RESULTS: Compared with ApoE-/- mice, ApoE-/-MHCII-/- mice had reduced levels of CD4+ T cells, immunoglobulin G and M levels, and Th1 and Th2 cytokines in plasma. CD8+ T cells were increased and regulatory T cells were reduced both in spleen and in lesions of ApoE-/-MHCII-/- mice. Decreased plasma levels of inflammatory cytokines in ApoE-/-MHCII-/- mice indicated reduced systemic inflammation. Despite this, ApoE-/-MHCII-/- mice had significantly more atherosclerosis as assessed by en face Oil Red O staining of the aorta (4.7±2.9% versus 1.9±1.3%; P<0.01) and cross-sectional area of subvalvular lesions (7.7±2.2×105 µm2 versus 4.6±2.8×105 µm2; P<0.05). Cell transfer and blocking antibody studies suggested that loss of regulatory T cells is the most important cause of aggravated atherosclerosis in ApoE-/-MHCII-/- mice. CONCLUSIONS: Our observations demonstrate that antigen presentation on MHCII has important protective functions in atherosclerosis and that this is primarily the result of activation of regulatory T cells. These findings have implications for understanding the possible risks and benefits of immunosuppressive therapy in patients with cardiovascular disease.

Interleukin-25 (IL-25) has a protective role in atherosclerosis development in the aortic arch in mice.

Atherosclerosis is a chronic inflammatory disease characterized by the entrapment of apolipoprotein B-containing lipoproteins in the arterial intima, leading to local inflammation. T helper (Th) cell 1-mediated immune responses have been associated with atherosclerosis, and the cytokine interleukin-25 (IL-25 or IL-17E) has been reported to potentially regulate Th1 cell- and Th17 cell-related immune responses. In this study, we evaluated the effects of complete IL-25 deficiency or of a temporal IL-25 blockade on atherosclerosis development in apolipoprotein E-deficient (Apoe-/-) mice. Mice deficient in both apolipoprotein E and IL-25 (Apoe-/-/IL-25-/-) had more Th1 cells in the spleen, along with elevated plasma levels of IL-17 and an increased release of splenic interferon-γ (INF-γ). In support of this observation, a 4-week-long treatment of young Apoe-/- mice (at 10-14 weeks of age) with an IL-25-blocking antibody increased the release of Th1/Th17-associated cytokines in the spleen. In both mouse models, these findings were associated with increased atherosclerotic plaque formation in the aortic arch. We conclude that complete IL-25 deficiency and a temporal IL-25 blockade during early plaque development aggravate atherosclerosis development in the aortic arch of Apoe-/- mice, accompanied by an increase in Th1/Th17-mediated immune responses. Our finding that endogenous IL-25 has an atheroprotective role in the murine aortic arch has potential implications for atherosclerosis development and management in humans.

Cartilage Oligomeric Matrix Protein Associates With a Vulnerable Plaque Phenotype in Human Atherosclerotic Plaques.

Background and Purpose- Extracellular matrix proteins are important in atherosclerotic disease by influencing plaque stability and cellular behavior but also by regulating inflammation. COMP (cartilage oligomeric matrix protein) is present in healthy human arteries and expressed by smooth muscle cells. A recent study showed that transplantation of COMP-deficient bone marrow to apoE-/- mice increased atherosclerotic plaque formation, indicating a role for COMP also in bone marrow-derived cells. Despite the evidence of a role for COMP in murine atherosclerosis, knowledge is lacking about the role of COMP in human atherosclerotic disease. Methods- In the present study, we investigated if COMP was associated with a stable or a vulnerable human atherosclerotic plaque phenotype by analyzing 211 carotid plaques for COMP expression using immunohistochemistry. Results- Plaque area that stained positive for COMP was significantly larger in atherosclerotic plaques associated with symptoms (n=110) compared with asymptomatic plaques (n=101; 9.7% [4.7-14.3] versus 5.6% [2.8-9.8]; P=0.0002). COMP was positively associated with plaque lipids (r=0.32; P=0.000002) and CD68 cells (r=0.15; P=0.036) but was negatively associated with collagen (r=-0.16; P=0.024), elastin (r=-0.14; P=0.041), and smooth muscle cells (r=-0.25; P=0.0002). COMP was positively associated with CD163 (r=0.37; P=0.00000006), a scavenger receptor for hemoglobin/haptoglobin and a marker of Mhem macrophages, and with intraplaque hemorrhage, measured as glycophorin A staining (r=0.28; P=0.00006). Conclusions- The present study shows that COMP is associated to symptomatic carotid atherosclerosis, CD163-expressing cells, and a vulnerable atherosclerotic plaque phenotype in humans.

