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Aim: To explore the influence of apical periodontitis (AP) on inflammatory markers in blood of otherwise healthy individuals and to depict the inflammatory profile of the healing after dental extraction. Methodology: This is a prospective case–control intervention study, during which, individuals with a diagnosis of AP of one affected tooth were included, along with a control group matched for age and gender. A broad panel of blood inflammatory mediators was examined longitudinally in all subjects during six visits. In the case of the AP subjects, the tooth with AP was extracted at the third visit. Results were analysed by linear regression analyses and linear mixed-model analyses. Results: A total of 53 subjects were included in the study, 27 with AP and 26 without. Fifteen females and 12 males were included in the AP group, and 14 females and 12 males in the control group. At baseline, granulocyte colony-stimulating factor (p

Objective: To determine whether MCP-1 (monocyte chemoattractant protein 1) levels in human atherosclerotic plaques associate with plaque vulnerability features. Approach and Results: We measured MCP-1 levels in human atherosclerotic plaque samples from 1199 patients in the Athero-EXPRESS Biobank who underwent endarterectomy for treatment of carotid stenosis. We explored associations with histopathologic and molecular features of plaque vulnerability, clinical plaque manifestations, and vascular events up to 3 years after endarterectomy. Following adjustments for age, sex, and vascular risk factors, MCP-1 plaque levels were associated with histopathologic markers of plaque vulnerability (large lipid core, low collagen content, high macrophage burden, low smooth muscle cell burden, intraplaque hemorrhage) and with a composite vulnerability index (range 0–5, β per SD increment in MCP-1, 0.42 [95% CI, 0.30–0.53], P =5.4×10 −13 ). We further found significant associations with higher plaque levels of other chemokines and proinflammatory molecules and markers of neovascularization and matrix turnover. When exploring clinical plaque instability, MCP-1 plaque levels were higher among individuals with symptomatic plaques as compared with those with asymptomatic plaques (odds ratio per SD increment in MCP-1, 1.36 [95% CI, 1.09–1.69]). MCP-1 levels were further associated with a higher risk of periprocedural major adverse vascular events and strokes occurring in the first 30 days after plaque removal. Conclusions: Higher MCP-1 plaque levels are associated with histopathologic, molecular, and clinical hallmarks of plaque vulnerability in individuals undergoing carotid endarterectomy. Our findings highlight a role of MCP-1 in clinical plaque instability in humans and complement previous epidemiological, genetic, and experimental studies supporting the translational perspective of targeting MCP-1 signaling in atherosclerosis.

Plasma osteoprotegerin (OPG) and vascular smooth muscle cell (VSMC) derived extracellular vesicles (EVs) are important regulators in the process of vascular calcification (VC). In population studies, high levels of OPG are associated with events. In animal studies, however, high OPG levels result in reduction of VC. VSMC-derived EVs are assumed to be responsible for OPG transport and VC but this role has not been studied. For this, we investigated the association between OPG in plasma and circulating EVs with coronary artery calcium (CAC) as surrogate for VC in symptomatic patients. We retrospectively assessed 742 patients undergoing myocardial perfusion imaging (MPI). CAC scores were determined on the MPI-CT images using a previously developed automated algorithm. Levels of OPG were quantified in plasma and two EV-subpopulations (LDL and TEX), using an electrochemiluminescence immunoassay. Circulating levels of OPG were independently associated with CAC scores in plasma; OR 1.39 (95% CI 1.17–1.65), and both EV populations; EV-LDL; OR 1.51 (95% CI 1.27–1.80) and EV-TEX; OR 1.21 (95% CI 1.02–1.42). High levels of OPG in plasma were independently associated with CAC scores in this symptomatic patient cohort. High levels of EV-derived OPG showed the same positive association with CAC scores, suggesting that EV-derived OPG mirrors the same pathophysiological process as plasma OPG.

Background: Monocyte chemoattractant protein-1 (MCP-1) is a chemokine recruiting monocytes to the atherosclerotic plaque. Experimental, genetic, and epidemiological data support a key role of MCP-1 in atherosclerosis. Yet, the translational potential of targeting MCP-1 signaling for lowering vascular risk is limited by the lack of data on plaque MCP-1 activity in human atherosclerosis. Methods: We measured MCP-1 levels in human plaque samples from 1,199 patients undergoing carotid endarterectomy from the Athero-Express Biobank. We explored associations of plaque MCP-1 levels with histopathological features of plaque vulnerability, clinical plaque instability (symptomatic vs. asymptomatic plaque), molecular markers of plaque inflammation and remodeling, and with incident vascular events up to three years after plaque removal. Results: MCP-1 plaque levels were associated with individual histopathological hallmarks of plaque vulnerability (large lipid core, low collagen, high macrophage burden, low smooth muscle cell burden, intraplaque hemorrhage), as well as with a cumulative vulnerability index (range 0-5, beta: 0.42, 95%CI: 0.30-0.53, p=5.4x10 -13 ) independently of age, sex, and conventional vascular risk factors. Furthermore, MCP-1 levels were higher among patients with symptomatic, as compared to asymptomatic plaques (p=0.0001) and were associated with the levels of pro-inflammatory cytokines involved in leukocyte adhesion, as well as with matrix metalloproteinase activity in the plaque. In the follow-up analyses, MCP-1 levels were associated with a higher risk of peri-procedural events (up to 30 days after surgery). Conclusions: Our findings highlight a role of MCP-1 in human plaque vulnerability, the leading mechanism underlying vascular events like stroke and myocardial infarction. As such, they suggest that interfering with MCP-1 signaling in patients with established atherosclerosis could lower vascular risk.

The clinical spectrum of COVID-19 varies and the differences in host response characterizing this variation have not been fully elucidated. COVID-19 disease severity correlates with an excessive proinflammatory immune response and profound lymphopenia. Inflammatory responses according to disease severity were explored by plasma cytokine measurements and proteomics analysis in 147 COVID-19 patients. Furthermore, peripheral blood mononuclear cell cytokine production assays and whole blood flow cytometry were performed. Results confirm a hyperinflammatory innate immune state, while highlighting hepatocyte growth factor and stem cell factor as potential biomarkers for disease severity. Clustering analysis revealed no specific inflammatory endotypes in COVID-19 patients. Functional assays revealed abrogated adaptive cytokine production (interferon-γ, interleukin-17, and interleukin-22) and prominent T-cell exhaustion in critically ill patients, whereas innate immune responses were intact or hyperresponsive. Collectively, this extensive analysis provides a comprehensive insight into the pathobiology of severe to critical COVID-19 and highlights potential biomarkers of disease severity.

The chronic inflammatory response plays an important role in adverse cardiac remodelling and the development of heart failure (HF). There is also evidence that in the pathogenesis of several cardiovascular diseases, chronic inflammation is accompanied by antibody and complement deposits in the heart, suggestive of a true autoimmune response. However, the role of antibody‐mediated immune responses in HF progression is less clear. We assessed whether immune cell infiltration and immunoglobulin levels are associated with HF type and disease stage, taking sex differences into account. We found IgG deposits and increased infiltration of immune cells in the affected myocardium of patients with end‐stage HF with reduced ejection fraction (HFrEF, n = 20). Circulating levels of IgG1 and IgG3 were elevated in these patients. Furthermore, the percentage of transitional/regulatory B cells was decreased (from 6.9% to 2.4%) compared with healthy controls (n = 5). Similarly, increased levels of circulating IgG1 and IgG3 were observed in men with left ventricular diastolic dysfunction (LVDD, n = 5), possibly an early stage of HF with preserved EF (HFpEF). In conclusion, IgG deposits and infiltrates of immune cells are present in end‐stage HFrEF. In addition, both LVDD patients and end‐stage HFrEF patients show elevated levels of circulating IgG1 and IgG3, suggesting an antibody‐mediated immune response upon cardiac remodelling, which in the early phase of remodelling appear to differ between men and women. These immunoglobulin subclasses might be used as marker for pre‐stage HF and its progression. Future identification of auto‐antigens might open possibilities for new therapeutic interventions.

