Regulation of atherosclerotic plaque inflammation.

The immune reactions that regulate atherosclerotic plaque inflammation involve chemokines, lipid mediators and costimulatory molecules. Chemokines are a family of chemotactic cytokines that mediate immune cell recruitment and control cell homeostasis and activation of different immune cell types and subsets. Chemokine production and activation of chemokine receptors form a positive feedback mechanism to recruit monocytes, neutrophils and lymphocytes into the atherosclerotic plaque. In addition, chemokine signalling affects immune cell mobilization from the bone marrow. Targeting several of the chemokines and/or chemokine receptors reduces experimental atherosclerosis, whereas specific chemokine pathways appear to be involved in plaque regression. Leukotrienes are lipid mediators that are formed locally in atherosclerotic lesions from arachidonic acid. Leukotrienes mediate immune cell recruitment and activation within the plaque as well as smooth muscle cell proliferation and endothelial dysfunction. Antileukotrienes decrease experimental atherosclerosis, and recent observational data suggest beneficial clinical effects of leukotriene receptor antagonism in cardiovascular disease prevention. By contrast, other lipid mediators, such as lipoxins and metabolites of omega-3 fatty acids, have been associated with the resolution of inflammation. Costimulatory molecules play a central role in fine-tuning immunological reactions and mediate crosstalk between innate and adaptive immunity in atherosclerosis. Targeting these interactions is a promising approach for the treatment of atherosclerosis, but immunological side effects are still a concern. In summary, targeting chemokines, leukotriene receptors and costimulatory molecules could represent potential therapeutic strategies to control atherosclerotic plaque inflammation.

Blocking CD40-TRAF6 interactions by small-molecule inhibitor 6860766 ameliorates the complications of diet-induced obesity in mice.

BACKGROUND: Immune processes contribute to the development of obesity and its complications, such as insulin resistance, type 2 diabetes mellitus and cardiovascular disease. Approaches that target the inflammatory response are promising therapeutic strategies for obesity. In this context, we recently demonstrated that the interaction between the costimulatory protein CD40 and its downstream adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes adipose tissue inflammation, insulin resistance and hepatic steatosis in mice in the course of diet-induced obesity (DIO). METHODS: Here we evaluated the effects of a small-molecule inhibitor (SMI) of the CD40-TRAF6 interaction, SMI 6860766, on the development of obesity and its complications in mice that were subjected to DIO. RESULTS: Treatment with SMI 6860766 did not result in differences in weight gain, but improved glucose tolerance. Moreover, SMI 6860766 treatment reduced the amount of CD45(+) leucocytes in the epididymal adipose tissue by 69%. Especially, the number of adipose tissue CD4(+) and CD8(+) T cells, as well as macrophages, was significantly decreased. CONCLUSIONS: Our results indicate that small-molecule-mediated inhibition of the CD40-TRAF6 interaction is a promising therapeutic strategy for the treatment of metabolic complications of obesity by improving glucose tolerance, by reducing the accumulation of immune cells to the adipose tissue and by skewing of the immune response towards a more anti-inflammatory profile.

Immunotherapy and cardiovascular diseases: novel avenues for immunotherapeutic approaches.

As current therapies for cardiovascular disease (CVD), predominantly based on lipid lowering, still face an unacceptable residual risk, novel treatment strategies are being explored. Besides lipids, inflammatory processes play a major role in the pathogenesis of atherosclerosis, the underlying cause of the majority of CVD. The first clinical trials targeting the interleukin-1ß-inflammasome axis have shown that targeting this pathway is successful in reducing cardiovascular events but did not decrease overall CVD mortality. Hence, novel and improved immunotherapeutics to treat CVD are being awaited.

Requirement for CD154 in the progression of atherosclerosis.

Atherosclerosis is a systemic disease of the large arteries, and activation of inflammatory pathways is important in its pathogenesis. Increasing evidence supports the importance of CD40-CD154 interactions in atherosclerosis, interactions originally known to be essential in major immune reactions and autoimmune diseases. CD40 is present on atheroma-derived cells in vitro and in human atheromata in situ. Ligation of CD40 on atheroma-associated cells in vitro activates the production of chemokines, cytokines, matrix metalloproteinases, adhesion molecules and tissue factor, substances responsible for lesion progression and plaque destabilization. Administration of antibody against CD154 to low-density lipoprotein receptor-deficient mice has been shown to reduce atherosclerosis and decrease T-lymphocyte and macrophage content; however, only initial lesions were studied. Here, we determined the effect of genetic disruption of CD154 in ApoE-/- mice in both initial and advanced atherosclerotic lesions. Plaque area was reduced 550%. In contrast to previous reports, initial lesion development was not affected. Advanced plaques in CD154-/-ApoE-/- mice had a less-lipid-containing, collagen-rich, stable plaque phenotype, with a reduced T-lymphocyte/macrophage content. These data indicate that CD40-CD154 signaling is important in late atherosclerotic changes, such as lipid core formation and plaque destabilization.