ILC2 transfers to apolipoprotein E deficient mice reduce the lipid content of atherosclerotic lesions.

BACKGROUND: Expansion of type 2 innate lymphoid cells (ILC2s) in hypercholesterolaemic mice protects against atherosclerosis while different ILC2 subsets have been described (natural, inflammatory) based on their suppression of tumorigenicity 2 (ST2) and killer-cell lectin like receptor G1 (KLRG1) expression. The aim of the current study is to characterize the interleukin 25 (IL25)-induced splenic ILC2 population (Lin-CD45+IL17RB+ICOS+IL7raintermediate) and address its direct role in experimental atherosclerosis by its adoptive transfer to hypercholesterolaemic apolipoprotein E deficient (apoE-/-) mice. RESULTS: Immunomagnetically enriched, FACS-sorted ILC2s from the spleens of IL-25 treated apoE-/- mice were stained for KLRG1 and ST2 directly upon cell obtainment or in vitro cell expansion for flow cytometric analysis. IL25-induced splenic ILC2s express high levels of both KLRG1 and ST2. However, both markers are downregulated upon in vitro cell expansion. In vitro expanded splenic ILC2s were intraperitoneally transferred to apoE-/- recipients on high fat diet. ApoE-/- mice that received in vitro expanded splenic ILC2s had decreased lipid content in subvalvular heart and brachiocephalic artery (BCA) plaques accompanied by increased peritoneal B1 cells, activated eosinophils and alternatively activated macrophages (AAMs) as well as anti-phosphorylcholine (PC) immunoglobulin (Ig) M in plasma. CONCLUSIONS: With the current data we designate the IL25-induced ILC2 population to decrease the lipid content of atherosclerotic lesions in apoE-/- mice and we directly link the induction of B1 cells and the atheroprotective anti-PC IgM antibodies with ILC2s.

Altered metabolism distinguishes high-risk from stable carotid atherosclerotic plaques.

Aims: Identification and treatment of the rupture prone atherosclerotic plaque remains a challenge for reducing the burden of cardiovascular disease. The interconnection of metabolic and inflammatory processes in rupture prone plaques is poorly understood. Herein, we investigate associations between metabolite profiles, inflammatory mediators and vulnerability in carotid atherosclerotic plaques. Methods and results: We collected 159 carotid plaques from patients undergoing endarterectomy and measured 165 different metabolites in a targeted metabolomics approach. We identified a metabolite profile in carotid plaques that associated with histologically evaluated vulnerability and inflammatory mediators, as well as presence of symptoms in patients. The distinct metabolite profiles identified in high-risk and stable plaques were in line with different transcription levels of metabolic enzymes in the two groups, suggesting an altered metabolism in high-risk plaques. The altered metabolic signature in high-risk plaques was consistent with a change to increased glycolysis, elevated amino acid utilization and decreased fatty acid oxidation, similar to what is found in activated leucocytes and cancer cells. Conclusion: These results highlight a possible key role of cellular metabolism to support inflammation and a high-risk phenotype of atherosclerotic plaques. Targeting the metabolism of atherosclerotic plaques with novel metabolic radiotracers or inhibitors might therefore be valid future approaches to identify and treat the high-risk atherosclerotic plaque.

Low Levels of Apolipoprotein B-100 Autoantibodies Are Associated With Increased Risk of Coronary Events.

