Therapy with high-dose statins reduces soluble P-selectin: The impact on plasma fibrin clot properties.

OBJECTIVE: Studies on the effect of statins on platelets in patients with coronary artery disease (CAD) yielded inconsistent results. We sought to investigate whether high-dose statin therapy reduces plasma concentrations of soluble P-selectin (sP-selectin), a well-established platelet activation marker and if such changes can affect fibrin clot properties, which are unfavorably altered in CAD patients. METHODS: We studied 130 consecutive patients with advanced CAD who did not achieve the target LDL cholesterol on statins. At baseline and after 6-12 months of treatment with atorvastatin 80 mg/day or rosuvastatin 40 mg/day, soluble plasma sP-selectin, along with plasma fibrin clot permeability (Ks), clot lysis time (CLT), thrombin generation and fibrinolysis proteins were determined. RESULTS: Before high-intensity statin treatment, lower Ks and longer CLT values were associated with increased sP-selectin (ß -0.27 [95% CI -0.44 to -0.10] and ß 0.21 [95% CI 0.01 to 0.41]; both p < 0.05, respectively) also after adjustment for potential confounders. sP-selectin, alongside fibrin features and other variables at baseline showed no association with lipid profile. On high-dose statin therapy, there was 32% reduction in sP-selectin levels (p < 0.001). On-treatment change (Δ) in sP-selectin correlated with ΔKs and ΔCLT (r = -0.32, p < 0.001 and r = 0.22, p = 0.011, respectively), but not with cholesterol and C-reactive protein lowering. We did not observe any associations between post-treatment sP-selectin levels and lipids, fibrin clot properties or thrombin generation. CONCLUSIONS: High-dose statin therapy reduces markedly sP-selectin levels in association with improved fibrin clot phenotype, which highlights the contribution of platelet-derived proteins to a prothrombotic state in hypercholesterolemia and statin-induced antithrombotic effects.

Intensive low-density lipoprotein cholesterol lowering improves fibrin clot properties: Association with lipoproteins and C-reactive protein.

OBJECTIVE: Dense fibrin networks resistant to lysis characterize coronary artery disease (CAD) patients. We investigated whether a statin-induced decrease of low-density lipoprotein cholesterol (LDL-C) could improve fibrin clot phenotype in CAD patients. METHODS: We recruited 130 consecutive patients with advanced CAD (baseline LDL-C of 4.4 [IQR, 3.8-4.8] mmol/L), who on statins did not achieve the LDL-C goal based on the 2016 ESC/EAS guidelines. On standard statin treatment and after 6-12 months of high-dose statin treatment (atorvastatin 80 mg/day or rosuvastatin 40 mg/day), plasma fibrin clot permeability (Ks), clot lysis time (CLT), thrombin generation, coagulation and fibrinolytic factors were determined. RESULTS: After a median high-dose statin therapy of 7 months there was 25% reduction in LDL-C associated with increased Ks and shorter CLT, together with lower thrombin activatable fibrinolysis inhibitor, factor VIII, D-dimer, and C-reactive protein (CRP); thrombin generation was unaltered. The patients who achieved the therapeutic goal (n = 49, 37.7%) had 29.2% increase in Ks and 16.3% shorter CLT compared with the standard therapy, while there were no similar changes in the remaining patients. After adjustment for potential confounders, including CRP, an increase in Ks (by 1 × 10-9 cm2) and decrease in CLT (by 10 min) were independently predicted by on-treatment LDL-C goal (odds ratio [OR] 6.23, 95% confidence interval [CI] 1.97-20.33 and OR 3.11, 95% CI 1.05-8.99, respectively). CONCLUSIONS: For the first time we showed that a decrease of LDL-C ≤ 1.8 mmol/L, or a reduction of at least 50% if the baseline LDL-C is between 1.8 and 3.5 mmol/L, is associated with favorable alterations to fibrin clot phenotype, with a stronger impact of lipoprotein reduction than CRP lowering, which might suggest that other potent cholesterol-lowering drugs can exert similar antithrombotic actions.

Inhibition of Atherosclerosis and Liver Steatosis by Agmatine in Western Diet-Fed apoE-Knockout Mice Is Associated with Decrease in Hepatic De Novo Lipogenesis and Reduction in Plasma Triglyceride/High-Density Lipoprotein Cholesterol Ratio.

