Search

The severity of the atherosclerotic burden is hardly quantifiable in subjects at high cardiovascular (CV) risk under intensive pharmacological therapy. Several molecules have been proposed as circulating biomarkers of atherosclerosis, but none has emerged as clinically meaningful.Circulating proteins involved in inflammation, plaque remodeling, smooth muscle cell migration, apoptosis and endothelial activity were measured by Proximity Extension Assay in the SUMMIT study cohort (n = 1500), including patients with type 2 diabetes (66%) and established CV disease (50%), who underwent ultrasound assessment of carotid atherosclerosis with total plaque area quantification.In patients with evidence of carotid artery atherosclerosis (n = 1174), seven biomarkers were identified as the more closely related to atherosclerosis extension. Compared with a multivariable model including major traditional CV risk factors, the percentage gain of explained variability in total plaque area was the greatest (33%) after inclusion of CD40 receptor (CD40R) ligand, followed by PDGF (30%), CD40R (26%), EGF (22%), CXCL1 (15%), HBEGF and MMP-17 (both 11%). The relationship of total plaque area with CD40R, PDGF was hyperbolic. In the whole study cohort, including subjects without carotid plaques, CD40R was the strongest predictor of the presence and extension of carotid atherosclerosis. Subjects in the third CD40R tertile had a more than two-fold greater atherosclerotic burden compared with lower CD40R tertiles, despite an only marginally higher load of CV risk factors.CD40R stands among an extended set of plausible atherosclerosis-related biomarkers as the most powerful predictor of carotid atherosclerosis burden in a high CV risk cohort.

Immune responses activated by LDL particles that have been trapped and oxidized in the arterial wall play an important role in atherosclerosis. Some of these immune responses are protective by facilitating the removal of pro-inflammatory and toxic lipid species formed as result of LDL oxidation. However, should these protective immune responses be insufficient, other more potent pro-inflammatory immune responses instead contributing to disease progression will gradually become dominant. The importance of the balance between protective and pathogenic immunity is particularly apparent when it comes to the adaptive immune system where pro-inflammatory T helper 1 (Th1) type T cells aggravate atherosclerosis, while regulatory T cells (Tregs) have an opposing role. As oxidized LDL is a key autoantigen in atherosclerosis, it has become an interesting possibility that immune-modulatory therapy that favors the activity of apolipoprotein B peptide-specific Tregs could be developed into a novel treatment strategy for prevention/stabilization of atherosclerosis and ischemic cardiovascular events. Indeed, several such oxidized LDL tolerance vaccines have shown promising results in animal models of atherosclerosis. This review will discuss the experimental background for development of atherosclerosis vaccines based on LDL-derived antigens as well as the challenges involved in translating these findings into clinical application.

Genetic loci associated with CHD show different relationships with intima-media thickness in the common carotid artery (IMT-CCA) and in the bulb (IMT-bulb). We evaluated if IMT-CCA and IMT-bulb differ also with respect to circulating protein profiles and risk of incident atherosclerotic disease.In three Swedish cohorts (MDC, IMPROVE, PIVUS, total n 7000), IMT-CCA and IMT-bulb were assessed by ultrasound at baseline, and 86 cardiovascular-related proteins were analyzed. In the PIVUS study only, IMT-CCA and IMT-bulb were investigated in relation to incident atherosclerotic disease over 10 years of follow-up.In a meta-analysis of the analysis performed separately in the cohorts, three proteins, matrix metalloproteinase-12 (MMP-12), hepatocyte growth factor (HGF) and N-terminal pro-B-type natriuretic peptide (NT-proBNP), were associated with IMT-CCA when adjusted for traditional cardiovascular risk factors. Five proteins were associated with IMT-bulb (MMP-12, growth/differentiation factor 15 (GDF-15), osteoprotegerin, growth hormone and renin). Following adjustment for cardiovascular risk factors, IMT-bulb was significantly more closely related to incident stroke or myocardial infarction (total number of cases, 111) than IMT-CCA in the PIVUS study (HR 1.51 for 1 SD, 95%CI 1.21-1.87, p 0.001 vs HR 1.17, 95%CI 0.93-1.47, p = 0.16). MMP-12 levels were related to this combined end-point (HR 1.30, 95%CI 1.08-1.56, p = 0.0061).Elevated levels of MMP-12 were associated with both IMT-CCA and IMT-bulb, but other proteins were significantly related to IMT in only one of these locations. The finding that IMT-bulb was more closely related to incident atherosclerotic disease than IMT-CCA emphasizes a difference between these measurements of IMT.