Anti-oxLDL antibody isotype levels, as potential markers for progressive atherosclerosis in APOE and APOECD40L mice.

In humans and animal models of atherosclerosis, antibodies against oxidized LDL have been associated with atherosclerotic lesion development. It has been suggested that IgM anti-oxLDL antibodies are anti-atherogenic, whereas IgG anti-oxLDL antibodies are pro-atherogenic. In this study, we examined the relation between IgM and IgG antibody levels and atherosclerosis severity in APOE(-/-)CD40L(-/-) mice, which are deficient for IgG and develop moderate advanced atherosclerosis, and compared results with mice developing severe (APOE(-/-)) or no atherosclerosis (C57Bl/6). Mice were followed in time for anti-oxLDL antibodies while on high-fat diet or normal chow. Anti-oxLDL antibody levels were determined by ELISA. Results revealed that 24-week-old APOE(-/-)CD40L(-/-) mice had enhanced IgM anti-oxLDL antibody levels when compared with wild-type mice, but similar levels to those of APOE(-/-) mice. As expected, IgG anti-oxLDL antibody levels were almost absent in APOE(-/-)CD40L(-/-) mice. The transition from early to advanced lesions in APOE(-/-) mice was reflected by elevated IgM anti-oxLDL antibody levels. IgM anti-oxLDL levels did not further increase during progression to more advanced lesions. No relation was found between IgG anti-oxLDL levels and atherosclerosis severity. In conclusion, the severity of advanced atherosclerosis in mice is not reflected by IgM and/or IgG anti-oxLDL antibody levels. Furthermore, less advanced atherosclerotic lesion development in APOE(-/-)CD40L(-/-) mice does not seem to be the result of higher levels of protective IgM anti-oxLDL antibodies. Therefore, our study does not support the idea that the previously observed inconsistency in the relation between anti-oxLDL and atherosclerosis severity is due to differences in antibody isotypes.

Disruption of the cathepsin K gene reduces atherosclerosis progression and induces plaque fibrosis but accelerates macrophage foam cell formation.

BACKGROUND: Cathepsin K (catK), a lysosomal cysteine protease, was identified in a gene-profiling experiment that compared human early plaques, advanced stable plaques, and advanced atherosclerotic plaques containing a thrombus, where it was highly upregulated in advanced stable plaques. METHODS AND RESULTS: To assess the function of catK in atherosclerosis, catK(-/-)/apolipoprotein (apo) E(-/-) mice were generated. At 26 weeks of age, plaque area in the catK(-/-)/apoE(-/-) mice was reduced (41.8%) owing to a decrease in the number of advanced lesions as well as a decrease in individual advanced plaque area. This suggests an important role for catK in atherosclerosis progression. Advanced plaques of catK(-/-)/apoE(-/-) mice showed an increase in collagen content. Medial elastin fibers were less prone to rupture than those of apoE(-/-) mice. Although the relative macrophage content did not differ, individual macrophage size increased. In vitro studies of bone marrow derived-macrophages confirmed this observation. Scavenger receptor-mediated uptake (particularly by CD36) of modified LDL increased in the absence of catK, resulting in an increased macrophage size because of increased cellular storage of cholesterol esters, thereby enlarging the lysosomes. CONCLUSIONS: A deficiency of catK reduces plaque progression and induces plaque fibrosis but aggravates macrophage foam cell formation in atherosclerosis.

Genome-wide expression studies of atherosclerosis: critical issues in methodology, analysis, interpretation of transcriptomics data.

