High Plasma Cholesterol Levels Research Articles

Identification by whole-genome resequencing of gene defect responsible for severe hypercholesterolemia

Whole-genome sequencing is a potentially powerful tool for the diagnosis of genetic diseases. Here, we used sequencing-by-ligation to sequence the genome of an 11-month-old breast-fed girl with xanthomas and very high plasma cholesterol levels (1023 mg/dl). Her parents had normal plasma cholesterol levels and reported no family history of hypercholesterolemia, suggesting either an autosomal recessive disorder or a de novo mutation. Known genetic causes of severe hypercholesterolemia were ruled out by sequencing the responsible genes (LDLRAP, LDLR, PCSK9, APOE and APOB), and sitosterolemia was ruled out by documenting a normal plasma sitosterol:cholesterol ratio. Sequencing revealed 3 797 207 deviations from the reference sequence, of which 9726 were nonsynonymous single-nucleotide substitutions. A total of 9027 of the nonsynonymous substitutions were present in dbSNP or in 21 additional individuals from whom complete exonic sequences were available. The 699 novel nonsynonymous substitutions were distributed among 604 genes, 23 of which were single-copy genes that each contained 2 nonsynonymous substitutions consistent with an autosomal recessive model. One gene, ABCG5, had two nonsense mutations (Q16X and R446X). This finding indicated that the infant has sitosterolemia. Thus, whole-genome sequencing led to the diagnosis of a known disease with an atypical presentation. Diagnosis was confirmed by the finding of severe sitosterolemia in a blood sample obtained after the infant had been weaned. These findings demonstrate that whole-genome (or exome) sequencing can be a valuable aid to diagnose genetic diseases, even in individual patients.

A twice-a-day feeding regimen optimizes performance in broiler breeder hens

To evaluate the effects of different methods of feed allocation on performance, plasma hormone concentration, and ovarian morphology, an experiment was conducted using 32 Cobb 500 broiler breeder hens. The experiment was started at 27 wk and lasted to 39 wk of age. Feeding regimens included: i) hens with an ad libitum feeding program, ii) hens that received their restricted feed once a day, iii) hens that received their restricted feed twice a day, and iv) hens that received their restricted feed 3 times a day. Each hen was assumed as an experimental unit and treatments were replicated 8 times. Daily egg production, BW, and egg and yolk weights were measured. Two blood samples were taken 3 and 6 h after the first feed allocation every 2 wk. Plasma samples were assayed for glucose, triacylglycerol (TAG), cholesterol, as well as leptin-like concentration, glucagon, triiodothyronine, thyroxine, progesterone, estradiol, and testosterone. Liver, abdominal fat pad, and ovary were collected at necropsy. Ovaries were weighed and follicles were characterized as large yellow follicles, small yellow follicles, and large white follicles. Results showed inferior egg production in ad libitum-fed birds along with high levels of plasma glucose, TAG, cholesterol, leptin-like concentration, and testosterone. Twice-a-day-fed birds produced more egg in the entire production period than once-a-day-fed birds. Better performance of twice-a-day-fed hens was associated with lower plasma glucose, TAG, and leptin-like concentration, whereas their estradiol and glucagon were higher than once-a-day-fed hens. Results obtained in our study suggest that allocation of restricted feed 2 times a day may alleviate or delay lipotoxicity development and improve reproductive performance in broiler breeder hens.

AAV/hSTAT3-gene delivery lowers aortic inflammatory cell infiltration in LDLR KO mice on high cholesterol