Atherosclerosis and NAFLD are the leading causes of death worldwide. The hallmark of NAFLD is triglyceride accumulation caused by an imbalance between lipogenesis de novo and fatty acid oxidation. Agmatine, an endogenous metabolite of arginine, exerts a protective effect on mitochondria and can modulate fatty acid metabolism. In the present study, we investigate the influence of agmatine on the progression of atherosclerotic lesions and the development of hepatic steatosis in apoE-/- mice fed with a Western high-fat diet, with a particular focus on its effects on the DNL pathway in the liver. We have proved that treatment of agmatine inhibits the progression of atherosclerosis and attenuates hepatic steatosis in apoE-/- mice on a Western diet. Such effects are associated with decreased total macrophage content in atherosclerotic plaque as well as a decrease in the TG levels and the TG/HDL ratio in plasma. Agmatine also reduced TG accumulation in the liver and decreased the expression of hepatic genes and proteins involved in lipogenesis de novo such as SREBP-1c, FASN and SCD1. In conclusion, agmatine may present therapeutic potential for the treatment of atherosclerosis and fatty liver disease. However, an exact understanding of the mechanisms of the advantageous actions of agmatine requires further study.

Diminazene Aceturate Stabilizes Atherosclerotic Plaque and Attenuates Hepatic Steatosis in apoE-Knockout Mice by Influencing Macrophages Polarization and Taurine Biosynthesis.

Atherosclerosis and nonalcoholic fatty liver disease are leading causes of morbidity and mortality in the Western countries. The renin-angiotensin system (RAS) with its two main opposing effectors, i.e., angiotensin II (Ang II) and Ang-(1-7), is widely recognized as a major regulator of cardiovascular function and body metabolic processes. Angiotensin-converting enzyme 2 (ACE2) by breaking-down Ang II forms Ang-(1-7) and thus favors Ang-(1-7) actions. Therefore, the aim of our study was to comprehensively evaluate the influence of prolonged treatment with ACE2 activator, diminazene aceturate (DIZE) on the development of atherosclerotic lesions and hepatic steatosis in apoE-/- mice fed a high-fat diet (HFD). We have shown that DIZE stabilized atherosclerotic lesions and attenuated hepatic steatosis in apoE-/- mice fed an HFD. Such effects were associated with decreased total macrophages content and increased α-smooth muscle actin levels in atherosclerotic plaques. Moreover, DIZE changed polarization of macrophages towards increased amount of anti-inflammatory M2 macrophages in the atherosclerotic lesions. Interestingly, the anti-steatotic action of DIZE in the liver was related to the elevated levels of HDL in the plasma, decreased levels of triglycerides, and increased biosynthesis and concentration of taurine in the liver of apoE-/- mice. However, exact molecular mechanisms of both anti-atherosclerotic and anti-steatotic actions of DIZE require further investigations.

The Influence of Trehalose on Atherosclerosis and Hepatic Steatosis in Apolipoprotein E Knockout Mice.

Atherosclerosis and nonalcoholic fatty liver disease (NAFLD) are frequent causes of death in the Western countries. Recently, it has been shown that autophagy dysfunction plays an important role in the pathogenesis of both atherosclerosis and NAFLD; thus, activators of autophagy might be useful for novel therapeutic interventions. Trehalose-a naturally occuring disaccharide present in plants, bacteria, fungi, insects, and certain types of shrimps-is a known inducer of autophagy. However, according to the literature, its anti-atherosclerotic and anti-steatotic potential seem to depend on the experimental setting. The aim of our study was to comprehensively describe the influence of a prolonged treatment with orally administered trehalose on the development of atherosclerotic lesions and hepatic steatosis in apolipoprotein E knockout (apoE-/-) mice in an experimental set up reflecting both moderate and severe proatherogenic conditions: male apoE-/- mice on a chow diet (CD) and female apoE-/- mice fed with a high-fat diet (HFD). We found that exogenous trehalose inhibited atherosclerosis and attenuated hepatic steatosis in apoE-/- mice. Such effects of trehalose were not associated with changes of plasma cholesterol, low-density lipoproteins (LDL), or high-density lipoproteins (HDL). Moreover, the anti-steatotic action of trehalose in the liver was associated with the induction of autophagy. The exact molecular mechanisms of both the anti-atherosclerotic action of trehalose and its inhibitory effect on liver steatosis require further clarification.