Atherosclerosis is the underlying syndrome of Cardiovascular Disease (CVD). Ischemic Heart Disease and Stroke are cardiovascular events both due to atherosclerosis that are the number 1 and 2 cause of death in the world and expect to increase especially in Asia. Ischemic heart disease (IHD) comprises 3 entities: stable coronary artery disease (SCAD), unstable angina (UA) and myocardial infarction (MI). Despite its large numbers, diagnosis IHD is challenging, as many patients present with atypical symptoms with women have a different symptom sensation than men. Troponins are the main diagnostic tool for detection of MI. Blood biomarkers for SCAD (typically causing stable angina) and UA, however, are not available. These diagnoses frequently require hospital visits/admissions for time-consuming and costly (non)invasive tests. Extracellular vesicles are composed of a lipid bilayer containing cytosolic, membrane and nuclear molecules derived from the cell and cell compartments of origin and released by the cell into extracellular biofluids. Using proteomics, we identified a plasma extracellular vesicle protein signature of inflammatory and coagulation proteins in different plasma vesicle subpopulations that can identify UA and SCAD patients. This signature was verified for UA in 60 acute chest pain patients (AUC 0.93), and for SCAD in 60 chest pain patients scheduled for heart perfusion imaging (AUC 0.86). This shows that plasma extracellular vesicle proteins can be used for early diagnosis of SCAD and UA and underlines the biological role for extracellular vesicles in atherosclerosis.

Statins are thought to have pleiotropic properties, including anticoagulant effects, in addition to reducing lipoprotein (LDL) levels. Plasma extracellular vesicles (EVs) are small bilayer membrane vesicles involved in various biological processes including coagulation. Since subsets of EVs in the LDL plasma fraction (LDL-EVs) correlate with thrombin activity, we hypothesized that changes in LDL-EVs after statin therapy may differ from that of serum levels of coagulation proteins, providing insight into the effects of statins on coagulation.The study was conducted in 666 subjects with available serum from the METEOR trial, a trial of the effect of rosuvastatin versus placebo in patients with subclinical atherosclerosis. Changes in protein levels of von Willebrand Factor (VWF), SerpinC1 and plasminogen were measured in serum and in LDL-EVs, and were compared between the rosuvastatin and placebo groups.LDL-EV levels of plasminogen and VWF increased with rosuvastatin treatment compared to placebo (mean change of 126 ± 8 versus 17 ± 12 μg/mL for plasminogen (p 0.001) and 310 ± 60 versus 64 ± 55 μg/mL for VWF (p = 0.015)). There was no difference between groups for change in LDL-EV-SerpinC1. In contrast, serum plasminogen levels increased to a lesser extent with rosuvastatin compared to placebo (23 ± 29 versus 67 ± 17 μg/mL, p = 0.024) and serum VWF levels showed no significant difference between both groups.Rosuvastatin increases LDL-EV coagulation proteins plasminogen and VWF in patients with subclinical atherosclerosis, an effect that is different from the effect of rosuvastatin on the same proteins in serum. This identifies LDL-EVs as a newly detected possible intermediate between statin therapy and coagulation.

Plasma extracellular vesicles (EVs) are lipid membrane vesicles involved in several biological processes including coagulation. Both coagulation and lipid metabolism are strongly associated with cardiovascular events. Lowering very-low- and low-density lipoprotein ((V)LDL) particles via dextran sulphate LDL apheresis also removes coagulation proteins. It remains unknown, however, how coagulation proteins are removed in apheresis. We hypothesize that plasma EVs that contain high levels of coagulation proteins are concomitantly removed with (V)LDL particles by dextran sulphate apheresis. For this, we precipitated (V)LDL particles from human plasma with dextran sulphate and analyzed the abundance of coagulation proteins and EVs in the precipitate. Coagulation pathway proteins, as demonstrated by proteomics and a bead-based immunoassay, were over-represented in the (V)LDL precipitate. In this precipitate, both bilayer EVs and monolayer (V)LDL particles were observed by electron microscopy. Separation of EVs from (V)LDL particles using density gradient centrifugation revealed that almost all coagulation proteins were present in the EVs and not in the (V)LDL particles. These EVs also showed a strong procoagulant activity. Our study suggests that dextran sulphate used in LDL apheresis may remove procoagulant EVs concomitantly with (V)LDL particles, leading to a loss of coagulation proteins from the blood. NMRC (Natl Medical Research Council, S’pore) Published version

Background— Recent studies found an immune regulatory role for Y chromosome and a relationship between loss of Y chromosome (LOY) in blood cells and a higher risk of cancer and mortality. Given involvement of immune cells in atherosclerosis, we hypothesized that LOY is associated with the severity of atherosclerotic plaque characteristics and outcome in men undergoing carotid endarterectomy. Methods and Results— LOY was quantified in blood and plaque from raw intensity genotyping data in men within the Athero-Express biobank study. Plaques were dissected, and the culprit lesions used for histology and the measurement of inflammatory proteins. We tested LOY for association with (inflammatory) atherosclerotic plaque phenotypes and cytokines and assessed the association of LOY with secondary events during 3-year follow-up. Of 366 patients with carotid endarterectomy, 61 exhibited some degree of LOY in blood. LOY was also present in atherosclerotic plaque lesions (n=8/242, 3%). LOY in blood was negatively associated with age (β=−0.03/10 y; r 2 =0.07; P =1.6×10 –7 ) but not with cardiovascular disease severity at baseline. LOY in blood was associated with a larger atheroma size (odds ratio, 2.15; 95% confidence interval, 1.06–4.76; P =0.04); however, this association was not significant after correction for multiple testing. LOY was independently associated with secondary major cardiovascular events (hazard ratio=2.28; 95% confidence interval, 1.11–4.67; P =0.02) in blood when corrected for confounders. Conclusions— In this hypothesis-generating study, LOY in blood is independently associated with secondary major cardiovascular events in a severely atherosclerotic population. Our data could indicate that LOY affects secondary outcome via other mechanisms than inflammation in the atherosclerotic plaque.

Toll-like receptors (TLR) provide a critical link between innate and adaptive immunity, both important players in atherosclerosis. Since evidence for the role of TLR5 is lacking, we aimed to establish this in the immune axis of atherosclerosis. We assessed the effect of the TLR5-specific ligand Flagellin on macrophage maturation and T-cell polarisation. Next, we generated TLR5−/−LDLr−/− chimeras to study the effect of hematopoietic TLR5 deficiency on atherosclerosis formation. Flagellin stimulation did not influence wildtype or TLR5−/− macrophage maturation. Only in wildtype macrophages, Flagellin exposure increased MCP-1 and IL6 expression. Flagellin alone reduced T-helper 1 proliferation, which was completely overruled in the presence of T-cell receptor activation. In vivo, hematopoietic TLR5 deficiency attenuated atherosclerotic lesion formation by ≈25% (1030*103 ± 63*103 vs. 792*103 ± 61*103 μm2; p = 0.013) and decreased macrophage area (81.3 ± 12.0 vs. 44.2 ± 6.6 μm2; p = 0.011). In TLR5−/− chimeric mice, we observed lower IL6 plasma levels (36.4 ± 5.6 vs. 15.1 ± 2.2 pg/mL; p = 0.003), lower (activated) splenic CD4+ T-cell content (32.3 ± 2.1 vs. 21.0 ± 1.2%; p = 0.0018), accompanied by impaired T-cell proliferative responses. In conclusion, hematopoietic TLR5 deficiency inhibits atherosclerotic lesion formation by attenuated macrophage accumulation and defective T-cell responsiveness.