Background and aimsExtracellular matrix (ECM) remodeling is one of the key components in the formation of vulnerable atherosclerotic plaques and cardiovascular events. We recently showed that the full-length ECM-proteoglycan osteoglycin was associated with plaque vulnerability and future cardiovascular events. In the present study, we aimed to investigate the association of cleaved osteoglycin with plaque phenotype.MethodsTwo-hundred human carotid plaques were analyzed by immunohistochemistry. Cleaved osteoglycin and active caspase-3 were assessed by ELISA. ECM components (collagen, elastin and glycosaminoglycans) were assessed by colorimetric assays in plaque tissue homogenates. Matrix metalloproteinases (MMPs) were assessed using Milliplex. MMP-cleavage of osteoglycin and its effect on apoptosis were studied in vitro. Cardiovascular events were recorded during follow-up using national registries.ResultsPlaque levels of cleaved osteoglycin were significantly higher in asymptomatic plaques and correlated to α-actin plaque area, collagen, elastin and inversely to lipids, active.caspase-3 and a histological vulnerability index. Cleaved osteoglycin correlated to several MMPs, especially MMP-12, which was also shown to cleave osteoglycin in vitro. In vitro cleavage of osteoglycin was also associated with less smooth muscle cell apoptosis. Patients with high plaque levels of cleaved osteoglycin had a significantly lower risk to suffer from future cardiovascular events.ConclusionsThe current study shows that cleaved osteoglycin is associated with a stable plaque phenotype and lower risk for future cardiovascular events. Potentially due to reduced cell apoptosis and ability to retain LDL. These results indicate that targeting the cleavage of osteoglycin may be a potential therapeutic strategy to stabilize plaques.

BACKGROUND AND AIMS After aortic coarctation (CoA) repair, patients still suffer from cardiovascular complications. The aim of this study was to measure cardiovascular markers, intima-media thickness (IMT) and plaques in controls and patients with CoA. METHODS Sixty-four patients with CoA (66% male, mean age 48 ± 15 years) and controls (54% men, mean age 47 ± 16 years) underwent ultrasound of their arteries. A multiplex platform to analyze circulating blood levels biomarkers reflecting inflammation, tissue remodeling and repair was used. RESULTS In men following CoA repair, a significantly increased carotid bulb IMT was observed in comparison to the control group (1.05 [0.72-1.24] vs. 0.67 [0.59-0.95] mm; p = 0.003). Median common carotid artery (CCA) IMT was increased in men compared to controls (0.82 [0.61-0.97] mm vs. 0.58 [0.53-0.76] mm, p

Background and aims: A vulnerable plaque is an atherosclerotic plaque that is rupture-prone with a higher risk to cause cardiovascular symptoms such as myocardial infarction or stroke. Mimecan or osteoglycin is a small leucine-rich proteoglycan, important for collagen fibrillogenesis, that has been implicated in atherosclerotic disease, yet the role of mimecan in human atherosclerotic disease remains unknown. Methods: 196 human atherosclerotic carotid plaques were immunostained for mimecan. Smooth muscle cells, macrophages and intraplaque haemorrhage were also measured with immunohistochemistry. Neutral lipids were stained with Oil Red O and calcium deposits were quantified. Plaque homogenate levels of MCP-1, IL-6 and MIP-1β were measured using a Proximity Extension Assay and MMP-9 levels were measured using Mesoscale. Glycosaminoglycans, collagen and elastin were assessed by colorimetric assays and TGF-β1, β2 and β3 were measured using a multiplex assay. Mimecan gene expression in THP-1 derived macrophages was quantified by qPCR and protein expression in vitro was visualized with immunofluorescence. Cardiovascular events were registered using medical charts and national registers during follow-up. Results: Mimecan correlated positively with plaque area of lipids, macrophages, intraplaque haemorrhage and inversely with smooth muscle cell staining. Mimecan also correlated positively with plaque levels of MMP-9 and MCP-1. Mimecan was upregulated in THP-1 derived macrophages upon stimulation with MCP-1. Patients with high levels of mimecan (above median) had higher risk for cardiovascular death. Conclusions: This study indicates that mimecan is associated with a vulnerable plaque phenotype, possibly regulated by plaque inflammation. In line, plaque levels of mimecan independently predict future cardiovascular death. (Less)

Background and aims The pro-inflammatory alarmin S100A12 (EN-RAGE) and the soluble form of its receptor, the receptor for advanced glycation endproducts (sRAGE), have diverging roles in cardiovascular disease. In experimental studies, S100A12 promoted atherosclerosis while sRAGE treatment was anti-atherogenic and reduced myocardial infarction size by scavenging RAGE ligands. Here, we aimed to explore the links between S100A12, sRAGE, and long-term prognosis after an acute coronary syndrome (ACS). Methods We measured S100A12 and sRAGE in 524 patients within 24 h after an ACS, and again 6 weeks later in a subgroup of 114 patients. This subgroup also completed a follow-up echocardiography after 1 year. The median follow-up time for recurrent major adverse cardiovascular events (MACE), defined as recurrent ACS or cardiovascular death, was 25.7 ± 12.6 months. Results In Cox proportional hazard analyses, baseline S100A12 and sRAGE were positively associated with the risk of MACE, independently of traditional cardiovascular risk factors. The association between sRAGE and MACE remained significant after additional adjustment for troponin T, NT-proBNP and hsCRP [HR 95%CI for highest versus lowest tertile 3.2 (1.5–6.5), p = 0.002]. High sRAGE was also associated with deteriorating left ventricular function and an increased rate of heart failure hospitalization post-discharge. In contrast, patients with increasing sRAGE at 6 weeks compared to baseline had lower incidence of recurrent ACS. Conclusions Our data suggest that sRAGE has a dual, phase-dependent association with residual cardiovascular risk after ACS. These findings are important for the design and interpretation of future studies on sRAGE as biomarker and potential treatment in ACS patients.