Atherosclerosis is an inflammatory disorder of arteries. Signal transducer and activator of transcription-3 (STAT3), an important signal transduction molecule, responds to a number of interleukins (IL) including IL-10, and has a significant immunosuppressive phenotype. Several studies have suggested a correlation of STAT3 expression with a lower state of inflammation. To investigate the contribution of STAT3 in regulating atherogenesis, we delivered full-length wild type human (h) STAT3 gene by adeno-associated virus type 8 (AAV8) via tail vein into low density lipoprotein knockout (LDLR KO) mice which were then fed high cholesterol diet (HCD). Compared to neomycin resistance (Neo) gene delivery-HCD, hSTAT3 delivery-HCD treatment did not result in significant changes in high plasma cholesterol levels. However, while vessel wall lipids were not directly measured, hSTAT3 delivery did result a significant reduction in aortic anomalies, as determined by larger aortic lumen size, thinner aortic wall thickness, and lower blood velocity than the Neo control (all statistically significant). Moreover, measurements of inflammation/monocyte/macrophage (Mo/MФ) burden, including CD68, ITGAM, EMR-1 and nitrotyrosine were reduced in hSTAT3-HCD-treated animals, while foxp3 (Tregs) and SOCS1 expression were increased. An advantage hSTAT3-gene therapy would have over IL-10 would be a reduced chance of systemic effects as STAT3 is not a secreted protein. While hSTAT3-inhibitory gene delivery has been performed by several groups, delivery of the wild type STAT3 gene has never been attempted before. These data strongly suggest, for the first time, that STAT3 gene delivery can down-regulate Mo/MФ burden and atherosclerosis. These data also suggest the possibility that STAT3 and IL-10 dual gene delivery may result in higher efficacy than either one alone.

대두단백질 및 카제인 섭취가 병아리의 혈액 Cholesterol 함량에 미치는 영향 비교

대두단백질(ISP)이 혈액 콜레스테롤 농도에 미치는 영향을 확인하기 위하여, 카제인, 어분, 쌀단백질, 옥수수 단백질(글루텐 밀) 등과 함께 이들을 각각 함유한 반정제(semi-purified) 사료를 2주간 병아리에게 급여하였다. 병아리는 쥐에 비해 혈액 콜레스테롤에 예민하게 반응하며, 인체의 지질 실험 모델로 더 적합한 동물이다. 각 단백질 사료 마다 NRC 요구량을 기준하여 부족한 필수아미노산(lysine, arginine 등)을 첨가하였다. 모든 실험 사료는 강제 급이 방식으로 1일 3회 급여하여 아미노산을 제외한 모든 영양소의 섭취량을 동일하게 하였다. 실험 2에서는 모든 사료에 콜레스테롤을 0.3% 수준으로 첨가하여 급여하였다. 카제인군의 병아리 증체량이 두 실험 모두 가장 저조하였고(P<0.05), 대두단백질과 어분군의 성장률이 가장 좋았다. 카제인군은 실험 1, 2에서 혈액 총콜레스테롤, non-HDL 콜레스테롤, TG 함량이 유의하게 가장 높았고, 대두단백질 군은 다른 단백질군(쌀 옥수수단백질, 어분)과 대체로 비슷한 수준을 보였다. 어분군은 실험 2에서 혈액 콜레스테롤이 대두 쌀 옥수수단백질군 보다 유의하게 더 높았다. 카제인군은 간 중량이 두 실험 모두 가장 무거웠고 간의 콜레스테롤 및 총지질 함량 도 가장 많았다. 반면에 어분군은 콜레스테롤 함량을 제외하고 대두 쌀 옥수수단백질 군과 같은 수준을 보였다. 카제인군에서 나타난 고콜레스테롤혈증(hypercholesterolemia)은 전형적인 Lys- Arg 길항 작용에 의한 결과이며, 카제인을 섭취한 대부분의 동물에서 나타나는 현상이다. 반면에 대두단백질과 쌀단백질, 옥수수단백질 및 어분군의 혈중 콜레스테롤 함량이 비슷하게 나타나는 것은 사료 Lys/Arg 비율이 카제인 만큼 심하게 높지 않기 때문이라고 생각된다. 결론적으로 대두단백질군의 저콜레스테롤 혈증(hypocholesterolemia)은 카제인에 비교할 때만 나타나는 상대적인 현상으로, 대두단백질 자체가 혈중 콜레스테롤을 저하시킬 수 있는 독자적인 기능을 갖고 있지 않다고 판단된다. Hypocholesterolemic effect of soy protein was examined in comparison with casein and three other dietary protein sources in chicks. In two feeding trials, 40 (Expt.1) or 50 (Expt. 2), three-day-old, male chicks were forced-fed each of five semi-purified-type diets containing isolated soy protein (ISP, cp 82%), casein (cp 92%), rice protein (RP, cp 70%), corn gluten meal (CGM, cp 65%) or fish meal (FM, cp 70%) for two weeks. The diets for Expt. 2 were supplemented with 0.3% cholesterol by replacing glucose. Each protein source was the only source of protein of each diet. Essential amino acids were added to the diets to satisfy their requirements according to NRC. The diets were forced-fed to equalize the intake of all nutrients except the amino acids which were inherently variable in the diets. Chicks fed casein showed lower body weight gain than those fed the other proteins in both experiments. Birds fed ISP and FM gained better body weight than the others. Chicks fed casein showed significantly higher levels of plasma total cholesterol, non-HDL cholesterol and triacylglycerol (TG) than those fed ISP and the other protein sources. Meanwhile, the chicks fed ISP, RP, CGM and FM showed comparable levels of plasma total cholesterol, non-HDL cholesterol and TG. In Expt. 2, the birds fed casein and FM showed markedly elevated plasma total cholesterol and non-HDL cholesterol levels. Liver weight and levels of total lipids and cholesterol of chicks fed casein appeared significantly higher than those of the other protein diets, whereas those of the chicks fed ISP, RP, CGM and FM appeared comparable except cholesterol in FM group. In conclusion, only the chicks fed casein diets in both experiments always showed significantly higher levels of plasma cholesterol and TG compared to those fed ISP and the other protein sources. These results support the views that casein, which has unique lysine-arginine ratio, is inherently hyper-cholesterolemic, and ISP is hypocholesterolemic only when compared to casein.