Objective Evidence is emerging that abdominal aortic aneurysm (AAA) formation cannot completely be explained by systemic atherosclerosis and is in part due to other pathophysiological mechanisms such as local immune reactions. The aim of the present study was to study variance in AAA wall inflammation, and relate that to clinical patient characteristics. Methods Ventral walls from 201 patients with intact AAAs undergoing open repair were prospectively collected and processed for histology and protein measurements. Patients were monitored for 3 years postoperatively. Results The amount of lymphocytic infiltrate was used to distinguish 96 lymphocyte-poor AAAs from 105 lymphocyte-rich AAAs. The walls of lymphocyte-rich AAAs had higher concentrations of various inflammatory markers, including interleukin (IL) 6, IL8, matrix metalloproteinase (MMP) 8; however, MMP9 levels were comparable. Patients with lymphocyte-poor AAAs had more atherosclerotic risk factors: type 2 diabetes (22% vs. 9%, P = .008), hypertension (81% vs 66%, P = .019), and serum cholesterol levels (mean[SD] 5.2[2.5] vs. 4.2[1.0] mmol/L, P = .023). Intimal lesions in the AAAs revealed more frequently an extracellular lipid pool in lymphocyte-poor AAAs (66% vs. 52%, P = .026). Lymphocyte poor AAAs were associated with a worse survival during 3 years of follow-up, although this association did not reach statistical significance when correcting for other cardiovascular predictors (24% vs. 14%; HR 1.9–2.3). Conclusion Low amount of inflammation in AAAs is associated with more atherosclerotic risk factors, more advanced local atherosclerotic lesions and more postoperative atherosclerotic adverse events. This observation supports the view that AAA development is a multi-factorial process in which part of the patient population has a closer relation with systemic atherosclerotic disease, while in other patients local inflammatory reactions might play a larger role.

BACKGROUND: SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma extracellular vesicle (EV) -levels of these proteins have been reported to be associated with systemic vascular events. Evidence is accumulating that inflammatory processes may play a pivotal role both in systemic vascular events and in heart failure. Therefore, we studied the association between plasma extracellular vesicle SerpinF2-, SerpinG1-, CystatinC and CD14-levels and the occurrence of acute heart failure in patients. METHODS AND RESULT: Extracellular vesicle protein levels of SerpinG1, SerpinF2, CystatinC and CD14 were measured in an observational study of 404 subjects presenting with dysponea at the emergency department (4B-cohort). Plasma extracellular vesicles were precipitated in a total extracellular vesicles (TEX)-fraction and in separate LDL- and HDL-subfractions. Extracellular vesicle protein levels were measured with a quantitative immune assay in all 3 precipitates. Out of 404 subjects, 141 (35%) were diagnosed with acutely decompensated heart failure. After correction for confounders (including comorbidities and medications), levels of CD14 in the HDL-fraction (OR 1.53, p = 0.01), SerpinF2 in the TEX-and LDL-fraction (ORs respectively 0.71 and 0.65, p

The innate immune response elicited by activation of TLRs (Toll-like receptors) plays an important role in the pathogenesis of atherosclerosis. We hypothesized that cardiovascular risk factors are associated with the activation status of the innate immune system. We therefore assessed the responsiveness of TLRs on circulating cells in two groups of patients with established atherosclerosis and related this to the presence of cardiovascular risk factors. TNF (tumour necrosis factor)-α release induced by TLR2 and TLR4 activation was measured in patients with established coronary [PCI (percutaneous coronary intervention) study, n=78] or carotid artery disease [CEA (carotid endarterectomy) study, n=104], by stimulating whole blood samples with lipopolysaccharide (TLR4 ligand) and Pam3CSK4 [tripalmitoylcysteinylseryl-(lysyl)4; TLR2 ligand]. As an early activation marker, CD11b expression was measured by flow cytometry on CD14+ cells. Obesity was the ‘only’ risk factor that correlated with the TLR response. In both studies, obese patients had significantly higher TNF-α levels after stimulation of TLR2 compared with non-obese patients [16.9 (7.7–49.4) compared with 7.5 (1.5–19.2) pg/ml (P=0.008) in coronary artery disease and 14.6 (8.1–28.4) compared with 9.5 (6.1–15.7) pg/ml (P=0.015) in carotid artery disease; values are medians (interquartile range)]. Similar results were obtained following TLR4 stimulation. The enhanced inflammatory state in obese patients was also confirmed by a significant increased expression of the activation marker CD11b on circulating monocytes. In conclusion, obesity is associated with an enhanced TLR response in patients suffering from established atherosclerotic disease.

Aims There is an increasing need for translational studies identifying molecular targets contributing to atherosclerotic plaque destabilization. Local molecular plaque markers that are related to plaque vulnerability may hold predictive value to identify patients who are at increased risk to suffer from cardiovascular events. Animal studies revealed that adipocyte fatty acid binding protein (FABP4) is associated with the progression of atherosclerosis; however, FABP4 expression studies in human atherosclerotic plaques are lacking. We investigated FABP4 expression in carotid atherosclerotic lesions in relation to plaque composition and future cardiovascular events. Methods and results Atherosclerotic plaques were obtained from 561 patients undergoing carotid endarterectomy (CEA). Plaques were analysed for the presence of macrophages, lipid core, smooth-muscle cells, collagen, calcification, and intraplaque haemorrhage. Patients were followed for 3 years after CEA. The primary outcome was defined as the composite of vascular death, vascular event, and surgical or percutaneous vascular intervention. Fatty acid binding protein levels correlated with unstable plaque characteristics and symptomatic lesions. Patients with increased FABP4 plaque levels showed a two-fold increased risk \[HR = 1.99, 95% confidence interval (95% CI) (1.30–3.04)\] ( P = 0.005) to reach the primary outcome during follow-up. Increased FABP4 levels related to primary outcome, independent from general cardiovascular risk factors \[HR = 1.33, 95% CI (1.08–1.65)\] ( P = 0.008). Conclusion FABP4 levels in atherosclerotic lesions are associated with an unstable plaque phenotype and an increased risk for cardiovascular events during follow-up. Besides risk stratification for adverse future cardiovascular events, the outcome of the present study supports the relevance of exploring FABP4 antagonists as a potential pharmaceutical intervention to treat atherosclerotic disease progression.

WOS: 000274658700038, PubMed ID: 20018935, Objective-Atherosclerotic cardiovascular disease is a major burden to health care. Because atherosclerosis is considered a systemic disease, we hypothesized that one single atherosclerotic plaque contains ample molecular information that predicts future cardiovascular events in all vascular territories. Methods and Results-AtheroExpress is a biobank collecting atherosclerotic lesions during surgery, with a 3-year follow-up. The composite primary outcome encompasses all cardiovascular events and interventions, eg, cardiovascular death, myocardial infarction, stroke, and endovascular interventions. A proteomics search identified osteopontin as a potential plaque biomarker. Patients undergoing carotid surgery (n=574) served as the cohort in which plaque osteopontin levels were examined in relation to their outcome during follow-up and was validated in a cohort of patients undergoing femoral endarterectomy (n=151). Comparing the highest quartile of carotid plaque osteopontin levels with quartile 1 showed a hazard ratio for the primary outcome of 3.8 (95% confidence interval, 2.6-5.9). The outcome did not change after adjustment for plaque characteristics and traditional risk factors (hazard ratio, 3.5; 95% confidence interval, 2.0-5.9). The femoral validation cohort showed a hazard ratio of 3.8 (95% confidence interval 2.0 to 7.4) comparing osteopontin levels in quartile 4 with quartile 1. Conclusion-Plaque osteopontin levels in single lesions are predictive for cardiovascular events in other vascular territories. Local atherosclerotic plaques are a source of prognostic biomarkers with a high predictive value for secondary manifestations of atherosclerotic disease. (Arterioscler Thromb Vasc Biol. 2010;30:612-619.), EUEuropean Union (EU) [FP6-2004-Mobility-6 021773, FP6-037400 Immunath-2006]; Ministry of Education of SingaporeMinistry of Education, Singapore [ARC: T206B3211]; UMCU strategic Investment Prevention, EU FP6-2004-Mobility-6 # 021773 (D.d.K.), Ministry of Education of Singapore (ARC: T206B3211 to S. K. S.), EU FP6-037400 Immunath-2006 (to D.d.K. and G. P.), and the UMCU strategic Investment Prevention (to G. P.).