Quantitative proteomics reveals decreased expression of major urinary proteins in the liver of apoE/eNOS-DKO mice.

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) plays an important role, not only in endothelium-dependent vasodilation but also in lipid and glucose homeostasis in the liver and exerts beneficial effects on mitochondrial biogenesis and respiration. Thus, the aim of our study was to use iTRAQ-based quantitative proteomics to investigate the changes in protein expression in the mitochondrial and cytosolic fractions isolated from the liver of the double (apolipoprotein E (apoE) and eNOS) knockout (apoE/eNOS-DKO) mice as compared to apoE KO mice (apoE-/- ) - an animal model of atherosclerosis and hepatic steatosis. Collectively, the deficiency of eNOS resulted in increased expression of proteins related to gluconeogenesis, fatty acids and cholesterol biosynthesis as well as the decreased expression of proteins participated in triglyceride breakdown, cholesterol transport, protein transcription & translation and processing in endoplasmic reticulum (ER). Moreover, one of the most downregulated proteins were major urinary proteins (MUPs), which are abundantly expressed in the liver and were shown to be involved in the regulation of lipid and glucose metabolism. The exact functional consequences of the revealed alterations require further investigation.

Anti-Atherosclerotic Action of Agmatine in ApoE-Knockout Mice.

Atherosclerosis is an inflammatory disease in which dysfunction of mitochondria play an important role, and disorders of lipid management intensify this process. Agmatine, an endogenous polyamine formed by decarboxylation of arginine, exerts a protective effect on mitochondria and modulates fatty acid metabolism. We investigated the effect of exogenous agmatine on the development of atherosclerosis and changes in lipid profile in apolipoprotein E knockout (apoE-/-) mice. Agmatine caused an approximate 40% decrease of atherosclerotic lesions, as estimated by en face and cross-section methods with an influence on macrophage but not on smooth muscle content in the plaques. Agmatine treatment did not changed gelatinase activity within the plaque area. What is more, the action of agmatine was associated with an increase in the number of high density lipoproteins (HDL) in blood. Real-Time PCR analysis showed that agmatine modulates liver mRNA levels of many factors involved in oxidation of fatty acid and cholesterol biosynthesis. Two-dimensional electrophoresis coupled with mass spectrometry identified 27 differentially expressed mitochondrial proteins upon agmatine treatment in the liver of apoE-/- mice, mostly proteins related to metabolism and apoptosis. In conclusion, prolonged administration of agmatine inhibits atherosclerosis in apoE-/- mice; however, the exact mechanisms linking observed changes and elevations of HDL plasma require further investigation.

Polymorphism rs7023923 and monocyte count in blood donors and coronary artery disease patients.

BACKGROUND: The importance of the role of monocytes in coronary artery disease (CAD) is well documented. An increased number of circulating monocytes is associated with higher incidence of CAD. Both environmental and genetic factors influence monocytosis. The latter have been extensively studied since the development of high-throughput genome-wide association studies. Several associations between polymorphisms and monocytosis were found among healthy individuals; the first example was rs7023923. The magnitude of the association of studied polymorphisms with the trait of interest is often confounded by environmental factors and may therefore differ between patient and healthy populations. It is very important to determine the magnitude of the association among patients to predict outcome of the disease, e.g. myocardial infarction. AIM: To determine whether the magnitude of association of rs7023923 with monocytosis, previously reported among healthy volunteers, is similar in patients in whom diagnosis of CAD was determined during elective coronarography. METHODS AND RESULTS: Leucocytosis and neutrophilocytosis were higher among patients with CAD, while thrombocytosis was lower. Monocyte count did not differ among the studied groups (p = 0.25). We confirmed the association of rs7023923 with monocytosis among healthy blood donors (p = 0.0156) but not among patients admitted for elective coronarography (p = 0.61). Inclusion of the age and sex of patients in the statistical model did not modify the results. CONCLUSIONS: Our data suggest that translation of the results of genetic association with the studied traits from healthy to patient population should be implemented with caution. It is possible that numerous environmental factors, which discriminate healthy volunteers from CAD patients, confound the magnitude of genetic associations and make interpretation of the data in patients less clear.