262 Are Zucker Obese Rats a Useful Model for Cardiovascular Complications in Metabolic Syndrome? Physical, Biochemical and Oxidative Stress Considerations

We wondered if Zucker obese (ZO) rats would be a good experimental model to evaluate cardiovascular complications of Metabolic Syndrome (MS). ZO rats were compared with both their littermate controls, Zucker lean (ZL) rats and to Wistar rats (reference strain). We designed this work 1) to measure certain physical and biochemical characteristics of MS 2) to evaluate coronary and cardiac function in isolated conditions and after ischemia 3) to study plasma and heart tissue oxidative stress markers. In vivo, ZO rats had higher levels of plasma glucose, cholesterol and triglycerides than their ZL littermates, but there was no difference between the groups for systolic arterial blood pressure and heart rate. In vitro, coronary endothelial function was notably impaired in ZO and ZL rats. After global ischemia, the worse ventricular recovery in ZO and ZL rats was associated with arrhythmias during reperfusion. We detected similar levels of plasma ascorbyl free radical, Oxygen Radical Absorbance Capacity and vitamin C concentrations in the three groups. DHE staining showed higher superoxide production in the coronary vessels of ZO rats than in ZL and Wistar rats. Our results show that ZO might only correspond to early-stage cardiovascular complications associated with MS.

Impact of Cholesterol on ABC and SLC Transporters Expression and Function and its Role in Disposition Variability to Lipid-Lowering Drugs

This report focuses on the effects of cholesterol on the expression and function of the ATP-binding cassette (ABCB1, ABCG2 and ABCC2) and solute-linked carrier (SLCO1B1 and SLCO2B1) drug transporters with a particular focus on the potential impact of cholesterol on lipid-lowering drug disposition. Statins are the most active agents in the treatment of hypercholesterolemia. However, considerable interindividual variation exists in the response to statin therapy. Therefore, it would be huge progress if factors were identified that reliably differentiate between responders and nonresponders. Many studies have suggested that plasma lipid concentrations can affect drug disposition of compounds, such as ciclosporin and amphotericin B. Both compounds are able to affect the expression and function of ABC transporters. Although still speculative, these effects might be owing to the regulation of drug transporters by plasma cholesterol levels. Studies with normo- and hyper-cholesterolemic individuals, before and after atorvastatin treatment, have demonstrated that plasma cholesterol levels are correlated with drug transporter expression, as well as being related to atorvastatin's cholesterol-lowering effect. The mechanism influencing the correlation between cholesterol levels and the expression and function of drug transporters remains unclear. Some studies provide strong evidence that nuclear receptors, such as the pregnane X receptor and the constitutive androstane receptor, mediate this effect. In the near future, pharmacogenomic studies with individuals in a pathological state should be performed in order to identify whether high plasma cholesterol levels might be a factor contributing to interindividual oral drug bioavailability.

