Cholesteryl ester transfer protein inhibition as a strategy to reduce cardiovascular risk

Human and rabbit plasma contain a cholesteryl ester transfer protein (CETP) that promotes net mass transfers of cholesteryl esters from high density lipoproteins (HDL) to other plasma lipoprotein fractions. As predicted, inhibition of CETP in both humans and rabbits increases the concentration of cholesterol in the potentially protective HDL fraction, while decreasing it in potentially proatherogenic non-HDL fractions. Inhibition of CETP in rabbits also inhibits the development of diet-induced atherosclerosis. However, use of the CETP inhibitor torcetrapib in humans did not reduce atheroma in three imaging trials and caused an excess of deaths and cardiovascular events in a large clinical outcome trial. The precise explanation for the harm caused by torcetrapib is unknown but may relate to documented, potentially harmful effects unrelated to inhibition of CETP. More recently, a trial using the weak CETP inhibitor dalcetrapib, which raises HDL levels less effectively than torcetrapib and does not lower non-HDL lipoprotein levels, was terminated early for reasons of futility. There was no evidence that dalcetrapib caused harm in that trial. Despite these setbacks, the hypothesis that CETP inhibitors will be antiatherogenic in humans is still being tested in studies with anacetrapib and evacetrapib, two CETP inhibitors that are much more potent than dalcetrapib and that do not share the off-target adverse effects of torcetrapib.     

Rescue of Mtp siRNA-induced hepatic steatosis by DGAT2 siRNA silencing

Microsomal triglyceride transfer protein (Mtp) inhibitors represent a novel therapeutic approach to lower circulating LDL cholesterol, although therapeutic development has been hindered by the observed increase in hepatic triglycerides and liver steatosis following treatment. Here, we used small interfering RNAs (siRNA) targeting Mtp to achieve target-specific silencing to study this phenomenon and to determine to what extent liver steatosis is induced by changes in Mtp expression. We observed that Mtp silencing led to a decrease in many genes involved in hepatic triglyceride synthesis. Given the role of diacylglycerol O-acyltransferase 2 (Dgat2) in regulating hepatic triglyceride synthesis, we then evaluated whether target-specific silencing of both Dgat2 and Mtp were sufficient to attenuate Mtp silencing-induced liver steatosis. We showed that the simultaneous inhibition of Dgat2 and Mtp led to a decrease in plasma cholesterol and a reduction in the accumulation of hepatic triglycerides caused by the inhibition of Mtp. Collectively, these findings provide a proof-of-principle for a triglyceride synthesis/Mtp inhibitor combination and represent a potentially novel approach for therapeutic development in which targeting multiple pathways can achieve the desired response.     

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.     

Potential association of Helicobacter cinaedi with atrial arrhythmias and atherosclerosis

Helicobacter cinaedi has been increasingly recognized as an emerging pathogen. Reports of recurrent bacteremia and isolation of H. cinaedi organisms from a patient with myopericarditis led us to postulate that H. cinaedi is associated with chronic inflammatory cardiovascular diseases such as atrial arrhythmias and atherosclerosis. To assess any association of H. cinaedi with atrial arrhythmias, a retrospective case-control study of patients attending Kumamoto University Hospital from 2005 to 2009 was performed. The arrhythmia status of these patients was determined from their electrocardiography and electrophysiological studies. Multiple logistic regression analysis was used to identify independent risk factors. In a comparison of case patients (n= 132) with control subjects (n= 137), H. cinaedi seropositivity was identified as an independent risk factor for atrial arrhythmia (odds ratio, 5.13; 95% confidence interval, 3.0-8.7; P < 0.001). There were no significant differences, however, between these two groups with respect to anti-H. pylori IgG concentrations, anti-Chlamydophila pneumoniae IgG concentrations, and other studied variables. IgG concentrations against H. cinaedi and H. pylori were inversely correlated, which suggests cross-immunity between these two bacteria. Also, to explore any association of H. cinaedi with atherosclerosis, immunohistochemical analysis of atherosclerotic aortic tissues collected post mortem from nine patients was performed. Immunohistochemistry of atherosclerotic aortic tissues from all nine patients detected H. cinaedi antigens inside CD68(+) macrophages. These findings provide the first evidence, to our knowledge, of a possible association of H. cinaedi with atrial arrhythmias and atherosclerosis.     

Dendritic Cells and Regulatory T Cells in Atherosclerosis

Although macrophages and other immune system cells, especially T cells, have been shown to play disease-promoting roles in atherosclerosis, less is known about the role of antigen presenting cells. Functional, immune stimulating dendritic cells (DCs) have recently been detected in aortic intima, the site of origin of atherosclerosis. We had compared DCs with macrophages in mice with experimental atherosclerosis, to clearly define cell types by developmental and functional criteria. This review summarizes recent advances in studies of DCs in humans and in mouse models of atherosclerosis, as well as providing a simple strategy to measure regulatory T (Treg) cells in the mouse aorta.     