Aims The small leucine-rich proteoglycans fibromodulin and lumican are functionally related extracellular matrix proteins involved in the regulation of collagen fiber formation. Fibromodulin-deficient apolipoprotein E-null mice have decreased vascular retention of lipids and reduced development of atherosclerosis suggesting that fibromodulin may influence the disease process. The aim of the present study was to investigate if fibromodulin and lumican are expressed in human carotid plaques and to determine if their expression is associated with the occurrence of preoperative symptoms and with risk for postoperative cardiovascular events. Methods and results 153 plaques (51% symptomatic) obtained by carotid endarterectomy were included in this study. Plaque content was analyzed by immunohistochemistry and plaque cytokine content by multiplex technology. Fibromodulin and lumican were widely expressed in plaques and fibromodulin expression was significantly higher in symptomatic plaques. Expression of fibromodulin was significantly higher in plaques obtained from patients with diabetes and a high fibromodulin expression was associated with a higher incidence of post-operative cerebrovascular events, whereas no such associations were seen for lumican. Fibromodulin expression also correlated with plaque lipids and several pro-inflammatory cytokines. In addition, fibromodulin expression correlated with low levels of smooth muscle cells and the anti-inflammatory cytokine IL-10. Conclusions These observations support previous experimental findings in mice for a role of fibromodulin in atherosclerosis and provide clinical evidence of the involvement of fibromodulin in the inflammatory processes that characterize atherosclerotic plaque vulnerability. They also suggest that this is of particular importance in diabetes.

Objectives Autoimmune responses against oxidized-LDL (oxLDL) have been suggested to modulate inflammation in atherosclerosis. Previous studies showed an association between autoantibodies against the apolipoprotein B (apoB) p210 antigen and a lower risk of cardiovascular (CV) events. In the present study we investigated if autoantibodies against p210 at the time of carotid endarterectomy (CEA) predict risk for future CV events. Methods Native (nat) and malondialdehyde (MDA)-modified apoB p210 autoantibodies (IgM-p210nat, IgG-p210nat, IgM-p210MDA and IgG-p210MDA) were analyzed by ELISA from plasma samples of 351 patients at the time they underwent CEA. The incidence of postoperative CV events was assessed using national registers. Results A total of 52 non-fatal and 15 fatal CV events were registered during the follow-up period (35.1 ± 16.7 months). Patients who suffered from a fatal CV event had significantly lower plasma levels of IgG-p210nat and IgG-p210MDA. Kaplan–Meier curves of event-free survival showed increased CV mortality in patients with levels of IgG-p210nat and IgG-p210MDA below the median (Log Rank 7.813, p .005 and 9.105, p .003 respectively). The association between low levels of p210 IgG and fatal post-operative CV events remained significant when adjusting for age, sex, total cholesterol, HDL cholesterol, smoking habits and hypertension in a Cox Proportional Hazard model (hazard ratios (HR) IgG-p210nat below median: HR 6.7 (95% C.I. 1.5–30.6, p .013) and IgG-p210MDA below median: HR 7.8 (95% C.I. 1.7–35.5, p .008). Conclusions The present findings support the notion that autoantibodies against LDL antigens are involved in the atherosclerotic disease process and suggest that CEA patients with low levels of IgG-p210nat and IgG-p210MDA have an increased risk of post-operative CV death.

Objective : To examine if overall diet quality is associated with cellular and soluble biomarkers of systemic inflammation in middle-aged individuals. Methods : A group of 667 individuals, aged 63–68 years, selected from the cardiovascular arm of the Malmo Diet and Cancer cohort, participated in this study. Baseline examinations consisted of an extensive socio-demographic questionnaire, anthropometric measurements, blood sampling and detailed dietary data. Mononuclear leukocytes frozen at baseline were thawed and analysed with flow cytometry to quantify monocyte subsets based on CD14 and CD16 expression. Plasma cytokines were measured using multiplexed immune assays. A diet quality index consisting of six components (saturated fatty acids, polyunsaturated fatty acids, fish and shellfish, dietary fibre, fruit and vegetables, and sucrose) was constructed to measure adherence to the Swedish Nutrition Recommendations/Dietary Guidelines. General linear models were used to investigate associations between index scores and several biomarkers of inflammation. Results : A higher percentage of women reported adherence to the nutritional recommendations and had better overall diet quality than men. Participants with higher diet quality were more likely to have a healthier lifestyle. The levels of high-sensitive CRP, S100A8/A9, TNF-α, white blood cells, neutrophils, lymphocytes and CD14 + CD16 ++ were lower in participants with higher index scores. The associations remained significant after adjustment for potential confounders. Conclusion : In this cross-sectional study, we found that a high diet quality is associated with lower systemic inflammation. As the incidence of cardiovascular disease and cancer is directly correlated with the levels of inflammation, our findings might indicate a protective role of high-quality diet.