Normal Hepatic Cell Surface Localization of the High Density Lipoprotein Receptor, Scavenger Receptor Class B, Type I, Depends on All Four PDZ Domains of PDZK1

PDZK1 is a four PDZ domain-containing scaffold protein that binds to scavenger receptor class B, type I (SR-BI), the high density lipoprotein receptor, by its first PDZ domain (PDZ1). PDZK1 knock-out mice exhibit a >95% decrease in hepatic SR-BI protein and consequently an approximately 70% increase in plasma cholesterol in abnormally large high density lipoprotein particles. These defects are corrected by hepatic overexpression of full-length PDZK1 but not the PDZ1 domain alone, which partially restores SR-BI protein abundance but not cell surface expression or function. We have generated PDZK1 knock-out mice with hepatic expression of four PDZK1 transgenes encoding proteins with nested C-terminal truncations: pTEM, which lacks the three C-terminal residues (putative PDZ-binding motif), and PDZ1.2, PDZ1.2.3, or PDZ1.2.3.4, which contain only the first two, three, or four N-terminal PDZ domains, respectively, but not the remaining C-terminal sequences. Hepatic overexpression of pTEM restored normal hepatic SR-BI abundance, localization, and function. Hepatic overexpression of PDZ1.2 or PDZ1.2.3 partially restored SR-BI abundance ( approximately 12 or approximately 30% of wild type, respectively) but did not (PDZ1.2) or only slightly (PDZ1.2.3) restored hepatic SR-BI cell surface localization and function. Hepatic overexpression of PDZ1.2.3.4 completely restored SR-BI protein abundance, cell surface expression, and function (normalization of plasma cholesterol levels). Thus, all four PDZ domains in PDZK1, but not PDZ1-3 alone, are sufficient for its normal control of the abundance, localization, and therefore function of hepatic SR-BI, whereas the residues C-terminal to the PDZ4 domain, including the C-terminal putative PDZ-binding domain, are not required.

Pharmacological activation of LXR in utero directly influences ABC transporter expression and function in mice but does not affect adult cholesterol metabolism

Cholesterol is critical for several cellular functions and essential for normal fetal development. Therefore, its metabolism is tightly controlled during all life stages. The liver X receptors-alpha (LXRalpha; NR1H3) and -beta (LXRbeta; NR1H2) are nuclear receptors that are of key relevance in coordinating cholesterol and fatty acid metabolism. The aim of this study was to elucidate whether fetal cholesterol metabolism can be influenced in utero via pharmacological activation of LXR and whether this would have long-term effects on cholesterol homeostasis. Administration of the LXR agonist T0901317 to pregnant mice via their diet (0.015% wt/wt) led to induced fetal hepatic expression levels of the cholesterol transporter genes Abcg5/g8 and Abca1, higher plasma cholesterol levels, and lower hepatic cholesterol levels compared with controls. These profound changes during fetal development did not affect cholesterol metabolism in adulthood nor did they influence coping with a high-fat/high-cholesterol diet. This study shows that the LXR system is functional in fetal mice and susceptible to pharmacological activation. Despite massive changes in fetal cholesterol metabolism, regulatory mechanisms involved in cholesterol metabolism return to a "normal" state in offspring and allow coping with a high-fat/high-cholesterol diet.

Screening phosphatidylcholine biomarkers in mouse liver extracts from a hypercholesterolemia study using ESI-MS and chemometrics

When fed a high-fat, high-cholesterol diet (HFD), homozygous LDL receptor knockout mice exhibit extremely high levels of plasma cholesterol that are expected to influence liver metabolism. One step in the investigation of potential hepatic alterations was the analysis of organic extracts of livers from these and control mice by electrospray mass spectrometry (ESI-MS). Chemometrics (bioinformatics) analysis shows that the sample spectra cluster into two groups: one from mice with plasma cholesterol levels in excess of 900 mg dL(-1) and one from animals with cholesterol levels of 60-250 mg dL(-1). The loadings plot of the first PC in the principal-components analysis (PCA) reveals the chemical basis for clustering, i.e., biomarkers present at different concentrations in the different groups. The exact masses of the key peaks in this loadings plot indicate these species are phosphatidylcholines (PtdChos). This assignment is confirmed by tandem MS. Partial least-squares (PLS) with variable selection shows that the spectra are well correlated with plasma total cholesterol, HDL cholesterol, and triglyceride (TG) levels.

Effects of atorvastatin on progression-regression of renal injury in hyperlipidemic chickens.