OBJECTIVE: Previous smaller studies have indicated inverse associations between autoantibodies to oxidized low-density lipoprotein epitopes, and cardiovascular disease. The present study investigated associations between autoantibodies against the apolipoprotein B-100 peptides p45 and p210, respectively, and risk of incident cardiovascular disease in a large population-based cohort. APPROACH AND RESULTS: Apolipoprotein B-100 autoantibodies were analyzed by ELISA in a prospective study, including 5393 individuals (aged 46-68 years) belonging to the cardiovascular arm of the Malmö Diet and Cancer study with a follow-up time of >15 years. Subjects that suffered an acute coronary event during follow-up (n=382) had lower levels at baseline of IgM autoantibodies recognizing the native and malondialdehyde-modified apolipoprotein B-100 peptides p45 and p210 and also lower IgG levels recognizing native p210, whereas no association was found with risk for stroke (n=317). Subjects in the highest compared with lowest tertile of IgM-p45MDA (hazard ratio [95% confidence interval]: 0.72 [0.55, 0.94]; P=0.017) and IgG-p210native (hazard ratio [95% confidence interval]: 0.73 [0.56, 0.97]; P=0.029) had lower risk for incident coronary events after adjustment for cardiovascular risk factors in Cox proportional hazard regression models. Moreover, subjects with high levels of IgG-p210native were less likely to have carotid plaques as assessed by ultrasonography at baseline (odds ratio=0.81, 95% confidence interval 0.70-0.95, P=0.008 after adjustment for risk factors). CONCLUSIONS: This large prospective study demonstrates that subjects with high levels of apolipoprotein B-100 autoantibodies have a lower risk of coronary events supporting a protective role of these autoantibodies in cardiovascular disease.

Low Levels of IgM Antibodies against an Advanced Glycation Endproduct-Modified Apolipoprotein B100 Peptide Predict Cardiovascular Events in Nondiabetic Subjects.

Increased glucose levels are associated with the generation of advanced glycation endproduct (AGE) modifications. Interaction between AGE-modified plaque components and immune cells is believed to have an important role in the development of vascular complications in diabetes. Methylglyoxal (MGO) is one type of reactive aldehyde that gives rise to AGE modification. The present study analyzed whether autoantibodies against MGO-modified epitopes of the low-density lipoprotein apolipoprotein B (apoB) 100 predict cardiovascular events. A library consisting of 302 peptides comprising the complete apoB100 molecule was screened to identify peptides targeted by MGO-specific autoantibodies. Peptide (p) 220 (apoB amino acids 3286-3305) was identified as a major target. Baseline IgM and IgG against MGO-peptide 220 (p220) were measured in 700 individuals from the Malmö Diet and Cancer Cohort. A total of 139 cardiovascular events were registered during the 15-y follow-up period. Controlling for major cardiovascular risk factors demonstrated that subjects in the lowest tertile of MGO-p220 IgM had an increased risk for cardiovascular events (hazard ratio [95% confidence interval]: 2.07 [1.22-3.50]; p(trend) = 0.004). Interestingly, the association between MGO-p220 IgM and cardiovascular events remained and even tended to become stronger when subjects with prevalent diabetes were excluded from the analysis (2.51 [1.37-4.61]; p(trend) = 0.002). MGO-p220 IgM was inversely associated with blood glucose, but not with oxidized low-density lipoprotein. Finally, we demonstrate that anti-MGO-p220 IgM is produced by B1 cells. These data show that subjects with low levels of IgM recognizing MGO-modified p220 in apoB have an increased risk to develop cardiovascular events and that this association is present in nondiabetic subjects.

Apolipoprotein B-100 Antibody Interaction With Atherosclerotic Plaque Inflammation and Repair Processes.

BACKGROUND AND PURPOSE: Treatment with IgG against the malondialdehyde (MDA)-modified apolipoprotein B-100 epitope p45 reduces atherosclerosis in experimental models. This study investigated the association between p45 IgG autoantibodies and plaque inflammation in subjects with advanced cardiovascular disease. METHODS: Native and MDA-p45 IgG levels were analyzed by ELISA in 349 carotid endarterectomy patients. In a subcohort of 195 subjects, endarterectomy samples were analyzed by immunohistochemistry and ELISA to determine plaque constituents and inflammation. Peripheral blood mononuclear cells were isolated from healthy donors. RESULTS: Patients with preoperative events of neurological ischemia had lower levels of native p45 IgG. Low levels of MDA-p45 IgG were associated with increased risk of postoperative cardiovascular death during a mean follow-up of 54 months. High plasma levels of native p45 IgG were associated with increased plaque content of collagen and smooth muscle cell growth factors, as well as with lower levels of proinflammatory cytokines. Exposure of peripheral blood mononuclear cells from healthy donors to recombinant MDA-p45 IgG in presence of oxidized low-density lipoprotein reduced the expression of tumor necrosis factor-α and stimulated release of smooth muscle cell growth factors. CONCLUSIONS: This study confirms previous experimental findings of anti-inflammatory properties of apolipoprotein B-100 p45 antibodies and provides the first clinical evidence of associations between p45 IgG autoantibody levels and atherosclerotic plaque inflammation, plaque repair as well as prevalent and incident cardiovascular events in carotid endarterectomy patients. These findings suggest the possibility that treatment with anti-p45 antibodies may have beneficial effects in advanced cardiovascular disease.