Background and aims There is convincing evidence that adaptive immune responses affect the development of atherosclerosis and thrombosis and several autoimmune diseases are associated with increased cardiovascular risk. However, our understanding of the underlying mechanisms remains limited. We investigated how biomarkers reflecting four aspects of autoimmunity: apoptosis, inflammation, tissue degradation and repair, associate with cardiovascular disease (CVD) in subjects with systemic lupus erythematosus (SLE). Methods We investigated 484 well-characterized SLE patients, 69 of whom had CVD (coronary artery disease, cerebrovascular disease or peripheral artery disease), and 253 controls. Occurrence of carotid plaques was investigated with ultrasound. Plasma levels of biomarkers reflecting apoptosis (Fas, TNF receptor 1, TRAIL receptor 2), inflammation (IL-6, IL-8, monocyte chemotactic protein-1), tissue degradation (matrix metalloproteinase (MMP)-1, MMP-3, MMP-7), and tissue repair (platelet-derived growth factor, epidermal growth factor and stem cell factor) were analyzed by Proximity Extension Assay. Results Subjects with SLE had markedly elevated plasma levels of biomarkers reflecting apoptosis, inflammation and tissue degradation as compared to controls. SLE patients with CVD had higher levels of Fas, TNF receptor 1, TRAIL receptor 2, MMP-1 and -7 than those without CVD. The same associations were found for the presence of a carotid plaque. When controlling for the factors included in the Framingham risk score, all biomarkers except MMP-1 remained associated with the presence of a carotid plaque, while only TRAIL receptor 2 levels remained significantly associated with CVD. Conclusions Our findings argue that the cardiovascular risk in SLE is associated with increased cell death by apoptosis and tissue degradation.

Objective: The immune system plays an important regulatory role in the development of atherosclerotic plaques and neointima formation following various types of angioplasty. In the present study we investigated the effect of antibodies against aldehyde-modified apolipoprotein B-100 (apoB-100), a component of oxidized LDL, on atherosclerosis and response to arterial injury in mice. Methods: The ability of a high affinity human recombinant antibody (21303), specific for malondialdehyde-modified apoB-100, to influence formation of atherosclerosis as well as remodelling and neointima formation after a collar-induced injury of the carotid artery was studied in LDL receptor(-/-) mice over-expressing human apoB-100. Results: The antibody recognized epitopes present in mouse plasma and reduced the plasma level of oxidized LDL by 34%. Antibody treatment inhibited injury-induced restrictive vascular remodelling but did not influence the size of the neointima. Atherosclerosis in the uninjured contra lateral carotid artery was determined by computerized image analysis and the mean plaque area in animals given control IgG1 was 7608 +/- 10,336 mu m(2). In contrast, essentially no plaques were present in animals treated with the 2DO3 antibody (397 +/- 235 mu m(2), P < 0.0 1 versus control IgG 1). Conclusions: Treatment with antibodies against aldehyde-modified apoB-100 dramatically reduces atherosclerosis and inhibits restrictive vascular remodelling in mice expressing human apoB-100. (c) 2006 Elsevier Ireland Ltd. All rights reserved. (Less)

Background The leading cause of morbidity and mortality worldwide is atherosclerotic cardiovascular disease, most commonly caused by rupture of a high-risk plaque and subsequent thrombosis resulting in stroke, myocardial infarction or sudden death depending on the affected arterial territory. Accurate, non-invasive methods to identify such lesions known as vulnerable or high-risk plaques are currently sub-optimal. Our aim was to validate a new non-invasive ultrasound method to identify high-risk carotid plaques. Methods We evaluated a new method based on the center frequency shift (CFS) of the ultrasound radio frequency data obtained from carotid plaques compared to a reference phantom. We evaluated the method both ex vivo , on 157 sections from 18 plaques, and in vivo , in 39 patients 1-day prior to carotid plaque removal, and correlated the data with histology. Results The CFS correlated with a plaque vulnerability index based on histological areas stained for lipids, macrophages, hemorrhage, smooth muscle cells and collagen (r = −0.726, P = 1.7 × 10 −8 ). Plaques with CFS below median had larger cores, more macrophages and were less rich in collagen in agreement with the definition of rupture-prone plaques. The accuracy to detect plaques with high vulnerability index was 78% (confidence interval (CI) 61–89%), with sensitivity 77% (CI 61–89%) and specificity 78% (CI 62–89%). Conclusions Our method is the first to characterize atherosclerotic plaque components that affect plaque vulnerability using CFS.

High levels of the triacylglycerol-rich lipoproteins, very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) have been identified as independent risk factors for coronary heart disease, and inflammation is thought to contribute to atherosclerosis and its complications. To understand how dyslipidemia promotes inflammation, we have characterised the effects of VLDL treatment on production of tumor necrosis factor-alpha (TNF) by human monocyte-derived macrophages. VLDL strongly potentiated lipopolysaccharide (LPS)-induced expression of TNF mRNA and secretion of TNF protein. VLDL activated mitogen-activated protein kinase-ERK kinase 1/2 (MEK1/2), and potentiated LPS-induced MEK1/2 activation. The MEK1/2 inhibitor U0126 strongly diminished TNF expression, indicating that MEK1/2 plays a central role in the regulation of TNF expression. VLDL did not activate transcription factors NF-kappaB and PPAR-gamma, but it activated AP-1 at least as potently as LPS, and potentiated LPS-induced activation of AP-1. The inhibitor U0126 completely prevented this potentiation. Inhibition of AP-1 by decoy oligonucleotides abolished potentiation of TNF secretion by VLDL. In conclusion, VLDL treatment potentiates TNF expression in macrophages by activation of MEK1/2 and AP-1. These findings suggest that triacylglycerol-rich lipoproteins are involved in inflammatory processes associated with atherosclerosis.