Background: Toll like receptors (TLR) have been recognized for their role in atherosclerotic lesion development and progression. Endogenous TLR ligands that are also expressed in atherosclerotic tissues have been shown to promote atherosclerosis in mice. Since repetitive stimulation of TLR induces an attenuated inflammatory response, we hypothesized that the TLR response is altered during atherosclerosis development, due to chronic exposure to endogenous ligands. Methods and Results: We examined five groups of both ApoE-/- and C57B1/6 mice aged 5, 10, 15, 25 and 40 weeks. In ApoE-/- mice with advanced stages of atherosclerosis, levels of mRNA encoding TLR2 and TLR4, the endogenous TLR ligands EDA and hsp60 as well as intracellular TLR-regulating mediators, like IRAK-M, were increased. Systemic TLR cell surface expression on circulating monocytes and EDA plasma levels were significantly increased in ApoE-/- mice with advanced atherosclerosis. We also observed that the endogenous TLR ligand EDA was capable of activating the TLR-signaling pathway in white blood cells. During the plaque progression stage however, stimulation of TLR2 and TLR4 in blood samples attenuated MIP-1 alpha and RANTES release in atherosclerotic mice. Conclusion: During atherosclerotic lesion development, TLR2 and TLR4 expression increases in atherosclerotic plaques and on circulating blood cells. However, with advanced stages of atherosclerotic disease, circulating blood cells become less responsive to TLR ligation, which may be due to chronic TLR engagement by endogenous EDA. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Background The eicosanoid genes ALOX5 , ALOX5AP and LTA4H have been implicated in atherosclerosis. We assessed the impact of common variants in these genes on gene expression, circulating protein levels, and atherosclerotic plaque phenotypes. Methods We included patients from the Stockholm Atherosclerosis Gene Expression study (STAGE, N = 109), and the Athero-Express Biobank Study (AE, N = 1443). We tested 1453 single-nucleotide variants (SNVs) in ALOX5 , ALOX5AP and LTA4H for association with gene expression in STAGE. We also tested these SNVs for association with seven histologically defined plaque phenotypes in the AE (which included calcification, collagen, cellular content, atheroma size, and intraplaque vessel density and hemorrhage). Results We replicate a known cis -eQTL (rs6538697, p = 1.96 × 10 −6 ) for LTA4H expression in whole blood of patients from STAGE. We found no significant association for any of the SNVs tested with serum levels of ALOX5 or ALOX5AP (p > 5.79 × 10 −4 ). For atherosclerotic plaque phenotypes the strongest associations were found for intraplaque vessel density and smooth muscle cells in the ALOX5AP locus (p > 1.67 × 10 −4 ). Conclusions We replicate a known eQTL for LTA4H expression in whole blood using STAGE data. We found no associations of variants in and around ALOX5 , ALOX5AP and LTA4H with serum ALOX5 or ALOX5AP levels, or plaque phenotypes. On the supposition that these genes play a causal role in atherosclerosis, these results suggest that common variants in these loci play a limited role (if any) in influencing advanced atherosclerotic plaque morphology to the extent that it impacts atherosclerotic disease.

Background and Purpose— We studied matrix metalloproteinases (MMP) 2, 8, and 9 and extracellular matrix metalloproteinase inducer (EMMPRIN) levels in relation to carotid atherosclerotic plaque characteristics. Methods— Carotid atherosclerotic plaques (n=150) were stained and analyzed for the presence of collagen, smooth muscle cell (SMC), and macrophages. Adjacent segments were used to isolate total protein to assess MMP-2 and MMP-9 activities and gelatin breakdown, MMP-8 activity, and EMMPRIN levels. Results— Macrophage-rich lesions revealed higher MMP-8 and MMP-9 activities, whereas SMC-rich lesions showed higher MMP-2 activity. The levels of less glycosylated EMMPRIN-45kD were higher in SMC-rich lesions and lower in macrophage-rich plaques. EMMPRIN-45kD was associated with MMP-2 levels, whereas EMMPRIN-58kD was related to MMP-9 levels. Conclusions— MMP-2, MMP-8, and MMP-9 activities differed among carotid plaque phenotypes. Different EMMPRIN glycosylation forms are associated with either MMP-2 or MMP-9 activity, which suggests that EMMPRIN glycosylation may play a role in MMP regulation and plaque destabilization.

Objective: Atherosclerosis may lead to acute clinical events by rupture of a vulnerable atherosclerotic plaque. Previously, we demonstrated that peptidoglycan (PGN), a major cell wall component of gram-positive bacteria that induces production of proinflammatory cytokines through TLR2 and CD14, is prevalent in atherosclerotic lesions with histological features associated with plaque vulnerability. We hypothesized that in atherosclerotic patients antibody levels against PGN may differ compared with matched controls. Methods and results: ELISA was performed to measure immunoglobulin levels against PGN in sera of 80 atherosclerotic patients versus 77 control patients with an increased cardiovascular risk, frequency-matched for age, sex and risk factors for atherosclerotic disease. In all patients and controls, intima-media (IMT) thickness was assessed using an array transducer. Significantly lower levels of IgM directed against PGN were found in atherosclerotic patients compared with the control patients without clinically manifested disease (P=0.02). The IgM levels against PGN decreased with increasing mean common carotid IMT thickness (P=0.006). Conclusions: These results show that patients suffering from atherosclerotic disease have decreased IgM levels against PGN. The data suggest that an antibody response against PGN could have a protective effect against the development or activity of atherosclerotic disease.

Background: Atherosclerotic luminal narrowing is caused by plaque growth and arterial remodeling. In peripheral arteries, a role for constrictive remodeling in luminal narrowing has been recognized, but the impact on lumen decrease has not yet been assessed. We studied to what extent arterial remodeling and plaque formation contribute to luminal narrowing in the superficial femoral artery. Methods: Elderly subjects (n = 79) were studied. Post mortem, pressure-fixed femoral arteries (n = 125) were dissected and divided into 0.5-cm segments (n = 3266). In each cross section, we measured lumen area, plaque area, and the area encompassed by the internal elastic lamina (IEL area). For each artery, the cross section with the least amount of plaque was considered the reference segment. In cross sections with a decrease in lumen area compared with the reference, we determined the contributions of both plaque increase and IEL area change. Results: A decrease in lumen area was found in 2193 cross sections. In cross sections with >50% lumen stenosis, plaque increase (accompanied by IEL area increase) fully explained lumen decrease in 80 of 280 cross sections (29%). In the remaining 200 of 280 cross sections (71%), both plaque increase and IEL area decrease contributed to lumen stenosis. In 57 of 280 cross sections (20%), IEL area decrease was the major determinant of lumen decrease, dominating over plaque increase. In 143 of 280 cross sections (51%), plaque increase was the major determinant, dominating over IEL area decrease. Conclusion: The results of this post mortem study suggest that in a substantial part (20%) of severely stenotic lesions in the femoral artery, constrictive remodeling, not plaque size, is the major determinant of lumen decrease. Further serial studies are needed to confirm these results. (J Vasc Surg 2002;36:1194-7.)

Background—Inflammation plays an important role in atherogenesis. The toll-like receptor 4 (TLR4) is the receptor for bacterial lipopolysaccharides and also recognizes cellular fibronectin and heat shock protein 60, endogenous peptides that are produced in response to tissue injury. To explore a possible role for this receptor in arterial obstructive disease, we determined the expression of TLR4 in the atherosclerotic arterial wall, including adventitia, and studied the effect of adventitial TLR4 activation on neointima formation in a mouse model.Methods and Results—Localization of TLR4 was studied in human atherosclerotic coronary arteries by immunohistochemistry and detected in plaque and adventitia. In the adventitia, not all TLR4-positive cells colocalized with macrophages. In primary human adventitial fibroblasts, expression of TLR4 was demonstrated by immunofluorescence, Western blot, and reverse transcriptase-polymerase chain reaction. Adding lipopolysaccharide to these fibroblasts induced activation of NF-κB and an increase of mRNAs of various cytokines. The effect of adventitial stimulation of TLR4 was studied in a mouse model. A peri-adventitial cuff was placed around the femoral artery. Application of lipopolysaccharide between cuff and artery augmented neointima formation induced by the cuff (intimal area±SEM, 9134±1714 versus 2353±1076 μm2,P2,P=0.02 versus wild type).Conclusions—A functional TLR4 is expressed in human adventitial fibroblasts and macrophages. Adventitial TLR4 activation augmented neointima formation in a mouse model. These results provide evidence for a link between the immune receptor TLR4 and intimal lesion formation.