Mitochondrial aldehyde dehydrogenase activation by Alda-1 inhibits atherosclerosis and attenuates hepatic steatosis in apolipoprotein E-knockout mice.

BACKGROUND: Mitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE(-/-)) mice. METHODS AND RESULTS: Alda-1 caused decrease of atherosclerotic lesions approximately 25% as estimated by "en face" and "cross-section" methods without influence on plasma lipid profile, atherosclerosis-related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda-1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda-1-treated apoE(-/-) mice. Alda-1 attenuated formation of 4-hydroxy-2-nonenal (4-HNE) protein adducts and decreased triglyceride content in liver tissue. Two-dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda-1 treatment in liver of apoE(-/-) mice, mostly proteins related to metabolism and oxidative stress. The most up-regulated were the proteins that participated in beta oxidation of fatty acids. CONCLUSIONS: Collectively, Alda-1 inhibited atherosclerosis and attenuated NAFLD in apoE(-/-) mice. The pattern of changes suggests a beneficial effect of Alda-1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda-1 action require further investigation.

Inactivation of heparin by cationically modified chitosan.

This study was performed to evaluate the ability of N-(2-hydroxypropyl)-3-tri methylammonium chitosan chloride (HTCC), the cationically modified chitosan, to form biologically inactive complexes with unfractionated heparin and thereby blocking its anticoagulant activity. Experiments were carried out in rats in vivo and in vitro using the activated partial thromboplastin time (APTT) and prothrombin time (PT) tests for evaluation of heparin anticoagulant activity. For the first time we have found that HTCC effectively neutralizes anticoagulant action of heparin in rat blood in vitro as well as in rats in vivo. The effect of HTCC on suppression of heparin activity is dose-dependent and its efficacy can be comparable to that of protamine-the only agent used in clinic for heparin neutralization. HTCC administered i.v. alone had no direct effect on any of the coagulation tests used. The potential adverse effects of HTCC were further explored using rat experimental model of acute toxicity. When administered i.p. at high doses (250 and 500 mg/kg body weight), HTCC induced some significant dose-dependent structural abnormalities in the liver. However, when HTCC was administered at low doses, comparable to those used for neutralization of anticoagulant effect of heparin, no histopathological abnormalities in liver were observed.

The effect of AVE 0991, nebivolol and doxycycline on inflammatory mediators in an apoE-knockout mouse model of atherosclerosis.

BACKGROUND: The aim of this study was to investigate whether the 3 different substances that can decrease the development of atherosclerosis--nebivolol, AVE 0991 and doxycycline--could at the same time diminish the level of inflammatory indicators interleukin-6 (IL-6), interleukin-12 (IL-12), serum amyloid A (SAA), and monocyte chemotactic protein-1 (MCP-1). MATERIAL/METHODS: Forty 8-week-old female apoE-knockout mice on the C57BL/6J background were divided into 4 groups and put on chow diet for 4 months. Three experimental groups received the same diet as a control group, mixed with AVE 0991 at a dose 0.58 µmol per kg of body weight per day, nebivolol at a dose 2.0 µmol per kg of body weight per day, and doxycycline at a dose 1.5 mg per kg of body weight per day. At the age of 6 months, the mice were sacrificed. RESULTS: All inflammatory indicators (MCP-1, IL-6, IL-12 and SAA) were diminished by AVE 0991. There was also a tendency to lower MCP-1, IL-6, IL-12 and SAA levels by nebivolol and doxycycline; however, it did not reach statistical significance. CONCLUSIONS: Of the 3 presented substances, only AVE 0991 was able to diminish the rise of inflammatory markers. Therefore, drug manipulations in the renin-angiotensin-aldosterone axis seem to be the most promising in the future treatment of atherogenesis.

The role of an experimental model of atherosclerosis: apoE-knockout mice in developing new drugs against atherogenesis.