Inflammation is a key factor in the development of plaque rupture and acute cardiovascular events. Although imaging techniques can be used to identify vulnerable atherosclerotic plaques, we are lacking non-invasive methods, such as plasma markers of plaque inflammation that could help to identify presence of vulnerable plaques. The aim of the present study was to investigate whether increased plasma levels of pro-inflammatory cytokines reflects inflammatory activity within atherosclerotic plaques.Cytokines were measured using Luminex immunoassay in 200 homogenized plaque extracts and plasma, obtained from 197 subjects undergoing carotid surgery. Plasma levels of macrophage inflammatory protein-1β (MIP-1β), tumor necrosis factor- α (TNF-α) and fractalkine correlated significantly, not only with plaque levels of the same cytokines but also with the abundance of several pro-inflammatory and atherogenic cytokines assessed in plaque tissue. High plasma levels (upper tertile) of MIP-1β, TNF-α and fractalkine identified the presence of a plaque with high inflammation (above median of a score based on the plaque content of MIP-1β, TNF-α, interferon-γ (IFN-γ) and fractalkine) with a sensitivity between 65 and 67% and a specificity between 78 and 83%. Furthermore, this study shows that high plasma levels of MIP-1β, TNF-α and fractalkine predict future transient ischemic attacks.Our findings show that the plasma levels of MIP-1β, TNF-α and fractalkine reflect the levels of several pro-atherogenic cytokines in plaque tissue and might be possible plasma markers for a vulnerable atherosclerotic disease. We thereby propose that these cytokines can be used as surrogate markers for the identification of patients with high-risk plaques.

We studied the effect of arterial balloon injury on nuclear factor-kappaB (NF-kappaB) mobilization and ICAM-1 expression in untreated rats and rats treated with aspirin. Baseline NF-kappaB nuclear binding in smooth muscle cells (SMC) increased two-fold within 6 h after balloon injury. The binding returned to baseline 3 days after injury. Consistently nuclear staining of p65 active subunit increased in the medial SMC following balloon injury. There was no baseline ICAM-1 expression. Within 3 days after balloon injury there was marked medial ICAM-1 expression, that localized to neointima 7 days after injury and to regrowing endothelial cells 14 days after injury. Treatment with aspirin inhibited NF-kappaB nuclear translocation and binding and was associated with reduction of ICAM-1 expression, SMC proliferation and neointimal thickening following balloon injury. These data suggest that transient mobilization of NF-kappaB in vascular SMC after balloon injury mediates ICAM-1 expression and is involved in arterial response to balloon injury.

Relatively little is known about the biological mechanisms by which lipoproteins promote atherogenesis. It has, however, been shown that structural modification of low density lipoprotein (LDL), such as by oxidation, results in their uptake and degradation by intimal macrophages and consequently leads to formation of lipid-rich atherosclerotic lesions. The aim of the present investigation was to study the influence of dietary intake of fat, lipoprotein lipid composition, smoking and gender on macrophage degradation of LDL before and after oxidation. The study group consisted of 48 males and 56 females with hyperlipidemia taking part in the open prerandomization phase of the Probucol Quantitative Regression Swedish Trial (PQRST). Analysis including lipoprotein determinations, dietary and smoking habit interviews, LDL degradation by macrophages, LDL receptor binding and LDL thiobarbituric acid reactive substance (TBARS) levels before and after copper ion-induced oxidation was done during the pre-andomization phase of the study. Increased plasma and very low density lipoprotein (VLDL) triglyceride levels were associated with an increased macrophage degradation of native LDL, whereas no such association was found after oxidation of LDL. The dietary intake of polyunsaturated fatty acids (PUFA) was also inversely related to the degradation of native LDL by macrophages, but increased the rate at which oxidized LDL was degraded. Smoking and gender did not influence the rate of macrophage degradation of native or oxidized LDL. It is concluded that hypertriglyceridemia is associated with an increased macrophage degradation of LDL. This may represent a mechanism by which hypertriglyceridemia promotes atherosclerosis.

OBJECTIVES: To examine whether the decrease in IMT progression rate in the carotid bulb induced by metoprolol CR/XL treatment (25mg once daily) observed in the β-blocker Cholesterol-lowering Asymptomatic Plaque Study (BCAPS) was accompanied by an effect on carotid plaque echogenicity. METHODS: Gray scale median (GSM) in carotid plaques, used as a score of echogenicity, was measured at baseline and after 36 months in those 341 subjects (aged 49-69 years) with an asymptomatic moderate- to large-sized carotid plaque present at baseline and at follow-up. Participants were in a factorial design assigned to treatment with metoprolol CR/XL (25mg once daily), fluvastatin (40mg once daily) or corresponding placebo. RESULTS: After 36 months plaques were more echogenic in participants treated compared to those not treated with metoprolol CR/XL (57.3±16.8 versus 51.8±20.0, p=0.006). GSM had increased more from baseline in the metoprolol CR/XL treated subjects (25±15 versus 18±20, p