Endarterectomy patients with elevated levels of circulating IL-16 have fewer cardiovascular events during follow-up.

BACKGROUND AND PURPOSE: Increased interleukin 16 (IL-16) levels in carotid plaques have been associated with reduced incidence of cardiovascular (CV) events during follow-up in patients who underwent carotid endarterectomy (CEA). In the present study we aimed to determine whether high circulating levels of IL-16 also are associated with a decreased risk of CV events after CEA. METHODS: Patients, who had their carotid plaques surgically removed (n=473), were followed for a mean follow-up time of 3.1years. Plasma levels of IL-16 the day before surgery were analyzed by proximity extension assay (PEA) and associated with the occurrence of CV events during follow-up (n=98). RESULTS: High levels of circulating IL-16 were independently associated with a decreased risk of CV events when comparing the highest versus the lowest IL-16 tertile (hazard ratio [HR] 0.47; 95% CI 0.27-0.81; P=0.007), as well as with CV deaths (HR 0.25; 95% CI 0.09-0.70; P=0.008). CONCLUSION: These present findings indicate an association between IL-16 and less clinical complications of atherosclerosis in a population with known advanced carotid disease.

Genetic loci on chromosome 5 are associated with circulating levels of interleukin-5 and eosinophil count in a European population with high risk for cardiovascular disease.

IL-5 is a Th2 cytokine which activates eosinophils and is suggested to have an atheroprotective role. Genetic variants in the IL5 locus have been associated with increased risk of CAD and ischemic stroke. In this study we aimed to identify genetic variants associated with IL-5 concentrations and apply a Mendelian randomisation approach to assess IL-5 levels for causal effect on intima-media thickness in a European population at high risk of coronary artery disease. We analysed SNPs within robustly associated candidate loci for immune, inflammatory, metabolic and cardiovascular traits. We identified 2 genetic loci for IL-5 levels (chromosome 5, rs56183820, BETA=0.11, P=6.73E(-5) and chromosome 14, rs4902762, BETA=0.12, P=5.76E(-6)) and one for eosinophil count (rs72797327, BETA=-0.10, P=1.41E(-6)). Both chromosome 5 loci were in the vicinity of the IL5 gene, however the association with IL-5 levels failed to replicate in a meta-analysis of 2 independent cohorts (rs56183820, BETA=0.04, P=0.2763, I(2)=24, I(2)-P=0.2516). No significant associations were observed between SNPs associated with IL-5 levels or eosinophil count and IMT measures. Expression quantitative trait analyses indicate effects of the IL-5 and eosinophil-associated SNPs on RAD50 mRNA expression levels (rs12652920 (r2=0.93 with rs56183820) BETA=-0.10, P=8.64E(-6) and rs11739623 (r2=0.96 with rs72797327) BETA=-0.23, P=1.74E(-29), respectively). Our data do not support a role for IL-5 levels and eosinophil count in intima-media thickness, however SNPs associated with IL-5 and eosinophils might influence stability of the atherosclerotic plaque via modulation of RAD50 levels.

High Plasma Levels of Galectin-3 Are Associated with Increased Risk for Stroke after Carotid Endarterectomy.

BACKGROUND: Galectin-3 (Gal-3) has been suggested to have both pro- and anti-atherogenic properties. High plasma Gal-3 levels are associated with increased risk for cardiovascular (CV) death. However, it has so far not been investigated if plasma Gal-3 levels can predict the risk for future stroke in patients suffering from carotid atherosclerosis. The aim of this study was to investigate whether Gal-3 could be used as a marker to predict postoperative cerebrovascular ischemic events among patients who underwent carotid endarterectomy (CEA). METHODS: Plasma samples were obtained from 558 CEA patients and Gal-3 levels were analyzed by the proximity extension assay technique. The Swedish national in-patient health register was used to identify postoperative cerebrovascular events during the follow-up period (42.6 ± 26.2 months). RESULTS: Plasma Gal-3 was increased in patients treated for a symptomatic carotid stenosis (p = 0.013). Patients with Gal-3 levels above the median value had an increased incidence of stroke as shown by Kaplan-Meier curves of event-free survival (p = 0.007). Gal-3 was a predictor of postoperative stroke among women (hazard ratio 15.1, 95% CI 1.3-172.2; p = 0.028) even after correction for traditional CV risk factors. CONCLUSIONS: This study is the first to show that increased plasma levels of Gal-3 can help in predicting the occurrence of postoperative strokes among female subjects who undergo CEA, independently of traditional risk factors for cerebrovascular disease. This finding suggests that Gal-3 could be used as a marker to identify patients in need of intensified postoperative medical care.