Peptidoglycan (PG) is a major component of the cell wall of gram-positive bacteria that is abundantly present in all human mucosa. PG is a functional lipopolysaccharide analog that binds to CD14 on macrophages and induces proinflammatory cytokine production and metalloproteinases. We investigated the hypothesis that bacterial PG is present in atherosclerotic tissue. In addition, plaque phenotypes were characterized in relation to presence of PG. Immunohistology of carotid (n = 15) and femoral (n = 6) endarterectomy specimens revealed the presence of PG in the cytoplasm of cells located in plaques. PG was detected in 14 of 15 carotid arteries and 5 of 6 femoral arteries. From the 14 coronary arteries, 31 atherosclerotic segments were selected. PG was detected within 19 of 31 of these coronary segments. Western blot demonstrated the presence of the toll-like receptor (TLR-2), the co-receptor for PG, in coronary artery tissue. The number of PG-containing cells in coronary arteries was significantly higher when the histologic features of plaque vulnerability were evident. Inflammation of the cap or shoulder was observed in 11 of 19 PG-positive versus 2 of 12 PG-negative segments (p = 0.023). More than 50% of the plaque area consisted of atheroma in 7 of 19 PG-positive segments and 0 of 12 PG-negative segments (p = 0.025). Heavy smooth muscle cell staining occurred in the plaque cap and shoulder in 3 of 19 PG-positive segments versus 9 of 12 PG-negative segments. Proinflammatory bacterial PG and its co-receptor have been observed in atherosclerotic arteries, in association with the vulnerable plaque phenotype.

Background Atherosclerosis is a process that begins in childhood, develops over decades and underlies the majority of cardiovascular events in adulthood. Previously, we demonstrated in adults with cardiovascular disease that levels of extracellular vesicle (EV) proteins CD14, Serpin F2 and cystatin C predict vascular outcome. Here, we study for the first time whether these EV proteins are related to vascular characteristics in healthy, young children. Methods and results In 141 eight-year old children of the Wheezing-Illnesses-Studie-LEidsche-Rijn birth cohort, anthropometrics and blood pressure were measured. In addition, common carotid intima-media thickness, carotid distensibility and carotid Young's elastic modulus were obtained non-invasively using ultrasound imaging. A fasting lipid spectrum was obtained and EVs were isolated from plasma. Levels of EV proteins CD14, Serpin F2 and cystatin C were measured using a multiplex assay. In a multivariable linear regression model we assessed the relation between these EV proteins and the selected vascular characteristics. Of the studied EV proteins, CD14 levels were positively related to common carotid intima-media thickness (log transformed, beta = 7.31 ln(mm)/(ng/mg) (1.24, 13.38), p = 0.02). EV proteins Serpin F2 and cystatin C were not related to common carotid intima-media thickness. In addition, we found no relation between all three EV proteins and carotid distensibility or carotid Young's elastic modulus. Conclusion In healthy eight-year old children, extracellular vesicle protein CD14 levels seem positively related to common carotid intima-media thickness. This would point towards inflammatory vascular alterations inflicted by extracellular vesicle protein CD14 already in early life and warrants further investigation.

The presence of Chlamydia pneumoniae in the human arterial system has mainly been determined in atherosclerotic plaque, whereas the adventitia has remained relatively unexplored. We assessed the presence of C. pneumoniae in all three vessel wall layers of coronary (n=72) and brachial (n=48) arteries in relation to local atherosclerosis. Immunohistochemical staining of C. pneumoniae was observed in plaque and adventitia. Cells stained for C. pneumoniae were detected in the same areas as cells stained for macrophages in adjacent sections. C. pneumoniae staining in the adventitia was associated with the extent and severity of atherosclerosis. Coronary sections with C. pneumoniae staining in both adventitia and plaque more often contained advanced atherosclerosis than sections with staining only in the adventitia. Staining was observed more often in the coronary artery than in the brachial artery (24/72 vs. 5/48 and 51/72 vs. 8/48 for plaque and adventitia, respectively, P=0.004 and P

Intravascular ultrasound elastography is a method for measuring the local elastic properties using intravascular ultrasound (IVUS). The elastic properties of the different tissues within the atherosclerotic plaque are measured through the strain. Knowledge of these elastic properties is useful for guiding interventional procedures (balloon dilatation, ablation) and detection of the vulnerable plaque. In the last decade, several groups have applied elastography intravascularly with various levels of success. In this paper, the approaches of the different research groups will be discussed. The focus will be on our approach to the application of intravascular elastography. Elastograms were acquired in vitro and in vivo using the relative local displacements between IVUS images acquired at two levels of intravascular pressure with a 30 MHz mechanical or a 20 MHz array echo catheter. These displacements were estimated from the time shift between gated radiofrequency echo signals using cross-correlation algorithms with interpolation around the peak. Experiments on gel-based phantoms mimicking atherosclerotic vessels demonstrated the capability of elastography to identify soft and hard tissues independently of the echogenicity contrast. In vitro experiments on human arteries have demonstrated the potential of intravascular elastography to identify different plaque types based on their mechanical properties. These plaques could not be identified using the IVUS image alone. In vivo experiments revealed that reproducible elastograms could be obtained near end-diastole. Partial validation using the echogram was performed. Intravascular elastography provides information that is frequently unavailable or inconclusive from the IVUS image and which may therefore assist in the diagnosis and treatment of atherosclerotic disease.

Atherosclerotic luminal narrowing is determined by plaque mass and the mode of geometrical remodeling. Recently, we reported that the type of atherosclerotic remodeling is associated with the presence of histological markers for plaque vulnerability. Inflammation and matrix degrading proteases (MMPs) may play a role in both plaque vulnerability and in expansive arterial remodeling. The aim of the present study was to investigate the association between the remodeling mode and the localization of macrophages and MMPs in coronary atherosclerotic segments. From 36 atherosclerotic coronary arteries, 45 and 51 segments were selected with a vessel area that was >10% smaller and larger compared with the adjacent segments, respectively. No significant difference in staining for macrophages was observed between segments with expansive and constrictive remodeling. More MMP-2 and MMP-9 staining was observed in plaques of expansively remodeled segments compared with constrictively remodeled segments. In general, MMP-staining was less evident in the adventitial layer compared with the plaque. Zymography revealed more active MMP-2 in expansively remodeled segments compared with constrictively remodeled segments (340+/-319 vs. 199+/-181 (adjusted counts/mm(2)), respectively, P=0.019). Zymography did not show differences in inactive MMP-2 or MMP-9 among groups. It might be postulated that MMPs within the plaque play a causal role not only in plaque vulnerability but also in de novo atherosclerotic remodeling.

Abstract —Retrospectively, plaque rupture is often colocalized with inflammation of the cap and shoulder of the atherosclerotic plaque. Local inflammation is therefore considered a potential marker for plaque vulnerability. However, high specificity of inflammation for plaque rupture is a requisite for application of inflammation markers to detect rupture-prone lesions. The objective of the present study was to investigate the prevalence and distribution (local versus general) of inflammatory cells in nonruptured atherosclerotic plaques. The cap and shoulder of the plaque were stained for the presence of macrophages and T lymphocytes in 282 and 262 cross sections obtained from 74 coronary and 50 femoral arteries, respectively. From most cases, 2 atherosclerotic arteries were studied to gain insight into the local and systemic distribution of the inflammatory process. In 45% and 41% of all cross sections, staining for macrophages was observed in the femoral and coronary arteries, respectively. Rupture of the fibrous cap was observed in 2 femoral and 3 coronary artery segments and was always colocalized with inflammatory cells. At least 1 cross section stained positively for CD68 or acid phosphatase in 84% and 71% of all femoral and coronary arteries, respectively. Only 1 femoral and 6 coronary arteries revealed a positive stain for CD68 in all investigated segments. Inflammation of the cap and shoulder of the plaque is a common feature, locally observed, in atherosclerotic femoral and coronary arteries. The high prevalence of local inflammatory responses should be considered if they are used as a diagnostic target to detect vulnerable, rupture-prone lesions.