Although atherosclerosis was previously thought to be mainly a degenerative disease, it is now well ascertained that its pathogenesis is inflammatory. There was a pivotal role of apoE-knockout mice in understanding the inflammatory background of atherosclerosis. Currently, atherosclerosis is known as a chronic inflammatory disease, in most cases initiated by hypercholesterolemia. Recently, the mouse has become the best model for experimental atherosclerosis. It was in 1992 that the first line of gene targeted mice, namely apolipoprotein E-knockout mice was developed. The apoE-deficient model develops extensive atherosclerotic lesions on a chow diet. The LDL receptor - deficient model has elevated LDL levels, but no lesions, or only very small lesions, form on the chow diet. However, robust lesions do form on the westerntype diet. The creation of apoE- knockout mice has changed the face of atherosclerosis research. Gene-targeted mouse models has changed the face of atherosclerotic research and helped in creation of the new theory of atherosclerosis: as an inflammatory disease. Recently, the mouse has become the best model for experimental atherosclerosis. It was in 1992 that the first line of gene targeted mice, namely apolipoprotein E-knockout mice was developed. The apoE-deficient model develops extensive atherosclerotic lesions on a chow diet. It is also the model in which the lesions have been characterized most thoroughly. The lesions develop into fibrous plaques; however, there is no evidence that plaque rupture occurs in this model. The LDL receptor - deficient model has elevated LDL levels, but no lesions, or only very small lesions, form on the chow diet. However, robust lesions do form on the western-type diet. The creation of apoE-knockout mice has changed the face of atherosclerosis research. Gene-targeted mouse models has changed the face of atherosclerotic research and helped in creation of the new theory of atherosclerosis: as an inflammatory disease. Nowadays, apoE- knockout mice model is therefore used in developing new drugs against atherosclerosis. This review describes how new groups of agents are searched.

Atherosclerosis in 2012: what is new?

Recently, two important issues concerning atherogensis have been raised: first - the role of hemostasis in the progression of atherosclerosis, and second - how the results of experimental animal studies can be translated into humans. There is no direct clinical evidence for the role of the coagulation system in the progression of atherosclerosis, but ample experimental data indicate that platelets and coagulation factors have an important role in the progression of both atherosclerosis and thromboembolism. A new scientific approach is thus needed to assess the actual effect of the hemostatic system on molecular and cellular responses in the vasculature. Although experimental studies helped to unravel numerous factors underlying the pathophysiology of atherosclerosis, there is still a significant gap in the translation of the experimental results to the clinic, and this gap needs to bridged to achieve reliable data from scientific research. Direct translation of the results from mouse studies to human is problematic. Clinical trials should be used more often as an early scientific probe, not just as a pathway to the commercialization of pharmaceuticals or for evaluating comparative efficacy of the agents in clinical use.

Increase in plasma sCD23 levels precedes immunoglobulin E elevation after coronary artery bypass graft surgery.

INTRODUCTION: Recent reports have confirmed an increase in plasma immunoglobulin E (IgE), which had been previously observed in clinical situations associated with tissue injury. Studies on the regulation of IgE levels have pointed to the role of low-affinity IgE receptor, i.e., sFcεRII (soluble CD23 [sCD23]). OBJECTIVES: The aim of the study was to assess the changes in the levels of this receptor in response to surgical injury during coronary artery bypass grafting. PATIENTS AND METHODS: The study group consisted of 33 patients (28 men and 5 women, aged 45-75 years). Blood samples were obtained from all patients before surgery and 24, 48, 72, and 120 hours after the surgery. The expression of FcεRII on B cells was measured using flow cytometry and plasma levels of sCD23 were determined by an enzyme-linked immunosorbent assay. RESULTS: We observed a significant increase in the total number of leukocytes and a significant decrease in the total number of lymphocytes with a simultaneous increase in the proportion of B cells (P <0.001). At the same time, the percentage of CD23-positive B cells (CD19/23+) decreased significantly (P <0.001) at 24 hours after surgery and remained low over the period of 72 hours. The plasma levels of sCD23 increased significantly (P <0.05) at 24 hours after surgery and remained elevated until the end of follow-up. All the above changes in the immune status preceded an increase in plasma IgE levels (P <0.001), which reached peak values on the fifth day after surgery (120 h). CONCLUSIONS: Surgical procedures are associated with a transient increase in plasma IgE levels, which is preceded by an increase in the level of sCD23 and a simultaneous decrease in the expression of CD23 on B cells. FcεRII (CD23) and sFcεRII (sCD23) may be involved in the regulation of IgE levels after trauma.