Complex interrelationships exist between hyperlipidemia and the progression of renal injury. The aim of this study was to evaluate the impact of high plasma cholesterol and triglyceride levels on renal structure and the effects of atorvastatin on progression-regression of renal injury. One-hundred chickens were divided into five groups: Group A: Standard diet (SD) for 6 months; Group B: Hyperlipidemic diet (HD) for 6 months; Group C: HD for three months and SD during the next 3 months; Group D: HD for 3 months and SD during the next 3 months, when they received oral atorvastatin (3 mg/kg/d); Group E: HD for the whole 6 months, and atorvastatin (3 mg/kg/d) during the last 3 months. Increased alpha-actine immunostaining was found in glomeruli of groups B and C. An important decrease of immunostaining was observed in glomeruli of atorvastatin treated groups. Group D showed the lowest value for presence of lipids, and significant differences were found with respect to the rest of the groups. The glomeruli of group B presented the highest damage grades and those of group D showed the lowest grades and presented significant differences from the rest of the groups. The combination of atorvastatin therapy and proper diet proved to be effective in promoting renal disease regression. However, the study of several parameters indicates that neither only diet nor atorvastatin in the progression group resulted completely effective in decreasing the progression of the disease.

The Cholesterol Paradox Revisited: Heart Failure, Systemic Inflammation, and Beyond

The pathophysiologic understanding of chronic heart failure has made significant advances over the last decades. Counterintuitively, high levels of plasma cholesterol are associated with better survival, perhaps because plasma lipoproteins are able to scavenge lipopolysaccharide, a cell-wall component from gram-negative bacteria. A number of similar features are present in patients who have sepsis. This article explores the cholesterol paradox in patients who have chronic heart failure and extends this view to patients who have sepsis. Also discussed is the potential of statins, which might be able to exert beneficial effects in both clinical conditions, despite lowering plasma cholesterol values.

Atherosclerosis prevention by a fish oil-rich diet in apoE −/− mice is associated with a reduction of endothelial adhesion molecules

Dietary intake of long-chain n-3 polyunsaturated fatty acids (PUFA) reduces the risk for atherosclerosis. Here we examine the effect of a fish oil (FO)-rich diet on the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE −/−) mice, which are vulnerable because of their high plasma cholesterol and triacylglycerol levels, focusing on the expression of endothelial adhesion molecules. Mice were fed semi-purified diets containing 5% corn oil (CO), rich in n-6 PUFA or menhaden oil as FO, rich in long-chain n-3 PUFA and 0.15% cholesterol after reaching 4 weeks of age, and they were killed when they were 4 weeks, 12 weeks, 18 weeks or 24 weeks old. Oxidative stress in plasma and aortic tissue was not increased in mice fed the FO-rich diet, despite its high peroxidizability index. A reduction of stenosis and intrusion at the aortic root, a decrease in the surface area of atherosclerotic lesions at the aorta and a decrease in P-selectin, vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression were observed in FO-fed mice compared to CO-fed mice. It seems likely that the reduced expression of VCAM-1 and ICAM-1 could be transcriptionally regulated by nuclear factor-κB in the aortic root. The protective effect of FO against atherosclerosis was more evident at early ages. In conclusion, FO reduces adhesion molecule expression in lesions in apoE −/− mice. Because these molecules are involved in lesion progression the effect of FO may explain the observed decrease in atherogenesis.

Cholesterol Suppresses Cellular TGF‐beta Responsiveness by Increasing TGF‐beta Receptor Accumulation in Lipid raft/Caveolae Microdomains and Alterring TGF‐beta Binding to TGF‐beta Receptors

TGF‐β responsiveness in cultured cells can be modulated by TGF‐β partitioning between lipid raft/caveolae‐ and clathrin‐mediated endocytosis pathways. The ratio of TGF‐β binding to TβR‐II and TβR‐I on the cell surface has recently been found to be a signal that controls TGF‐β partitioning between these pathways. Since cholesterol is a structural component in lipid rafts/caveolae, we have studied the effects of cholesterol on TGF‐β binding to TGF‐β receptors and TGF‐β responsiveness in cultured cells and in animals. Here we demonstrate that treatment with cholesterol decreases the TβR‐II/TβR‐I binding ratio of TGF‐β while treatment with cholesterol‐lowering or cholesterol‐depleting agents increases the TβR‐II/TβR‐I binding ratio of TGF‐β in all cell types studied. Cholesterol treatment increases accumulation of the TGF‐β receptors in lipid rafts/caveolae microdomains of the plasma membrane. Cholesterol/LDL suppresses TGF‐β responsiveness and statins/β‐CD enhances it, as measured by the levels of P‐Smad2 and PAI‐1 expression. Furthermore, the cholesterol effects observed in cultured cells are also found in the aortic endothelium of atherosclerotic ApoE‐null mice fed a high cholesterol diet. These results indicate that high plasma cholesterol levels contribute to the pathogenesis of atherosclerosis, at least in part, by suppressing TGF‐β responsiveness.