Objective. To relate local arterial geometry with markers that are thought to be related to plaque rupture. Background. Plaque rupture often occurs at sites with minor luminal stenosis and has retrospectively been characterized by colocalization of inflammatory cells. Recent studies have demonstrated that luminal narrowing is related with the mode of atherosclerotic arterial remodeling. Methods. We obtained 1,521 cross section slices at regular intervals from 50 atherosclerotic femoral arteries. Per artery, the slices with the largest and smallest lumen area, vessel area and plaque area were selected for staining on the presence of macrophages (CD68), T-lymphocytes (CD45RO), smooth muscle cells (alpha-actin) and collagen. Results. Inflammation of the cap or shoulder of the plaque was observed in 33% of all cross sections. Significantly more CD68 and CD45RO positive cells, more atheroma, less collagen and less alpha-actin positive staining was observed in cross sections with the largest plaque area and largest vessel area vs. cross sections with the smallest plaque area and smallest vessel area, respectively. No difference in the number of inflammatory cells was observed between cross sections with the largest and smallest lumen area. Conclusion. Intraindividually, pathohistologic markers previously reported to be related to plaque vulnerability were associated with a larger plaque area and vessel area. In addition, inflammation of the cap and shoulder of the plaque was a common finding in the atherosclerotic femoral artery.

Abstract Luminal stenosis can be based on large atherosclerotic plaques in compensatory enlarged segments or on relatively little plaques in shrunken segments. In the present study, the contribution of plaque formation and remodeling to luminal narrowing was compared among six types of arteries prone to symptomatic atherosclerosis. Cross-sections (n=5195) were obtained at regular intervals from 329 arteries. For each artery, the cross-section that contained the least amount of plaque was considered to be the reference. For each cross-section, the percentage of lumen area decrease was expressed as a percentage of the lumen area at the reference site (luminal stenosis). Similarly, the area encompassed by the internal elastic lamina (IEL area) was expressed as a percentage of the IEL area at the reference site (relative IEL area). All cross-sections were categorized in three groups: relative IEL area >105% (enlargement), 95% to 105% (no remodeling), and P

The abundant presence of macrophages and activated T cells in atherosclerotic plaques suggests an active involvement of the immune system in this cardiovascular disease. However, insight into cellular interactions inducing relevant effector functions is still limited. Here, Jon Laman and colleagues propose that local interactions between CD40 and CD40 ligand may induce multiple activities by different cell types contributing to atherosclerosis. Chemicals/CAS: Antigens, CD40; CD40 Ligand, 147205-72-9; Ligands; Membrane Glycoproteins

Objective: Atherosclerosis is associated with increased levels of plasma cytokines and expression of Toll-like receptors (TLRs). Yet, little is known about the potential use of TLR ligand induced cytokine release as a biomarker of coronary artery disease (CAD). In this study, we investigated whether TLR ligand induced cytokine release is associated with atherosclerotic disease severity and its predictive value for future cardiovascular events. Methods: Blood samples were obtained from 260 patients with stable angina and 15 healthy controls. Cytokine levels of TNFα, IL-8 and IL-6 were measured after 2h of whole blood stimulation with 10ng/ml lipopolysaccharide (LPS, TLR4 ligand) and P3C 500ng/ml (TLR2 ligand). In a subgroup, dose-response curves were created using additional LPS concentrations. Results: LPS induced whole blood release of TNFα and IL-6, but not IL-8, was significantly higher in patients compared to healthy controls. Among CAD patients, TLR responses did hardly differ when associated with the presence of traditional risk factors and atherosclerotic disease severity (number of diseased vessels and coronary stenosis degree). Patients with secondary events during follow-up showed a trend towards an increased TLR response. Furthermore, positive associations were found between CRP levels and TLR-induced TNFα (CRP2: 2364pg/ml) and IL-6 production (CRP2: 2250pg/ml). Conclusion: In conclusion, TLR-induced whole blood cytokine release in patients with stable angina indicates the presence of coronary atherosclerosis but does not reflect its severity.

BackgroundLeukotriene B4 (LTB4) has been associated with the initiation and progression of atherosclerosis and abdominal aortic aneurysm (AAA) formation. However, associations of LTB4 levels with tissue characteristics and adverse clinical outcome of advanced atherosclerosis and AAA are scarcely studied. We hypothesized that LTB4 levels are associated with a vulnerable plaque phenotype and adverse clinical outcome. Furthermore, that LTB4 levels are associated with inflammatory AAA and adverse clinical outcome.MethodsAtherosclerotic plaques and AAA specimens were selected from two independent databases for LTB4 measurements. Plaques were isolated during carotid endarterectomy from asymptomatic (n = 58) or symptomatic (n = 317) patients, classified prior to surgery. LTB4 levels were measured without prior lipid extraction and levels were corrected for protein content. LTB4 levels were related to plaque phenotype, baseline patient characteristics and clinical outcome within three years following surgery. Seven non-diseased mammary artery specimens served as controls. AAA specimens were isolated during open repair, classified as elective (n = 189), symptomatic (n = 29) or ruptured (n = 23). LTB4 levels were measured similar to the plaque measurements and were related to tissue characteristics, baseline patient characteristics and clinical outcome. Twenty-six non-diseased aortic specimens served as controls.ResultsLTB4 levels corrected for protein content were not significantly associated with histological characteristics specific for vulnerable plaques or inflammatory AAA as well as clinical presentation. Moreover, it could not predict secondary manifestations independently investigated in both databases. However, LTB4 levels were significantly lower in controls compared to plaque (p = 0.025) or AAA (p = 0.017).ConclusionsLTB4 levels were not associated with a vulnerable plaque phenotype or inflammatory AAA or clinical presentation. This study does not provide supportive evidence for a role of LTB4 in atherosclerotic plaque destabilization or AAA expansion. However, these data should be interpreted with care, since LTB4 measurements were performed without prior lipid extractions.

Background and Objectives Microvesicles (MVs) are small membrane vesicles that are involved in atherotrombotic processes. In the present study, we evaluated the risk of MV protein levels on the occurrence of new vascular events in patients with clinically manifest vascular disease. Methods In this cohort study 1060 patients were prospectively followed for the occurrence of a new vascular event or death (median follow up 6.4years, interquartile range 5.2–7.3years). MVs were isolated from plasma and MV protein levels of Cystatin C, Serpin G1, Serpin F2 and CD14 were measured. Multivariable Cox proportional hazards models were used to estimate the risk for new vascular events, vascular mortality and all-cause mortality. During follow up 136 vascular events occurred, 65 vascular mortality and 114 all-cause mortality. Results An increase in 1 standard deviation (SD) of Cystatin C MV level was related to an increased risk for myocardial infarction (HR 1.49; 95%CI 1.20–1.86), vascular mortality (HR 1.48; 95%CI 1.17–1.86), vascular events (HR 1.27; 1.07–1.52) and all-cause mortality (HR 1.41; 95%CI 1.18–1.69). Serpin F2 MV levels were related to an increased risk for myocardial infarction (HR 1.22; 95%CI 1.00–1.51), vascular mortality (HR 1.25; 95%CI 1.00–1.56), and all-cause mortality (HR 1.22; 95% CI 1.03–1.45). CD14 MV levels were related to an increased risk for myocardial infarction (HR 1.55; 95%CI 1.27–1.91), vascular mortality (HR 1.37; 95%CI 1.10–1.70), vascular events (HR 1.32; 95%CI 1.12–1.55), all-cause mortality (HR 1.36; 95%CI 1.15–1.62) and occurrence of ischemic stroke (HR 1.32; 95%CI 1.00–1.74). Conclusions Cystatin C, Serpin F2 and CD14 MV levels are related to an elevated risk for future vascular events and mortality in patients with clinically manifest vascular disease.