Proteomic analysis of changes in protein expression in liver mitochondria in apoE knockout mice.

The involvement of both apolipoprotein E (apoE) and mitochondria in lipid metabolism is widely recognized, however there is surprisingly scarce data about the putative mitochondrial action(s) of this protein. The aim of the study was to screen the alterations in liver mitochondrial proteome caused by apoE deficiency. We applied 2DE-LC-MS/MS methodology to investigate the changes in liver mitochondrial protein expression in 6-months old apoE(-/-) mice as compared to C57BL/6J controls. ApoE(-/-), but not C57BL/6J mice developed visible atherosclerotic changes in aorta and mild, diffuse steatosis of the liver. Collectively, 18 differentially expressed proteins were identified in mitochondria, related to apoptosis, antioxidant and detoxifying mechanisms of mitochondria, as well as lipid metabolism and transport. In conclusion, differential proteomic approach revealed several lines of proteomic evidence that mitochondrial function in the liver of apoE(-/-) mice could be altered as a result of overlapping of pathological and compensatory changes in expression of proteins.

The putative role of leukotrienes in experimental atherogenesis.

Since inflammation plays an important role in atherogenesis, during recent years it has become apparent that the 5-lipoxygenase (5-LO) pathway may take a significant part in the pathogenesis of atherosclerosis. These data raised the possibility that antileukotriene drugs may be an effective treatment regimen in atherosclerosis. This review describes the research performed on the apolipoprotein E/low-density lipoprotein receptor-double knockout mice as a model of atherosclerosis. The study has shown that 5-LO activating protein inhibitors and leukotriene receptor blockers decrease atherosclerosis in atherosclerotic mice. The article also discusses the importance of these findings for the future use in the clinic.

Triple immunofluorescence labeling of atherosclerotic plaque components in apoE/LDLR -/- mice.

This paper presents a simple and reliable method of triple immunofluorescence staining that allows simultaneous detection of various cell types present in atherosclerotic plaque of apolipoprotein E and LDL receptor-double knockout (apoE/LDLR -/-) mice. We used combined direct and indirect procedures applying commercially available primary antibodies raised in different species to detect smooth muscle cells (Cy3-conjugated mouse anti-smooth muscle actin, SMA), macrophages (rat anti-CD68) and T lymphocytes (rabbit anti-CD3). Fixation of the material in acetone and modified incubation protocol employing nonfat dry milk in preincubation and incubation media significantly increased the intensity of labeling and effectively quenched the background. Our method offers an efficient way to detect qualitative as well as quantitative changes of macrophages, T lymphocytes and smooth muscle cells in atherosclerotic plaque of apoE/LDLR -/- mice during atherosclerosis development or in response to pharmacological treatment.

New insights into immunological aspects of atherosclerosis.

Although atherosclerosis was previously thought to be mainly a degenerative disease, it is now well ascertained that its pathogenesis is inflammatory. This review describes the history of a new atherogenetic concept, including the pivotal role of apoE-knockout mice in understanding the inflammatory background of atherosclerosis. There has been lack of unequivocal evidence of an important inflammatory component in atherogenesis. This evidence was delivered by a new technique--gene targeting, for the invention of which Mario R. Capecchi, Martin J. Evans and Oliver Smithies received in 2007 the Nobel Prize in Physiology or Medicine. The pivotal stage of atherogenesis is the antigen presentation by macrophages to T lymphocytes. This antigen could be a fragment of oxidized low-density lipoproteins "digested" by macrophage, heat shock protein 60, beta2-glycoprotein I or fragments of bacterial antigens. For interaction between the immunological cells a presence of CD40 receptor on macrophages and its ligand CD40L on the surface of T lymphocytes are necessary. During the interaction between these cells an immunological type T helper 1 (Th1--cellular) or T helper 2 (Th2--humoral) response arises. Th1 response and its mediators: interferon gamma, tumor necrosis factor alpha, interleukin-1, interleukin-12 and interleukin-18 enhance atherogenesis, whereas Th2 response and its mediators: interleukin-4, interleukin-5, interleukin-10 and interleukin-13 inhibit the development of atherosclerosis. Atherosclerosis is therefore a chronic inflammatory disease, in most cases initiated by hypercholesterolemia. Nowadays, hypercholesterolemia and inflammation are considered as "partners in crime". The concept of atherosclerosis as inflammatory disease is fairly new, however, it is already considered as an undisputable achievement of science which have particular therapeutic consequences.