High plasma levels of heparin-binding epidermal growth factor are associated with a more stable plaque phenotype and reduced incidence of coronary events.

OBJECTIVE: Rupture of atherosclerotic plaques is the major cause of acute coronary events (CEs). Plaque destabilization is the consequence of an imbalance between inflammatory-driven degradation of fibrous tissue and smooth muscle cell-dependent tissue repair. Proinflammatory factors have been documented extensively as biomarkers of cardiovascular risk but factors that contribute to stabilization of atherosclerotic plaques have received less attention. The present study aimed to investigate whether plasma levels of the smooth muscle cell growth factor epidermal growth factor (EGF), heparin-binding-EGF (HB-EGF), and platelet-derived growth factor correlate with plaque phenotype and incidence of CEs. APPROACH AND RESULTS: HB-EGF, EGF and platelet-derived growth factor were measured in plasma from 202 patients undergoing carotid endarterectomy and in 384 incident CE cases and 409 matched controls recruited from the Malmö Diet and Cancer cohort. Significant positive associations were found between the plasma levels of all 3 growth factors and the collagen and elastin contents of the removed plaques. CE cases in the Malmö Diet and Cancer cohort had lower levels of HB-EGF in plasma, whereas no significant differences were found for EGF and platelet-derived growth factor. After adjusting for cardiovascular risk factors in a Cox proportional hazard model, the hazard ratio for the highest HB-EGF tertile was 0.61 (95% confidence interval, 0.47-0.82; P<0.001). CONCLUSIONS: The associations between high levels of smooth muscle cell growth factors in plasma and a more fibrous plaque phenotype as well as the association between low levels of HB-EGF and incident CEs point to a potential clinically important role for factors that contribute to plaque stabilization by stimulating smooth muscle cells.

Elevated Plasma Levels of MMP-12 Are Associated With Atherosclerotic Burden and Symptomatic Cardiovascular Disease in Subjects With Type 2 Diabetes.

OBJECTIVE: Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins and play important roles in development and tissue repair. They have also been shown to have both protective and pathogenic effects in atherosclerosis, and experimental studies have suggested that MMP-12 contributes to plaque growth and destabilization. The objective of this study was to investigate the associations between circulating MMPs, atherosclerosis burden, and incidence of cardiovascular disease with a particular focus on type 2 diabetes mellitus. APPROACH AND RESULTS: Plasma levels of MMP-1, -3, -7, -10, and -12 were analyzed by the Proximity Extension Assay technology in 1500 subjects participating in the SUMMIT (surrogate markers for micro- and macrovascular hard end points for innovative diabetes tools) study, 384 incident coronary cases, and 409 matched controls in the Malmö Diet and Cancer study and in 205 carotid endarterectomy patients. Plasma MMP-7 and -12 were higher in subjects with type 2 diabetes mellitus, increased with age and impaired renal function, and was independently associated with prevalent cardiovascular disease, atherosclerotic burden (as assessed by carotid intima-media thickness and ankle-brachial pressure index), arterial stiffness, and plaque inflammation. Baseline MMP-7 and -12 levels were increased in Malmö Diet and Cancer subjects who had a coronary event during follow-up. CONCLUSIONS: The plasma level of MMP-7 and -12 are elevated in type 2 diabetes mellitus, associated with more severe atherosclerosis and an increased incidence of coronary events. These observations provide clinical support to previous experimental studies, demonstrating a role for these MMPs in plaque development, and suggest that they are potential biomarkers of atherosclerosis burden and cardiovascular disease risk.

Association between renin and atherosclerotic burden in subjects with and without type 2 diabetes.

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