Aims: Biomarkers are essential in the early detection of acute coronary syndromes (ACS). Serum extracellular vesicles are small vesicles in the plasma containing protein and RNA and have been shown to be involved in ACS-related processes like apoptosis and coagulation. Therefore, we hypothesized that serum extracellular vesicle protein levels are associated with ACS. Methods and results: Three serum extracellular vesicle proteins potentially associated with ACS were identified with differential Q-proteomics and were evaluated in 471 frozen serum samples of ACS-suspected patients presenting to the emergency department (30% of whom had an ACS). Protein levels were measured after vesicle isolation using ExoQuick. Mean serum extracellular vesicle concentration of the different proteins was compared between ACS and non-ACS patients. Selected proteins were tested in a univariate logistic regression model, as well as in a multivariate model to adjust for cardiovascular risk factors. A separate analysis was performed in men and women. In the multivariate logistic regression analysis, polygenic immunoglobulin receptor, (pIgR; OR 1.630, p=0.026), cystatin C (OR 1.641, p=0.021), and complement factor C5a (C5a, OR 1.495, p=0.025) were significantly associated with ACS, while total vesicle protein concentration was borderline significant. The association of the individual proteins with ACS was markedly stronger in men. Conclusions: These data show that serum extracellular vesicle pIgR, cystatin C, and C5a concentrations are independently associated with ACS and that there are pronounced gender differences. These observations should be validated in a large, prospective study to assess the potential role of vesicle content in the evaluation of patients suspected of having an ACS.

Objective Angiogenesis and inflammation are important features in atherosclerotic plaque destabilization. The transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) is a key regulator of angiogenesis and is also involved in inflammatory reactions. We studied HIF-1α expression in different atherosclerotic plaque phenotypes. Methods and results HIF-1α expression was observed in 18/37 (49%) carotid and in 9/15 (60%) femoral endarterectomy specimens. Expression of HIF-1α was associated with the presence of a large extracellular lipid core (P = 0.03) and macrophages (P = 0.02). HIF-1α co-localized with vascular endothelial growth factor (VEGF), an important downstream target of HIF-1α. In addition, a strong association was observed between expression levels of HIF-1α and VEGF (P = 0.001). The average number of plaque microvessels was higher in plaques with no or minor HIF-1α staining than in plaques with moderate or heavy HIF-1α staining (P = 0.03). In human macrophages, lipopolysaccharide activation induced HIF-1α expression. In embryonic fibroblasts derived from wild-type mice, lipopolysaccharide activation induced an increase in HIF-1α mRNA, whereas in Toll-like receptor 4 defective embryonic fibroblasts no effect was observed after lipopolysaccharide stimulation. Conclusions In atherosclerotic plaque, the transcription factor HIF-1α is associated with an atheromatous inflammatory plaque phenotype and with VEGF expression. HIF-1α expression is upregulated in activated macrophages under normoxic conditions.

Background and Purpose— Anti-inflammatory qualities are held partially responsible for the reduction of cardiovascular events after statin treatment. We examined the phenotype of carotid atherosclerotic plaques harvested during carotid endarterectomy in relation to the previous use of different statins prescribed in clinical practice. Methods— Three hundred and seventy-eight patients were included. Atherosclerotic plaques were harvested, immunohistochemically stained and semiquantitively examined for the presence of macrophages (CD68), smooth muscle cells, collagen and fat. Adjacent atherosclerotic plaques were used to study protease activity and interleukin levels. Patients’ demographics were recorded and blood samples were stored. Results— Serum cholesterol, low-density lipoprotein, apolipoprotein B, and C-reactive protein levels were lower in patients treated with statins compared with patients without statin treatment. Atheromatous plaques were less prevalent in patients receiving statins compared with patients without statin therapy (29% versus 42%, P =0.04). An increase of CD68 positive cells was observed in patients receiving statins compared with nonstatin treatment ( P =0.05). This effect was specifically related to atorvastatin treatment. In patients treated with atorvastatin, the increased amount of CD68 positive cells were not associated with increased protease activity. In contrast, a dose-dependent decrease in protease activity was shown in the atorvastatin group. Interleukin 6 expression was lower in plaques obtained from patients treated with statins ( P =0.04). Conclusions— Statin use may exert pleiotropic effects on plaque phenotype. However, not the presence of macrophages but activation with subsequent protease and cytokine release may be attenuated by statin use.

Atherosclerotic carotid artery disease is responsible for a variety of clinical presentations, ranging from asymptomatic to cerebral ischemic events. Considering the upcoming use of noninvasive imaging modalities, plaque characteristics could serve as a marker in the selection of patients eligible for carotid endarterectomy (CEA). This would be more likely if characteristics corresponded with clinical manifestations and were predictive of future events. In this study, we hypothesized that plaque characteristics correlate with the clinical presentation of carotid artery disease.We included 404 patients undergoing a carotid endarterectomy (CEA). Ipsilateral clinical symptoms and duplex measurements were recorded. Patients could be asymptomatic (23.5%) or symptomatic with stroke (26.5%), transient ischemic attack (TIA) (36.1%), or amaurosis fugax (AFX) (13.9%). Plaques were stained and semi-quantitatively analyzed for the presence of macrophages, smooth muscle cells, collagen, calcifications, and thrombus. Plaques were categorized in three phenotypes by their overall presentation and the amount of fat. In addition, plaque matrix metalloproteinase (MMP) activity and cytokines expressions were measured.Fibrous, fibro-atheromatous, and atheromatous plaques were observed in 30.2%, 35.6%, and 34.2%, respectively. Atheromatous plaques were more prevalent in patients with stroke and TIA compared with asymptomatic patients or patients with AFX (P = .001). Collagen staining was less evident in patients with TIA and stroke compared with asymptomatic patients or patients with AFX (P.001). Plaques of patients with TIA and stroke showed significantly higher activity levels of MMP-8 and MMP-9 and higher levels of interleukin-8 compared with asymptomatic and AFX patients.Plaque phenotype of patients with TIA is comparable to that of patients with stroke; whereas, the plaque phenotype of patients with AFX resembles the plaque phenotype of asymptomatic patients. Follow-up studies should be encouraged to determine whether plaque characteristics visualized by imaging techniques might help to identify patients most likely to benefit from CEA.

The extent of atherosclerotic plaque burden and the incidence of atherosclerosis-related cardiovascular events accelerate with increasing age. The composition of the plaque is associated with plaque thrombosis and acute coronary occlusion. Surprisingly, however, the relation between advancing age and atherosclerotic plaque composition is still unclear. In the present study, we investigated the association between plaque characteristics and advancing age in a population of patients with haemodynamically significant carotid artery stenosis.Patients (N=383), ages 39-89 years, underwent carotid endarterectomy (CEA). Morphometric analysis was performed on the dissected atherosclerotic plaques to study the prevalence of fibrous and atheromatous plaques. Picro sirius red, haematoxylin eosin, alfa actin and CD68 stainings were performed to investigate the extent of collagen, calcification, smooth muscle cells and macrophages in carotid plaques, respectively. The presence of metalloproteinases-2 and -9 was assessed by ELISA.With aging, a decrease in fibrous plaques and an increase in atheromatous plaques were observed. This was accompanied by an age-associated decrease in smooth muscle cell content in carotid plaques. Macrophage content slightly increased with age. In addition, total matrix metalloprotease (MMP)-2 was negatively and MMP-9 positively related with age. Differences in plaque phenotype were most prominent for the youngest age quartile compared with older age quartiles.With increasing age, the morphology of atherosclerotic plaques from patients with carotid artery stenosis changes. Plaques become more atheromatous and contain less smooth muscle cells with increasing age. Local inflammation and MMP-9 levels slightly increased with age in plaques obtained from patients suffering from haemodynamically significant advanced atherosclerotic lesions.