PURPOSE: Accumulation of oxidized LDL in the arterial wall is believed to play a key role in the development of atherosclerosis. Experimental studies have identified the presence of immune responses against epitopes in oxidized LDL that protects against atherosclerosis. We have produced human recombinant IgG against one of these epitopes (aldehyde-modified apoB-100 amino acids 661-680) and demonstrated that treatment with this human IgG1 2D03 antibody markedly reduces atherosclerosis in hypercholesterolemic mice. METHODS: In the present study, we screened a panel of 25 carotid plaques associated with clinical symptoms and 26 clinically silent plaques obtained at surgery for presence of the aldehyde-modified apoB-100 peptide defined by the 2D03 antibody and compared the expression of this epitope with other plaque constituents, plasma lipoproteins levels, plasma oxidized LDL and autoantibodies against apoB-100 peptides. RESULTS: We demonstrated that the epitope is commonly expressed in human atherosclerotic plaques and that plaques associated with clinical symptoms have an almost three-fold higher content of this epitope (8.6+/-4.9% versus 22.1+/-12.2% immunostaining of total plaque area, p

Matrix remodelling plays an important role in regulating plaque stability. Cystatin C, an inhibitor of the elastin-degrading cysteine proteases of the cathepsin family, is believed to be one of the key protease inhibitors in this process. The aim of the present study was to investigate the role of leukocyte-specific cystatin C expression under conditions that favour plaque regression. Apolipoprotein E-deficient mice (apoE-/-) were given a Western-type diet 15 weeks prior transplantation with bone marrow from mice lacking cystatin C (cysC-/-) or cystatin C positive (cysC+/+) mice, in both cases apoE+/+ to create conditions favouring plaque regression. Transplantations were verified with PCR and Western analyses. Transplanted mice showed a 70% decrease in lipid content and reduction in plaque area compared to baseline ApoE-/- mice, demonstrating plaque regression due to apoE expression in macrophages. apoE-/- mice transplanted with cysC-/- bone marrow were then compared to mice transplanted with cysC+/+ bone marrow. Mice receiving cysC-/- bone marrow had a 30% larger plaque area, despite absence of significant differences in plasma cholesterol and lipid contents in plaque. Unexpectedly, mice transplanted with cystatin C-deficient bone marrow cells had increased elastin and collagen content in lesions. These observations suggest that leukocyte-specific expression of cystatin C is actively involved in matrix remodelling associated with plaque regression.

Objectives: To evaluate the importance of different autoantibodies against modified low-density lipoprotein (LDL) in patients with coronary artery disease (CAD). Background: Previous studies of autoantibodies against LDL have shown that patients with CAD have increased titers of autoantibodies against LDL modified by copper and malondialdehyde (MDA), whereas there is a lack of information about autoantibody titers against LDL modified by hypochlorite (HOCl). Studies of autoantibodies in relation to severity of atherosclerosis are few and have reached divergent results. Furthermore, no data exist on the relationship between autoantibody titers and prognosis. Methods: Titers of autoantibodies against copper-, MDA- and HOCl-modified LDL were determined in serum by ELISA. Autoantibody titers in young male survivors of a first myocardial infarction were compared with those of healthy controls and related to coronary angiographic findings and to prognosis during I I years of follow-up. Results: Patients had higher titers of autoantibodies against LDL modified by copper and MDA than controls. In contrast, no consistent associations were found between autoantibody titers and global severity of coronary atherosclerosis or number and severity of coronary stenoses and prognosis. Conclusions: The prognostic value of autoantibodies against modified LDL is limited in young postinfarction patients despite the fact that autoantibody titers against copper- and NIDA-modified LDL are raised compared with healthy controls. Furthermore, the results indicate that autoantibodies against modified LDL are not protective in later stages of coronary atherosclerosis. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved. (Less)

Background: Immunization of animals with LDL reduces atherosclerosis. However, whether the timing of immunization affects its efficacy is not known. In this study, we evaluated the influence of timing of immunization on the athero-protective effects of LDL immunization in apo E (-/-) mice. Methods and results: Hypercholesterolemic apo E (-/-) mice were immunized with native LDL (nLDL) at age of 6-7 weeks old or at 20 weeks old. Compared to adjuvant group, mice that were immunized at the age of 6-7 weeks developed significantly smaller aortic sinus plaques with reduced gelatinolytic activity and increased collagen content. This was associated with an increase of oxidized LDL (oxLDL) antibody titer and a marked decrease in splenic IL-4 mRNA expression. Immunization at 20 weeks of age also increased oxLDL antibody titer but did not reduce plaque size, gelatinolytic activity or collagen content but resulted in a modest decrease in macrophage infiltration. Late immunization did not alter splenic IL-4 mRNA expression. Conclusions: Our findings demonstrate that, only early nLDL immunization modulates humoral and cellular immune responses and affects plaques size and composition in apo E (-/-) mice, indicating the critical importance of timing of immunization for its antiatherogenic efficacy. (C) 2004 Elsevier Ireland Ltd. All rights reserved. (Less)