During the past 6 years, gene expression profiling of atherosclerosis has been used to identify genes and pathways relevant in vascular (patho)physiology. This review discusses some critical issues in the methodology, analysis, and interpretation of the data of gene expression studies that have made use of vascular specimens from animal models and humans. Analysis of gene expression studies has evolved toward the genome-wide expression profiling of large series of individual samples of well-characterized donors. Despite the advances in statistical and bioinformatical analysis of expression data sets, studies have not yet fully exploited the potential of gene expression data sets to obtain novel insights into the molecular mechanisms underlying atherosclerosis. To assess the potential of published expression data, we compared the data of a CC chemokine gene cluster between 18 murine and human gene expression profiling articles. Our analysis revealed that an adequate comparison is mainly hindered by the incompleteness of available data sets. The challenge for future vascular genomic profiling studies will be to further improve the experimental design, statistical, and bioinformatical analysis and to make data sets freely accessible.

Is there more than C-reactive protein and fibrinogen? The prognostic value of soluble CD40 ligand, interleukin-6 and oxidized low-density lipoprotein with respect to coronary and cerebral vascular disease.

Incidence of atherosclerosis and atherosclerosis-related complications will increase significantly in the coming decennia. Research identified many serum and plasma markers that are associated with cardiovascular disease. However, little is known about the prognostic value of these markers to identify patients at risk for future cardiovascular events. Therefore, we aimed to investigate the prognostic value of three of these markers (soluble CD40 ligand (sCD40L), interleukin-6 (IL-6) and oxidized low-density lipoprotein (oxLDL)) with respect to coronary vascular disease and stroke. For this reason the Medline database was searched for the period January 1999-January 2005. To be selected in our study, concentration of the marker had to be determined at baseline, follow-up period had to be longer than 3 months and an estimate of relative risk had to be available. Based on these criteria, 4 studies for sCD40L, 10 for IL-6 and 2 for oxLDL were selected. Relative risk estimates adjusted for potential confounders varied between 1.9 and 2.8 for sCD40L, between 1.1 and 3.1 for IL-6 and between 1.9 and 3.2 for oxLDL. In conclusion, this systematic review shows that sCD40L, IL-6 and oxLDL are associated with an increased relative risk of developing cardiovascular disease.

Hypoxia induces aortic hypertrophic growth, left ventricular dysfunction, and sympathetic hyperinnervation of peripheral arteries in the chick embryo.

BACKGROUND: Low birth weight is associated with an increased incidence of cardiovascular diseases, including hypertension, later in life. This suggests that antenatal insults program for fetal adaptations of the circulatory system. In the present study, we evaluated the effects of mild hypoxia on cardiac function, blood pressure control, and arterial structure and function in near-term chick embryos. METHODS AND RESULTS: Chick embryos were incubated under normoxic (21% O2) or hypoxic (15% O2) conditions and evaluated at incubation day 19 by use of histological techniques, isolated heart preparations, and in vivo measurements of sympathetic arterial tone and systemic hemodynamics. Chronic hypoxia caused a 33% increase in mortality and an 11% reduction in body weight in surviving embryos. The lumen of the ascending aorta in hypoxic embryos was 23% smaller. Left ventricular systolic pressure was 22% lower, and heart weight/body weight ratio was 14% higher. In resistance arteries of hypoxic embryos, in vivo baseline tone was 23% higher, norepinephrine sensitivity was similar, and norepinephrine release from sympathetic nerves increased 2-fold, indicating sympathetic hyperinnervation. Mean arterial pressure and heart rate were similar under resting conditions, but chronically hypoxic embryos failed to maintain blood pressure during acute stress. CONCLUSIONS: This study indicates that mild hypoxia during embryonic development induces alterations in cardiac and vascular function and structure and affects hemodynamic regulation. These findings reveal that antenatal insults have profound effects on the control and design of the circulatory system that are already established at birth and may program for hypertension and heart failure at a later age.

Atherosclerosis in APOE*3-Leiden transgenic mice: from proliferative to atheromatous stage.