Background— Toll-like receptor 4 (Tlr4) is the receptor for exogenous lipopolysaccharides (LPS). Expression of endogenous Tlr4 ligands, heat shock protein 60 (Hsp60) and extra domain A of fibronectin, has been observed in arthritic and oncological specimens in which matrix turnover is an important feature. In atherosclerosis, outward remodeling is characterized by matrix turnover and a structural change in arterial circumference and is associated with a vulnerable plaque phenotype. Since Tlr4 ligands are expressed during matrix turnover, we hypothesized that Tlr4 is involved in arterial remodeling. Methods and Results— In a femoral artery cuff model in the atherosclerotic ApoE3 (Leiden) transgenic mouse, Tlr4 activation by LPS stimulated plaque formation and subsequent outward arterial remodeling. With the use of the same model in wild-type mice, neointima formation and outward remodeling occurred. In Tlr4-deficient mice, however, no outward arterial remodeling was observed independent of neointima formation. Carotid artery ligation in wild-type mice resulted in outward remodeling without neointima formation in the contralateral artery. This was associated with an increase in Tlr4 expression and EDA and Hsp60 mRNA levels. In contrast, outward remodeling was not observed after carotid ligation in Tlr4-deficient mice. Conclusions— These findings provide genetic evidence that Tlr4 is involved in outward arterial remodeling, probably through upregulation of Tlr4 and Tlr4 ligands.

Collagen turnover and cell migration are fundamental aspects of arterial restructuring. To identify mRNAs involved in blood flow-induced arterial restructuring, we performed subtraction polymerase chain reaction and found expression of haptoglobin mRNA in adventitial fibroblasts of rabbit arteries. Haptoglobin is highly expressed in liver, but its arterial expression and function are unknown. In vitro studies revealed that stimulation of haptoglobin expression by lipopolysaccharides in mice fibroblasts stimulated migration of wild-type fibroblasts but had no effect on migration of haptoglobin knockout fibroblasts. In vivo studies showed that flow-induced arterial restructuring was delayed in haptoglobin knockout mice. This new function of haptoglobin might be explained by facilitating cell migration through accumulation of a temporary gelatin matrix because cell culture showed that haptoglobin is involved in the breakdown of gelatin. We conclude that haptoglobin is highly expressed in arterial tissue and is involved in arterial restructuring. This new haptoglobin function may also apply to other functional and pathological restructuring processes such as angiogenesis, tissue repair, and tumor cell invasion.

The purpose of the present study was to obtain insight into the natural development of adaptive intimal thickening and atherosclerosis in the arterial tree of human species. The morphometry and composition of the intimal layer were studied in the arterial system of elderly individuals. Post mortem, a total of 703 arterial segments were dissected from 24 subjects (age 81.9 +/- 9.9 years). From each subject, segments were dissected from 31 different arteries. Area stenosis [(plaque area/vessel area) x 100%] was determined in each segment. By (immuno)histochemistry, lipid content and the presence of inflammatory cells (macrophages) were assessed in the coronary, common carotid, brachial, radial and internal iliac arteries. Adaptive intimal thickening or advanced atherosclerosis was observed in all studied artery types. Area stenosis was highest in the coronary arteries (median, 30%) and lowest in the arteries supplying the brain (median,or = 7%). Plaques hiding a lipid-rich core and plaques with macrophage infiltration were observed in all five selected artery types. In summary, the present observation demonstrates that intimal thickening is a systemic process involving most artery types. Within elderly humans, features of advanced atherosclerotic disease, a lipid-rich core and macrophages, can be observed in the intimal layer of artery types that are recognised for their relation with clinical syndroms as well as artery types that remain clinically silent.

Matrix metalloproteinase (MMP) activation is an essential feature of pathological and physiological arterial enlargement or shrinkage. Recently, furin-activated membrane type-1 MMP (MT1-MMP) was identified as the in vivo activator of MMP2 in mice. Although arterial enlargement and shrinkage are important in several pathological processes, this proprotein convertase–MT1-MMP axis has not been described during arterial remodeling. In rabbit femoral and carotid arteries, we report an increase in furin and MT1-MMP mRNA levels before and at the onset of arterial remodeling followed by an increase in activated MMP2. This reveals the presence of the proprotein convertase–MT1-MMP axis in flow-induced arterial remodeling and identifies furin as a possible target for local intervention in pathological arterial remodeling.

Background — Chlamydia pneumoniae has been suggested to play a role in the origin of atherosclerosis. We studied the prevalence of C pneumoniae at multiple locations in the arterial system within the same individual. Studying the association between atherosclerosis and C pneumoniae within the individual excludes confounding by interindividual variability. Methods and Results —Postmortem, the presence in the intima/plaque and media of C pneumoniae membrane protein was determined by use of a C pneumoniae –specific monoclonal antibody. In 24 individuals, 33 arterial locations were studied (n=738 segments). Area stenosis was determined in adjacent cross sections. In all individuals, immunostaining of C pneumoniae was observed in ≥1 artery. The highest prevalences were observed in the abdominal aorta (67%), internal and common iliac arteries (41%), and coronary arteries (33%). The lowest prevalences were observed in the radial (0%) and cerebral (2%) arteries. Within the individual, area stenosis was larger in cross sections with immunoreactivity compared with cross sections without immunoreactivity (31.0±11.9% versus 14.3±6.1%, respectively; P C pneumoniae was associated with the average area stenosis throughout the arterial system ( r 2 =0.56, P Conclusions — C pneumoniae was mostly observed at locations that are related to clinically relevant features. Within the individual, the distribution of C pneumoniae is associated with the distribution of atherosclerosis. The role of the microorganism in atherosclerotic disease remains to be elucidated.

The atherosclerotic carotid artery is easily accessible for non-invasive duplex investigation. The aim of the present post mortem study was to examine whether plaque accumulation and luminal stenosis in the common carotid artery is representative for atherosclerotic plaque accumulation and luminal stenosis in other peripheral arteries. A total of 3765 cross-sections were obtained at regular intervals from 240 arteries (24 individuals). Five types of peripheral arteries were investigated: common carotid, femoral, common iliac, external iliac and renal arteries. In each cross-section, the lumen area, vessel area, plaque area and maximal plaque thickness was measured. For each location, the percentage luminal stenosis and relative plaque area was calculated. Relative plaque area was defined as the percentage of the vessel area which was occupied by plaque. Weak correlations (r=0.41-0.59) were observed between percentage relative plaque area or maximal plaque thickness in the common carotid artery and percentage relative plaque area in other peripheral arteries. Neither plaque accumulation nor luminal stenosis in the common carotid artery correlated with the percentage luminal stenosis in other peripheral arteries (P > 0.05). We conclude that plaque area in the common carotid artery is weakly correlated with plaque area and not correlated with luminal stenosis in other peripheral arteries.

Aim: Atherosclerotic plaque characteristics may vary among individuals, in part due to heritable factors. The genetic architecture of plaque phenotypes is largely unknown. A common variant (rs17398575-A) near PIK3CG on chromosome 7 has previously been associated with carotid plaque presence. Animal models suggest that PIK3CG may play a role in plaque formation through neovascularization. We hypothesized that the PIK3CG variant is associated with intraplaque hemorrhage and vessel density in human plaque tissue. Methods: We collected 831 patients in the Athero-Express Biobank Study, all of whom underwent carotid endarterectomy and genotyped them using Affymetrix SNP 5.0. We tested PIK3CG variants for association to intraplaque hemorrhage and vessel density using logistic and linear regression model, respectively, correcting for age, gender and principal components. We used the BiKE cohort to assess the effect of PIK3CG variants on PIK3CG expression in circulating monocytes (N=95) and in carotid plaques (N=126). Results: The reported PIK3CG variant, rs17398575 (risk allele A, frequency = 0.72), was nominally significantly associated with intraplaque hemorrhage (OR=1.40 [1.10-1.69 95% CI], p=0.0271) and vessel density (β=0.095 [0.0415 s.e.m.], p=0.0221). We also report another nearby variant, rs849429 (risk allele A, frequency=0.49), but uncorrelated to rs17398575, associated with intraplaque vessel density (β=-0.164 [0.0361 s.e.m.], p=6.70×10-6). The SNP dependent PIK3CG mRNA expression demonstrated a differential effect in the vascular wall (p=0.783 for rs17398575; p=0.0198 for rs849429) compared to circulating monocytes (p=0.0261 for rs17398575; p=0.350 for rs849429). Conclusion: To our knowledge this is the first report involving the association of common genetic variants to histological plaque phenotypes. These results require further replication in independent cohorts. Further research should focus on elucidating the underlying mechanisms leading to plaque vessel formation and intraplaque hemorrhage, as both have been demonstrated to associate with secondary cardiovascular disease.