In the present study, we characterize the early cytotoxic effects of 7beta-hydroxycholesterol, a major cytotoxin in oxidized LDL, in human aortic smooth muscle cells. Within a few minutes after addition, 7beta-hydroxycholesterol induced Ca(2+) oscillations with a frequency of approximately 0.3-0.4 min(-1). A few hours later, thapsigargin-sensitive Ca(2+) pools were depleted, indicating that 7beta-hydroxycholesterol perturbs intracellular Ca(2+) homeostasis. The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (but not JNK) were activated within 5 min after addition of 7beta-hydroxycholesterol. The side-chain hydroxylated oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol were more potent in inducing apoptosis than 7beta-hydroxycholesterol and cholesterol-5alpha,6alpha-epoxide, as determined by TUNEL staining. Addition of TNFalpha (10 ng/ml) and IFNgamma (20 ng/ml) enhanced the cytotoxicity of oxysterols and potentiated apoptosis. The cytokines alone were not toxic to smooth muscle cells at these concentrations. 25-Hydroxycholesterol and 7beta-hydroxycholesterol but not cholesterol inhibited protein synthesis at 4-8 h as determined by [35S]methionine incorporation assay. Morphologically, oxysterol-induced cell death was characterized by disorganization of the ER and Golgi membranes. The Ca(2+) and ERK signals preceded the ultrastructural changes induced by 7beta-hydroxycholesterol.

The aim of the present study was to analyze the frequency and mechanism of cell death in atherosclerotic plaques with a recent history ( 70% diameter reduction) undergoing carotid endarterectomy. In situ tailing nick translation of fragmented DNA, agarose gel electrophoresis of plaque DNA and electron microscopy were used to identify cell death by apoptosis (programmed cell death) and necrosis. The mean number of cell containing fragmented DNA in the plaques was 12.7 ± 3.5% (n=15). Focal accumulations of cells with DNA fragmentation occurred in the fibrous cap, at sites of rupture, close to lipid deposits and necrosis and was always accompanied by the presence of inflammatory cells. Electrophoretic separation of DNA isolated form part of plaques where the presence of DNA fragmentation had previously been demonstrated by in situ DNA nick translation resulted in multiple ladders of 180–200 base pairs characteristic of apoptosis. Electron microscopic analysis revealed presence of cells with morphological signs of degeneration in a frequency even higher than that found by in situ nick translation. Some of these cells had a characteristic apoptotic appearance with condensed chromatin and cytoplasm, but the large majority of the cells had an ultrastructure typical for cells undergoing cell death by necrosis with membrane disruption and swollen, disintegrating organelles. Thus, although apoptosis clearly takes place in atherosclerotic plaques necrosis appears to be a much more common mechanism for cell death. This may have negative consequences for plaque stability since cell death by necrosis induces a stronger inflammatory reaction than apoptotic cell death.

The Probucol Quantitative Regression Swedish Trial (PQRST) investigated the effect of the lipid lowering and antioxidant drug probucol on the development of atherosclerosis in humans. 303 hypercholesterolemic patients were randomized to receive either probucol or placebo, in combination with dietary advice and cholestyramine for a three-year period. Probucol was not found to effect progression regression of femoral atherosclerosis significantly as assessed by quantitative arteriography. To evaluate the effectiveness of probucol as an antioxidant during the study period, detailed analyses were performed on 42 of the randomized patients. During the trial, probucol-treated patients (n = 26) had 15% lower total cholesterol (P < 0.01) and 35% lower high density lipoprotein (HDL) cholesterol (P < 0.0001) compared with controls (n = 16). Low density lipoprotein (LDL) from probucol treated individuals was more resistant to oxidation by Cu2+ as determined by the lag phase for the formation of conjugated dienes (220 +/- 8 vs. 82 +/- 7 min (mean +/- S.E)), showed a 13 times lower formation of lipid peroxides, a 97% reduction in macrophage degradation and close to 90% less decrease in LDL receptor binding following oxidation as compared with controls (P < 0.001 for all differences). The results demonstrate that although probucol provided a significant protection against Cu(2+)-induced oxidative modification of LDL, it lacked effect on the development of femoral atherosclerosis. The relevance of these observations for the proposed role of lipid oxidation in atherosclerosis is discussed.

In animals fed a hypercholesterolemic diet, development of atherosclerosis is preceded by attachment of mononuclear leukocytes to the arterial endothelium. Early lesions begin to develop as monocytes migrate into the intima and ingest lipids. A major part of these lipids is believed to be derived from oxidatively modified low density lipoprotein (LDL). In the present study we demonstrate that human mononuclear leukocytes exposed to low concentrations of copper-oxidized LDL secrete one or several factors that stimulate the expression of intercellular adhesion molecule-1 (ICAM-1, CD54), vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin-1, ELAM-1), whereas native LDL was found to be without effect. Exposure of endothelial cells to non-conditioned medium containing oxidized LDL did not influence the expression of adhesion molecules. Incubation of endothelial cells with conditioned medium from mononuclear cells grown in the presence of oxidized LDL also resulted in a three-fold increase in the binding of monocytoid U937 cells. The present findings suggest that mononuclear leukocytes exposed to oxidatively modified LDL in early atherosclerotic lesions may stimulate the recruitment of other leukocytes by secreting cytokines which induce the expression of adhesion molecules on the endothelium.