瑞舒伐他汀治疗颈动脉粥样硬化斑块的临床研究

目的:研究瑞舒伐他汀对颈动脉粥样硬化斑块的治疗效果。方法:将在本院接受治疗的250例颈动脉粥样硬化斑块患者随机分成治疗组125例和对照组125例,治疗组服用瑞舒伐他汀10mg/晚,对照组行其他非瑞舒伐他汀药物治疗,进行为期6个月的观察对比。结果:治疗组治疗后总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)水平显著下降,高密度脂蛋白(HDL)水平显著升高(P<0.05),颈动脉内膜-中层厚度(IMT)、斑块面积变小,与治疗前比较,差异有统计学意义(P<0.05);对照组治疗前后无显著性差异(P>0.05)。结论:瑞舒伐他汀对降低血脂、减缓不稳定型心绞痛早期动脉粥样硬化、稳定斑块和预防脑血管疾病起到非常重要的作用。     

C-reactive protein enhances macrophage lipoprotein lipase expression

High serum levels of C-reactive protein (CRP), a strong predictor of cardiovascular events, are documented in patients with type 2 diabetes. Accumulating evidence suggests that CRP could directly promote arterial damage. To determine the role of CRP in diabetic atherosclerosis, we examined the effect of CRP on the expression of macrophage lipoprotein lipase (LPL), a proatherogenic molecule upregulated in type 2 diabetes. Treatment of human macrophages with native CRP increased, in a dose- and time-dependent manner, LPL protein expression and secretion. Modified CRP reproduced these effects. Preincubation of human macrophages with antioxidants, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) inhibitors prevented CRP-induced LPL expression. Exposure of human macrophages to CRP further increased intracellular reactive oxygen species generation, classic PKC isozymes expression, and extracellular signal-regulated protein kinase 1/2 phosphorylation. In CRP-treated J774 macrophages, increased macrophage LPL mRNA levels and enhanced binding of nuclear proteins to the activated protein-1 (AP-1)-enhancing element were observed. These effects were prevented by antioxidants, as well as by PKC, MAPK, and AP-1 inhibitors. These data show for the first time that CRP directly increases macrophage LPL expression and secretion. Given the predominant role of macrophage LPL in atherogenesis, LPL might represent a novel factor underlying the adverse effect of CRP on the diabetic vasculature.     

Safety, pharmacokinetics, and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients

Patients with coronary heart disease or equivalent risk received a single dose of 30, 100, 300, or 500 mg of unformulated D-4F (n = 8, each dose) or placebo (n = 8) under fasting conditions. An additional 10 patients received 500 mg (n = 8) or placebo (n = 2) with a low-fat meal. There were no significant trends in any safety parameter. D-4F was detectable in plasma at all doses with a T(max) of 30 min, 1 h, and 2 h for 30, 100, and > or = 300 mg, respectively. The area under the curve((0-t)) was 27.81 ng/hr/ml and 54.71 ng/hr/ml for the 300 mg and 500 mg dose groups, respectively, and 17.96 ng/hr/ml for the 500 mg dose given with food. HDL from each time point for each subject was tested for its ability to inhibit LDL-induced monocyte chemotactic activity in cultures of human aortic endothelial cells. The values obtained were normalized to 1.0 for LDL alone to obtain the HDL inflammatory index. This index significantly improved at 4 h at the 300 mg dose and at 2 h at the 500 mg dose compared with placebo (P < 0.05). There were no changes in plasma lipid or lipoprotein levels. We conclude that unformulated D-4F has low bioavailability that is improved under fasting conditions, and that a single dose of D-4F is safe and well tolerated and may improve the HDL anti-inflammatory index.     

Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue

Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. Mouse arteries were dissected and placed in chloroform-methanol without tissue grinding. Extracts underwent hydrolysis of cholesteryl esters and derivatization of cholesterol followed by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We demonstrated that FC and CE could be quantitatively extracted without tissue grinding and that lipid extraction simultaneously worked for tissue fixation. Delipidated tissues can be embedded in paraffin, sectioned, and stained. Microscopic images obtained from delipidated arteries have not revealed any structural alterations. Delipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE(-/-) mice, LC/MS/MS revealed early antiatherosclerotic effects of dual PPARalpha,gamma agonist LY465606 in brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in CE and FC accumulation in atherosclerotic lesions was associated with the reduction of lesion size. Thus, a combination of LC/MS/MS measurements of CE and FC followed by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.     

A new mouse mutant for the LDL receptor identified using ENU mutagenesis

In an effort to discover new mouse models of cardiovascular disease using N-ethyl-N-nitrosourea (ENU) mutagenesis followed by high-throughput phenotyping, we have identified a new mouse mutation, C699Y, in the LDL receptor (Ldlr), named wicked high cholesterol (WHC). When WHC was compared with the widely used Ldlr knockout (KO) mouse, notable phenotypic differences between strains were observed, such as accelerated atherosclerotic lesion formation and reduced hepatosteatosis in the ENU mutant after a short exposure to an atherogenic diet. This loss-of-function mouse model carries a single base mutation in the Ldlr gene on an otherwise pure C57BL/6J (B6) genetic background, making it a useful new tool for understanding the pathophysiology of atherosclerosis and for evaluating additional genetic modifiers regulating hyperlipidemia and atherogenesis. Further investigation of genomic differences between the ENU mutant and KO strains may reveal previously unappreciated sequence functionality.