BACKGROUND: This study documents (1) the progression of atherosclerosis along the entire arterial tree in APOE*3-Leiden mice after 1, 4, 6, 9, and 12 months of a high-fat/high-cholesterol (HFC) diet and (2) the amount and phenotype of DNA-synthesizing and apoptotic cells in different lesion types after 6 months of HFC diet. METHODS AND RESULTS: Diet duration was correlated with a craniocaudal progression of lesion development and with an increase in severity of the lesion. Typically, the lesions contained smooth muscle cells, macrophages, and T lymphocytes and were covered by an intact endothelium. Whereas DNA synthesis (BrdU uptake) was usually elevated in type II lesions (8.6+/-0.8% versus 1.0+/-0.2% in the nondiseased arterial wall; P<0.05), apoptosis was found primarily in advanced lesions (type IV, 1.3+/-0.1% and type V, 1.2+/-0.2% versus 0.04+/-0.04% in the nondiseased arterial wall [P<0.05]). Cell phenotyping revealed that the majority of DNA synthesis and apoptosis was confined to the macrophage-derived foam cell (68.6+/-3. 0% and 82.2+/-4.6%, respectively). CONCLUSIONS: This study shows that in APOE*3-Leiden mice, duration of an HFC diet is associated with (1) a craniocaudal progression of lesion development and (2) an increased complexity of atherosclerotic lesions. Furthermore, DNA synthesis is predominant in early lesions, whereas apoptosis is present mainly in more advanced lesions. Both parameters of cell turnover are confined primarily to the macrophage-derived foam cell.

Leukocyte CD40L deficiency affects the CD25(+) CD4 T cell population but does not affect atherosclerosis.

Inhibition of CD40-CD40L interactions results in a reduction of innate regulatory T cells (Tregs) in CD40(-/-) mice and induces a stable plaque phenotype in atherosclerosis-prone mouse strains. Here we investigated the effects of leukocyte CD40L on the Treg population and on atherosclerosis. LDLR(-/-) mice were reconstituted with wild-type or CD40L(-/-) bone marrow (BM). These BM chimeras were analysed by flow cytometry for the presence of innate Tregs (CD45RB(low) CD25(+) CD4) in lymphoid organs and peripheral blood. As in CD40(-/-) mice, the CD45RB(high):CD45RB(low) CD4 T cell ratio significantly increased and the CD25(+) CD4(+) subpopulation significantly decreased in LDLR(-/-) mice receiving CD40L(-/-) BM compared to LDLR(-/-) mice receiving wild-type BM. However, atherosclerotic plaque progression and plaque phenotype did not change in LDLR(-/-) mice reconstituted with CD40L(-/-) BM. In conclusion, the present study shows that CD40-CD40L interactions on leukocytes are essential for the size of the CD45RB(low) CD25(+) CD4 Treg subpopulation. Nevertheless, CD40L deficiency on hemopoietic cells did not affect atherosclerosis, implying that CD40L expressing leukocytes alone are not responsible for the stable plaque phenotype observed after total CD40L blockade.

Compensatory enlargement and stenosis develop in apoE(-/-) and apoE*3-Leiden transgenic mice.

Atherosclerotic mouse models develop little ischemic organ damage and no infarctions, despite the presence of large atherosclerotic lesions. Therefore, we hypothesize that luminal changes do not follow atherosclerotic lesion development. Because a phenomenon that may explain the discrepancy between luminal changes and lesion size is vascular remodeling, we measured parameters of vascular remodeling in the carotid arteries (CAs), thoracic aorta (TA), and abdominal aorta (AA) of apolipoprotein E (apoE)-deficient (apoE(-/-)) and apoE*3-Leiden mice, 2 well-known mouse models of atherosclerosis. Atherosclerotic lesions were classified (American Heart Association [AHA] types II through V), and plaque thickness, compensatory enlargement versus constrictive remodeling, lumen diameter, stenosis, and media thickness were measured relative to the nondiseased arterial wall. In CAs, plaque thickness increased during atherogenesis. CAs showed compensatory enlargement (apoE(-/-) 55%, apoE*3-Leiden 38%). Regression analysis revealed a positive correlation between plaque and lumen area (for apoE(-/-), R=0.95; for apoE*3-Leiden, R=0.90). Medial thinning and elastolysis were also observed. During atherogenesis, lumen diameter decreased (apoE(-/-) -69%, apoE*3-Leiden -40%), and stenosis >70% developed. TA and AA showed similar features, but neither developed a progressive decrease in lumen diameter or stenosis >70%. In CAs, TA, and AA of apoE(-/-) and apoE*3-Leiden mice, atherogenesis is associated with compensatory enlargement, medial thinning, and elastolysis. A progressive decrease in lumen diameter and stenoses >70% occur only in CAs. Vascular remodeling is more prominent in apoE(-/-) mice.

Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques.