Turbulent blood flow plays an essential localizing role in the development of atherosclerotic lesions in experimentally induced hypercholesterolaemia in rats

Taking into account that atherosclerosis is a focal disease and high levels of plasma cholesterol are closely correlated with its pathogenesis, it is a challenge to explain how equal concentrations of cholesterol bathing the endothelium can produce local, rather than global, effects on arteries. The focal distribution of atherosclerotic lesions has been considered to be dependent, at least in part, on hydrodynamic factors. The present study was carried out to further test the hypothesis that these forces are an important localizing factor in rats feeding a hypercholesterolaemic diet and submitted to infra-diaphragmatic aortic constriction. These animals develop a normotensive prestenotic region with laminar blood flow that serves as control for a normotensive poststenotic region with turbulent blood flow. Our findings clearly demonstrated that the combination of turbulent blood flow and low wall shear stress (WSS) in the presence of hypercholesterolaemia and oxidative stress creates conditions to the formation of focally distributed incipient atherosclerotic lesions observed in the poststenotic segment. In contrast, only diffuse fatty streaks could be observed in the normotensive prestenotic segment with laminar blood flow and normal WSS in the presence of hypercholesterolaemia and oxidative stress. Although haemodynamic forces are not by themselves responsible for the pathogenesis of atherosclerosis, they prime the local vascular wall in which the lesion develop. Further studies are required to establish how haemodynamic forces are detected and transduced into chemical signalling by the cells of the artery wall and then converted into pathophysiologically relevant phenotypic changes.

Statin and endothelial cell-derived microparticles

Thromb Haemost 2008; 100: 377–378 Microparticles (MP) are small membrane vesicles that are released from many different cell types by a process of exocytic budding of the plasma membrane (1). Because MP disseminate various bioactive effectors originating from the parent cells, they can alter vascular function and may induce biological responses involved in the vascular system (2). For example, MP can modulate coagulation via direct and indirect mechanisms. Therefore, MP concentrations are documented in almost all thrombotic diseases occurring in venous or arterial beds (3–5). Furthermore, elevated levels of MP have been found in a number of conditions associated with inflammation, cellular activation and dysfunction, angiogenesis and transport (1, 6–8). Various factors are involved in the mechanisms of endothelial cell-derived MP (EDMP) generation (9, 10). TNFα, lipopolysaccharideor oxidized low-density lipoprotein (LDL)stimulation of cultured human umbilical vein endothelial cells (HUVEC) result in an increase in the release of EDMP expressing surface tissue factor (11, 12). Several studies report the existence of EDMP generated from apoptotic endothelial cells; for example, elevation of EDMP in patients following allogeneic stem cell transplantation is associated with some transplant-related complications that include the apoptotic mechanism of endothelial cells (13–15). Furthermore, the pattern of procoagulant MP released during acute allograft rejection suggests endothelial cell activation and Fas-mediated apoptosis (16); while procoagulant MP in pulmonary arterial hypertension also show apoptotic EDMP (17). However, the clinical significance of EDMP has not been fully revealed. A high plasma cholesterol level is a risk factor for the progression of atherosclerosis and cardiovascular disease. A high plasma level of LDL cholesterol may also promote the development of atherosclerotic disease (18). Large-scale clinical trials with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) have shown that lowering cholesterol levels decreases the incidence of cardiovascular events and the progression of atherosclerosis (19). Statins have additional pleiotropic effects, many of which are mediated by the vascular endothelium (20–22). These pleiotropic effects of statins, as well as their lipid lowering effects, may be responsible for the prevention of atherosclerosis. However, the distinct mechanisms of these effects of statins are poorly understood. In this issue of Thrombosis and Haemostasis, Diamant et al. (23) have partially resolved the above noted problems. They found that simvastatin promotes EDMP release during the detachment of endothelial cells undergoing apoptosis (induced by this drug), whereas the adherent cells showed no signs of simvastatin-induced apoptosis. The authors also concluded that statins improve the overall condition of the remaining vascular endothelium by facilitating detachment and EDMP release. Such a defense system of MP protects parent cells, as previously reported. The release of MP protects cells against complement-induced lysis (24). EDMP are likely to be generated to maintain endothelial homeostasis. The current view of the role of MP is changing. Freyssinet proposed that MP not only impose a potential environment threat, but may also be essential to maintain cellular homeostasis (25). Most MP in thrombotic diseases are considered to be disadvantageous because of their procoagulant and proadhesive characteristics (25, 26). However, the study by Diamant et al. (23) suggests that elevated numbers of EDMP in patients with diabetes or cardiovascular disease reflect an activated cellular mechanism to cope with the increased endothelial stress, rather than a mechanism to impose an environmental threat to other cells and tissues. Thus, their report revises our understanding about EDMP. It also suggests a new outlook for EDMP, since the detachment of endothelial cells could contribute to a better appreciation of the effect of statins on endothelial cells.