Adhesion of monocytes to the arterial endothelium is an important early event in atherosclerosis. Several lines of evidence have suggested that oxidation of low density lipoprotein (LDL) in the arterial wall may initiate the inflammatory-like process that generally is present in atherosclerotic lesions. In vitro, oxidation of LDL can be obtained both by exposure to divalent ions, such as Cu2+, or by incubation with different cell types, including monocytes and endothelial cells. The present study was designed to investigate the possible influence of oxidized LDL on the adhesive properties of endothelial cells. We report here that Cu(2+)-oxidized LDL is as effective as interleukin 1 beta in stimulating the ability of cultured human endothelial cells to bind U937 monocytic cells. The stimulation was inhibited by cycloheximide, indicating that de novo protein synthesis is required. Biologically modified LDL, obtained by incubation with human peripheral blood monocytes, also enhanced the adhesiveness of endothelial cells. This effect was not due to an increased secretion of interleukin 1 beta from the monocytes exposed to LDL. Treatment of endothelial cells for 24 h with native LDL was also found to increase the adhesion of U937 cells. Exposure of endothelial cells to LDL for 24 h resulted in an oxidative modification of LDL. Furthermore, the antioxidant butylated hydroxytoluene inhibited both the endothelial-dependent oxidation of LDL as well as the increased adhesion of U937 cells, suggesting a coupling between these two processes. The results indicate that LDL, modified by exposure to monocytes or endothelial cells in the arterial wall, may increase the adhesive properties of the endothelium.

Lipid accumulation in monocyte-originated macrophages in the subendothelial space is an important characteristic of atherosclerotic lesions. Several lines of evidence have indicated that this accumulation occurs as a result of lipid peroxidation. In the present study the ability of probucol to prevent oxidation of low density lipoprotein (LDL) was investigated in 20 hypercholesterolemic individuals taking part in the Probucol Quantitative Regression Swedish Trial (PQRST). The effect of Cu2(+)-induced oxidation of LDL on degradation by macrophages, binding to LDL receptors on fibroblasts and LDL TBARS content was analysed. With LDL isolated from patients on diet alone oxidation led to a 44.3% decreased binding to fibroblasts (P less than 0.001), a ninefold increased uptake in macrophages (P less than 0.001) and a twentyfold increase in TBARS content (P less than 0.001) as compared to native LDL. These values were essentially the same during treatment with cholestyramine alone. However, during treatment with probucol plus cholestyramine exposure of LDL to Cu2+ resulted in an increase in TBARS which was less than 50% (P less than 0.02) of that observed during the other two treatment periods. Furthermore, probucol treatment abolished more than 70% of the decrease in B,E receptor binding to fibroblasts (P less than 0.05) and more than 90% of the increased degradation by macrophages (P less than 0.001) of Cu2+ oxidatively modified LDL.

The effect of prostaglandins (PG) on initiation of DNA synthesis in arterial smooth muscle cells (SMC) stimulated with platelet-derived growth factor (PDGF) was examined. A concentration of 10 ng/ml PGE 1 inhibited DNA synthesis, measured as autoradiographically labeled nuclei, by about 70%. Similar results were obtained with PGE 2 and PGD 2 but at concentrations 10–20 times higher, whereas PGF 2α lacked effect. The inhibitory action of the prostaglandins was restricted to the first 6 h of the lag phase. Treatment with PGE 1 also raised the intracellular concentration of cyclic AMP, indicating that the inhibition may be mediated via changes in the levels of cyclic nucleotides.

The early state of atherosclerosis is characterized by a nodular proliferation of smooth muscle cells in the arterial intima. It has been suggested that this proliferation is initiated by platelet-derived growth factor (PDGF) released from aggregating platelets in connection with endothelial injury. In the present study platelet reactivity and mitogenic activity of plasma and serum were compared in young male survivors of myocardial infarction with angiographically demonstrable coronary atherosclerosis and in healthy subjects of similar age. Young post-infarction patients with coronary atherosclerosis had lower ED50 values of ADP-induced platelet aggregation. Furthermore plasma and serum from the patients contained increased amounts of mitogenic activity. Experiments using antibodies against platelet-derived growth factor indicated that the increase in mitogenic activity represented elevated concentrations of free PDGF growth factor in plasma. The results raise the possibility of a connection between increased levels of free PDGF and the proliferative reaction that characterizes early lesion progression.

Migration of smooth muscle cells from the media to the intima of the arterial wall and proliferation of intimal smooth muscle are major early events in the formation of an atherosclerotic lesion. The start of proliferation requires that the cells have passed through a modulation from contractile to synthetic phenotype and that they are stimulated with growth factors. Here, we have examined the effects of the calcium antagonist nifedipine on phenotypic modulation and growth of isolated rat arterial smooth muscle cells cultivated in vitro. The results indicate that micromolar concentrations of nifedipine slow down the rate of transformation of the cells from a contractile to a synthetic phenotype and inhibit initiation of DNA synthesis as well as cellular proliferation. The inhibitory effect on DNA synthesis was seen both in cells stimulated with whole blood serum and with purified platelet-derived growth factor. The results raise the possibility that nifedipine may be used to prevent atherogenesis and to inhibit progression of fibromuscular lesions by interfering with the proliferation of arterial smooth muscle cells.