Functional effects of eggs, naturally enriched with conjugated linoleic acid, on the blood lipid profile, development of atherosclerosis and composition of atherosclerotic plaque in apolipoprotein E and low-density lipoprotein receptor double-knockout mice (apoE/LDLR-/-).

The objective of this study was to evaluate potential anti-atherogenic properties of hen eggs enriched naturally with conjugated linoleic acid (CLA) isomers (cis-9, trans-11 and trans-10, cis-12). Eighteen apoE and LDL receptor double-knockout mice (apoE/LDLR- / - ), at the age of 4 months with pre-established atherosclerosis, were randomly assigned to three experimental groups (n 6) and fed AIN-93G-based diets for the next 2 months. The experimental diets were: AIN-93G+ CLA-free egg-yolk powder (control); AIN-93G+ CLA-free egg-yolk powder +0.1 % CLA (CLA-supplemented eggs); and AIN-93G+ CLA-enriched egg-yolk powder, providing 0.1 % CLA (CLA-enriched eggs). For assessment of anti-atherogenic properties of CLA-enriched or CLA-supplemented eggs the following criteria were used: (1) serum lipid profile; (2) development of atherosclerosis; and (3) composition of atherosclerotic plaque. CLA-enriched eggs, compared with CLA-supplemented eggs, reduced significantly (P < 0.05) total plasma cholesterol in the mice. At the same time, both CLA-supplemented eggs and CLA-enriched eggs tended to decrease the size of atherosclerotic plaque in aortic roots of mice. Most importantly, atherosclerotic plaques of mice fed CLA-enriched eggs showed significantly (P < 0.05) reduced number of atherogenic macrophages and increased area occupied by smooth muscle cells in atherosclerotic lesions. In conclusion, CLA-enriched eggs exerted an anti-inflammatory effect more effectively than CLA-supplemented eggs. This anti-inflammatory effect can be considered their major functional claim that warrants further exploitation.

Ticlopidine attenuates progression of atherosclerosis in apolipoprotein E and low density lipoprotein receptor double knockout mice.

Platelets are involved in the development of atherothrombosis. However, the anti-atherosclerotic effects of thienopiridines have not been, as yet, proven. We analyzed the effects of ticlopidine on atherogenesis in apolipoprotein E/low density lipoprotein receptor double knockout (apoE/LDLR(-/-)) mice. 2-month-old apoE/LDLR(-/-) mice fed a Western diet (21% fat, 0.15% cholesterol) were treated with ticlopidine (90 mg/kg/day) for a period of 4 months. In 6-month-old apoE/LDLR(-/-) mice treated with ticlopidine and in their non-treated counterparts we analyzed: cholesterol and triglyceride levels, the size of atherosclerotic plaques in aortic roots (oil red-O staining, cross-section method), and in the whole aorta (Sudan IV staining, en face method), the number of macrophages in atherosclerotic plaque (CD68 staining), as well as the endothelial function in the isolated thoracic aorta. Concentrations of total cholesterol and triglycerides in plasma were not altered by treatment with ticlopidine. However, the size of atherosclerotic plaques measured in aortic roots by the cross-section method and the number of macrophages estimated by anti-CD68 staining were significantly reduced by ticlopidine treatment. In contrast, the effect of ticlopidine on the area covered by plaques in the whole aorta (en face analysis) was not statistically significant. Importantly, acetylcholine-induced vasodilation in isolated aorta was improved in ticlopidine-treated apoE/LDLR(-/-) mice as compared to their non-treated counterparts. In conclusion, ticlopidine attenuates the progression of atherosclerosis and improves the endothelial function in apoE/LDLR(-/-) mice.