Cholesterol modulates cellular TGF‐β responsiveness by altering TGF‐β binding to TGF‐β receptors

Transforming growth factor-beta (TGF-beta) responsiveness in cultured cells can be modulated by TGF-beta partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. The TbetaR-II/TbetaR-I binding ratio of TGF-beta on the cell surface has recently been found to be a signal that controls TGF-beta partitioning between these pathways. Since cholesterol is a structural component in lipid rafts/caveolae, we have studied the effects of cholesterol on TGF-beta binding to TGF-beta receptors and TGF-beta responsiveness in cultured cells and in animals. Here we demonstrate that treatment with cholesterol, alone or complexed in lipoproteins, decreases the TbetaR-II/TbetaR-I binding ratio of TGF-beta while treatment with cholesterol-lowering or cholesterol-depleting agents increases the TbetaR-II/TbetaR-I binding ratio of TGF-beta in all cell types studied. Among cholesterol derivatives and analogs examined, cholesterol is the most potent agent for decreasing the TbetaR-II/TbetaR-I binding ratio of TGF-beta. Cholesterol treatment increases accumulation of the TGF-beta receptors in lipid rafts/caveolae as determined by sucrose density gradient ultracentrifugation analysis of cell lysates. Cholesterol/LDL suppresses TGF-beta responsiveness and statins/beta-CD enhances it, as measured by the levels of P-Smad2 and PAI-1 expression in cells stimulated with TGF-beta. Furthermore, the cholesterol effects observed in cultured cells are also found in the aortic endothelium of atherosclerotic ApoE-null mice fed a high cholesterol diet. These results indicate that high plasma cholesterol levels may contribute to the pathogenesis of certain diseases (e.g., atherosclerosis) by suppressing TGF-beta responsiveness.

Evaluation of Aortic Remodeling in Apolipoprotein E-deficient Mice and Renovascular Hypertensive Mice

The apolipoprotein E-deficient mouse (ApoE) spontaneously develops hypercholesterolemia and atherosclerotic lesions in large arteries. It is also known that angiotensin II-induced hypertension accelerates the development of atherosclerosis in ApoE mice. The objective of this study was to evaluate the aortic remodeling process in ApoE mice during the early phase of atherosclerosis in two-kidney one-clip hypertensive (2K1C) mice and in mice with the coexistence of atherosclerosis and arterial hypertension. Renovascular hypertension was induced in 8- to 9-week-old C57BL/6 (C57) and ApoE and compared to sham animals 28 days later. C57-2K1C and ApoE-2K1C mice showed hypertension, tachycardia, and cardiac hypertrophy of similar magnitude. ApoE and ApoE-2K1C mice showed high levels of plasma cholesterol (4.8- and 3.6-fold) and aorta lipid deposition (85- and 101-fold) compared to C57 mice. The aorta lumen area was increased in C57-2K1C and ApoE-2K1C mice (0.57 +/- 0.04 and 0.55 +/- 0.02 mm(2)) compared to C57 mice (0.50 +/- 0.02 mm(2), p <0.05). The aorta wall area was increased by 20% in C57-2K1C and by 12% in ApoE-2K1C mice compared to C57 and ApoE. The main finding of this study was the absence of aorta remodeling in ApoE mice at the early stage of atherosclerosis and an outward remodeling of similar magnitude in C57-2K1C and ApoE-2K1C mice.