In the present study, we examined the expression of regulators of bone formation and osteoclastogenesis in human atherosclerosis because accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. The most striking finding of this study was the constitutive immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein in nondiseased aortas and the absence of bone morphogenetic protein (BMP)-2, BMP-4, osteopontin, and osteonectin in nondiseased aortas and early atherosclerotic lesions. When atherosclerotic plaques demonstrated calcification or bone formation, BMP-2, BMP-4, osteopontin, and osteonectin were upregulated. Interestingly, this upregulation was associated with a sustained immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein. The 2 modulators of osteoclastogenesis (osteoprotegerin [OPG] and its ligand, OPGL) were present in the nondiseased vessel wall and in early atherosclerotic lesions. In advanced calcified lesions, OPG was present in bone structures, whereas OPGL was only present in the extracellular matrix surrounding calcium deposits. The observed expression patterns suggest a tight regulation of the expression of bone matrix regulatory proteins during human atherogenesis. The expression pattern of both OPG and OPGL during atherogenesis might suggest a regulatory role of these proteins not only in osteoclastogenesis but also in atherosclerotic calcification.

Chronic myocardial infarction in the mouse: cardiac structural and functional changes.

OBJECTIVES: We studied the effects of chronic left coronary artery ligation on cardiac structure and function in the mouse. METHODS: Morphometric studies of the left ventricle were performed in coronary artery-ligated and sham-operated animals at one, two, three and five weeks after surgery. The fraction of DNA-synthesizing cells was determined as the fraction of cells incorporating 5'-bromo-2'-deoxyuridine, which was infused by osmotic minipumps one week before sacrifice. Collagen content of the septum was determined morphometrically. Left ventricular pressure and its derivatives were measured in separate groups of animals at one and three weeks after surgery. RESULTS: Ligation of the main left coronary artery resulted in antero-apical infarction of the left ventricular wall, involving approximately 40% of left ventricular circumference. Infarction resulted in thinning of the infarcted area and left ventricular dilatation. DNA synthesis increased, peaking between one and two weeks in the border-zone of the infarct (22-fold), septum (ten-fold) and right ventricle (five-fold). At five weeks, DNA synthesis was still increased in the border zone of the infarct. Septal collagen content increased approximately eight-fold in infarcted mice at two weeks, and decreased thereafter; it was still significantly elevated at five weeks. Left ventricular systolic pressure, and maximal positive and negative dP/dt decreased following infarction; left ventricular end-diastolic pressure was elevated at three weeks, but this effect was not statistically significant. CONCLUSION: These data provide basic information on changes in cardiac structure and function in mice following chronic coronary artery ligation. They indicate the feasibility of induction of chronic myocardial infarction in this species. Furthermore, they show the similarity of cardiac structural and functional consequences of chronic myocardial infarction in mice to those previously described in rats.

Biphasic pattern of cell turnover characterizes the progression from fatty streaks to ruptured human atherosclerotic plaques.

OBJECTIVE: To study the amount and phenotype of DNA-synthesizing and apoptotic cells during atherogenesis. METHODS: Atherosclerotic lesions (n = 76), obtained at autopsy (N = 6) or during vascular surgery (N = 8), were classified [type I-VI; American Heart Association (AHA) classification], immunolabeled with MIB 1 or the TUNEL technique and double stained with cell-type-specific antibodies. Subsequently, the labeled fractions were quantified. RESULTS: In type II-VI lesions, intimal DNA synthesis was increased compared to that of the non-diseased (ND) arterial wall. DNA synthesis peaked in early type II lesions (2.7 +/- 0.5 vs. 0.02 +/- 0.02% in ND; p < 0.05), and declined to 0.7 +/- 0.2% in type V lesions (p < 0.05). Interestingly, a second peak of DNA synthesis of 1.7 +/- 0.1%, was observed in type VI (ruptured plaque) lesions. Double staining revealed that DNA synthesis was mostly confined to the macrophage-derived foam cell (51.9%). In type II lesions, 100.0% of all DNA-synthesizing cells were present in the intimal foam cell-rich area, while in advanced type III, IV and V lesions, DNA synthesis had shifted to the shoulder region (74.8, 78.5 and 68.1%, respectively). In type VI lesions, DNA synthesis was present in the area underlying the plaque rupture (52.7%). Apoptosis was only elevated in advanced type IV, V and VI lesions (0.8 +/- 0.1, 0.8 +/- 0.1 and 1.1 +/- 0.1%, respectively, vs. 0.0 +/- 0.0% in ND) and was predominant in the lipid core (90.5% in type IV lesions; 54.2% in type V lesions) or equally divided between the lipid core and the region underlying the plaque rupture (31.8 and 34.6% in type VI lesions). In type III-VI lesions, 50.0, 38.9, 42.6 and 42.8% of the TUNEL-positive cells were macrophages. CONCLUSIONS: In stable atherosclerotic lesions, DNA synthesis is an early event, while apoptosis is a late event. Ruptured plaques show a second peak of cell turnover. Lastly, cell turnover is mostly confined to the macrophage-derived foam cell.