Nutritional Control, Gene Regulation, and Transformation of Vascular Smooth Muscle Cells in Atherosclerosis

Contractile-state smooth muscle cells (SMC), the only cell type in the arterial media, undergoes migration to the intima, proliferation, and abundant extracellular matrix production during the early stages of atherosclerosis. This involves the ingestion of low-density lipoprotein (LDL) and modified or oxidised LDL by macrophages together with SMC by several pathways including a scavenger pathway leading to accumulation of cholesterol esters and formation of foam cells. High-plasma cholesterol levels constitute a major causative risk for atherosclerosis. The membrane-bound transcription factor called sterol regulatory element binding protein (SREBP) activates gene-encoding enzymes of cholesterol and fatty acid biosynthesis. The SREBP expression, in response to diet, shows that are involved in both lipogenesis and cholesterol homeostasis, moreover SREBPs are regulated directly by cholesterol. Animal models were used in trials of atherosclerosis, and cholesterol feeding has been described elsewhere as producing atherosclerotic lesions. We have examined the morphological, molecular and proliferative change in arterial SMC mimicking such a cholesterol diet, this transformed SMC is a good model to study the alterations of the differentiated state of SMC, and the transformation into foam cell, caused by cholesterol-rich diet. Despite the complexity of the interactions in atherosclerosis, there are many opportunities to affect the homeostatic balance of the artery wall at SMC levels. We have considered here some of the possible targets for intervention with promising strategies for the nutritional control of the genes, and, in a general way, the possibilities for modulating the expression of genes influencing atherosclerosis.

Analysis of sterols by high‐performance liquid chromatography/mass spectrometry combined with chemometrics

A newly developed high-performance liquid chromatography/mass spectrometry (HPLC/MS) method has been successfully used to analyze plasma concentrations of various phytosterols (cholestanol and beta-sitosterol) and cholesterol metabolites (desmosterol and lathosterol). This was based on an unusual solvent combination of water/methanol vs. methanol/acetone/n-hexane applied on a Purospher Star RP-18e (125 x 2 mm, 3 microm) column, which proved excellent for the separation, identification and quantification of plasma sterols. Simple solid-phase extraction preparation of plasma samples was performed, followed by the developed fast and robust HPLC separation. Results on four groups of people were compared, those with low, normal and high plasma cholesterol levels and those with high cholesterol levels on statin therapy, and the results were evaluated using linear discriminant analysis (LDA). Variable selection for LDA was achieved using backward removal selection. Highly discriminatory variables were the ratios of desmosterol to sitosterol and of lathosterol to total plasma cholesterol. The latter ratio was also excellent for distinguishing subjects on statin therapy. The success rate of classification was 100%. The present pilot study shows the potential of HPLC/MS analysis and chemometrics for studying cholesterol-related disorders and warrants future full-scale medical study.

Severity of Hyperlipidemia Does Not Affect Antiatherosclerotic Effect of an Angiotensin II Receptor Antagonist in Apolipoprotein E-deficient Mice

The purpose of this study was to clarify whether severity of hyperlipidemia affects the antiatherosclerotic effect of angiotensin II receptor blockers (ARBs). The effect of olmesartan medoxomil, an ARB, on atherosclerotic lesion was examined in apolipoprotein E-deficient (ApoEKO) mice fed a normal diet (ND) or a high-fat-supplemented diet (FD) for 25 weeks. ApoEKO mice have high plasma cholesterol levels, which were further increased by feeding of an FD. Both the atherosclerotic lesion area of the aortic luminal surface and the atherosclerotic lesion thickness in the aortic valves were significantly greater in the FD mice than in the ND mice. Olmesartan medoxomil did not affect the plasma cholesterol levels in either the ND or FD ApoEKO mice; however, it reduced effectively both the atherosclerotic lesion surface area and the lesion thickness even in FD ApoEKO mice. It is concluded that the antiatherosclerotic effect of ARBs is not weakened by the high plasma cholesterol level, suggesting the usefulness of ARBs in the treatment of atherosclerosis, even in a situation in which the plasma cholesterol level is not fully controlled.