An inflammatory link in atherosclerosis and obesity. Co-stimulatory molecules.

Atherosclerosis and obesity-induced metabolic dysfunction are lipid-driven inflammatory pathologies responsible for a major part of cardiovascular complications. Immune cell activation as well as interactions between the different immune cells is dependent on and controlled by a variety of co-stimulatory signals. These co-stimulatory signals can either aggravate or ameliorate the disease depending on the stage of the disease, the cell-types involved and the signal transduction cascades initiated. This review focuses on the diverse roles of the most established co-stimulatory molecules of the B7 and Tumor Necrosis Factor Receptor (TNFR) families, ie the CD28/CTLA4-CD80/CD86 and CD40L/CD40 dyads in the pathogenesis of atherosclerosis and obesity. In addition, we will explore their potential as therapeutic targets in both atherosclerosis and obesity.

Neutrophils in atherosclerosis. A brief overview.

Atherosclerosis is a chronic inflammation of the arterial wall and the continuous infiltration of leukocytes into the plaque enhances the progression of the lesion. Because of the scarce detection of neutrophils in atherosclerotic plaques compared to other immune cells, their contribution was largely neglected. However, in the last years studies have accumulated pointing towards the contribution of neutrophils to atherogenesis. In addition, studies are emerging implying a role for neutrophils in advanced atherosclerosis and/or plaque destabilization. Thus, this brief review delivers an overview of the role of neutrophils during early and late stage atherosclerosis.

The APO(*)E3-Leiden mouse as an animal model for basal laminar deposit.

AIM: To investigate the APO(*)E3-Leiden mouse as an animal model for age related maculopathy (ARM) related extracellular deposits. METHODS: Eyes were obtained from APO(*)E3-Leiden transgenic mice on a high fat/cholesterol (HFC) diet (n=12) or on a normal mouse chow (n=6), for 9 months. As controls, eyes were collected from APO-E knockout mice on the same diets. From each mouse one eye was processed for microscopic evaluation and immunohistochemistry with a polyclonal antibody directed against human apo-E. Electron microscopy was also performed. RESULTS: All 12 eyes of the APO(*)E3-Leiden mice on an HFC diet contained basal laminar deposit (BLD; class 1 to class 3), whereas two of six APO(*)E3-Leiden mice on normal chow showed BLD class 1. The ultrastructural aspects of this BLD were comparable with those seen in early BLD in humans, and BLD showed immunoreaction with anti-human-apo-E antibodies. No BLD was found in any of the control mice. Drusen were not detected in any of the mice. CONCLUSION: These results indicate that APO(*)E3-Leiden mice can be used as animal model for the pathogenesis of BLD, and that a HFC diet enhances the accumulation of this deposit. Furthermore, this study supports the previously suggested involvement of dysfunctional apo-E in the accumulation of extracellular deposits in ARM.

Leukocyte counts, myeloperoxidase, and pregnancy-associated plasma protein a as biomarkers for cardiovascular disease: towards a multi-biomarker approach.

We evaluated leukocyte counts and levels of CRP, fibrinogen, MPO, and PAPP-A in patients with stable and unstable angina pectoris, acute myocardial infarction, and healthy controls. All biomarkers were analyzed again after 6 months. Leukocyte counts and concentrations of fibrinogen, CRP, MPO, and PAPP-A were significantly increased in patients with acute myocardial infarction. Leukocyte counts and concentrations of MPO were significantly increased in patients with unstable angina pectoris compared with controls. After 6 months, leukocyte counts and MPO concentrations were still increased in patients with acute myocardial infarction when compared to controls. Discriminant analysis showed that leukocyte counts, MPO, and PAPP-A concentrations classified study group designation for acute coronary events correctly in 83% of the cases. In conclusion, combined assessment of leukocyte counts, MPO, and PAPP-A was able to correctly classify acute coronary events, suggesting that this could be a promising panel for a multibiomarker approach to assess cardiovascular risk.