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Journal of Atherosclerosis and Thrombosis  Vol.16, No.1

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Review

Apolipoprotein CⅢ Links Dyslipidemia with Atherosclerosis Akio Kawakami 1, 2 and Masayuki Yoshida 2 1 2

Department of Geriatrics and Vascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan Life Science and Bioethics Research Center, Tokyo Medical and Dental University, Tokyo, Japan

Plasma levels of lipoproteins that contain apolipoprotein (apo) CⅢ predict coronary heart disease (CHD), and associate with contributors to metabolic syndrome such as type 2 diabetes and hypertriglyceridemia. ApoCⅢ causes hypertriglyceridemia by inhibiting the catabolism and the clearance of TG-rich lipoproteins (TLRs), and the association of apoCⅢ with CHD has been commonly attributed to these properties; however, it has been untested whether apoCⅢ itself or in association with lipoproteins directly affects atherogenic mechanisms in vascular cells. This review describes the proatherogenic effect of apoCⅢ-containing lipoproteins. In brief, apoCⅢ-rich VLDL (VLDL CⅢ＋) increased the adhesion of human monocytes to vascular endothelial cells (ECs). ApoCⅢ alone also increased monocyte adhesion to vascular ECs. Interestingly, apoCⅢ-rich HDL did not reduce the adhesion of monocytes to vascular ECs, whereas HDL without apoCⅢ decreased their adhesion, suggesting that apoCⅢ in HDL counteracts the anti-inflammatory property of HDL. ApoCⅢ alone as well as VLDL CⅢ＋ also activated vascular ECs through the activation of NF-κB, and induced the recruitment of monocytes to vascular ECs. Moreover, apoCⅢ induced insulin resistance in vascular ECs and caused endothelial dysfunction. These findings indicate that apoCⅢ in TLRs not only modulates their metabolism, but also may directly contribute to the development of atherosclerosis by activating the proinflammatory signal transduction of vascular cells. Here, we propose a novel role for apoCⅢ that links dyslipidemia with atherosclerosis. J Atheroscler Thromb, 2009; 16:6-11. Key words; Inflammation, Monocyte-endothelial interaction, Hypertriglyceridemia, Metabolic syndrome

Introduction The human apoCⅢ gene is expressed in the liver and intestine, comprising a gene cluster together with apoAⅠ and apoAⅣ genes on the long arm of chromosome 11 1-3). ApoCⅢ is synthesized by the liver, and by the intestine to a lesser extent, as a 99-amino acid peptide. After removing the 20-amino acid peptide in the endoplasmic reticulum, a mature apoCⅢ protein is composed of 79 amino acids with a molecular mass of 8.8 kDa 1, 2). Several pathways regulate apoCⅢ gene expression. Insulin reduces the transcription of the apoCⅢ Address for correspondence: Akio Kawakami, Geriatrics and Vascular Medicine, Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan E-mail: [email protected] Received: July 17, 2008 Accepted for publication: September 9, 2008

gene via the promoter IRE (insulin-response element) 4). The expression and secretion of apoCⅢ increase in insulin-resistant states 5). Indeed, plasma apoCⅢ is elevated in metabolic syndrome and type 2 diabetes and correlates with BMI and HOMA-IR 6, 7). Transcription of the apoCⅢ gene is also down-regulated by PPARs, especially PPARα8). In contrast, the activation of NF-κB leads to up-regulation of the apoCⅢ gene expression 3). ApoCⅢ resides on the broad distribution of apoB lipoproteins such as chylomicron, VLDL, IDL, and LDL. It also resides on particles slightly larger than HDL. In the fasting state, apoCⅢ is mainly associated with HDL, whereas in the fed state, apoCⅢ preferentially redistributes to chylomicron and VLDL particles. In normotriglyceridemic patients, about half to two-thirds of VLDL and IDL particles have apoCⅢ. In contrast, apoCⅢ is contained in only about 10% of LDL particles 2).

ApoCⅢ Induces Atherosclerosis

ApoCⅢ Causes Hypertriglyceridemia ApoB lipoproteins with apoCⅢ are rich in both triglyceride (TG) and cholesterol ester, and are selectively elevated in patients with hypertriglyceridemia. Plasma total apoCⅢ concentration and apoCⅢ concentrations in apoB lipoproteins causally correlate with plasma TG concentrations 9). ApoCⅢ causes hypertriglyceridemia through several mechanisms. ApoCⅢ inhibits the catabolism and clearance of TG-rich lipoproteins (TRLs). Previous studies have demonstrated that apoCⅢ on chylomicrons and VLDL inhibits their uptake by hepatocytes in vitro and in vivo 10, 11). ApoCⅢ inhibits the binding of apoB lipoproteins to hepatic apoB/E receptor 12). This inhibitory action of apoCⅢ was due to a masking of the receptor domain of apoB or apoE 13). ApoCⅢ also inhibits LPL activity 14) and is one of the most specific inhibitors of LPL in hypertriglyceridemic patients. Subjects lacking apoCⅢ have low TRL levels 15); sera from these subjects are able to activate human milk LPL, whereas normal sera effectively inhibit LPL activity. These findings suggest that apoCⅢ contributes to the development of hypertriglyceridemia by inhibiting the LPL-mediated lipolysis of TRLs. More recently, apoCⅢ has been shown to stimulate VLDL synthesis in cultured hepatocytes 3). Several kinetic studies using an isotope tracer showed that elevated apoCⅢ concentrations associated with increased VLDL secretion as well as decreased VLDL catabolism in subjects with metabolic syndrome and hypertriglyceridemia 16). Studies using transgenic and knockout mouse models support the apoCⅢ effect on the metabolism of TRLs. Overexpression of human apoCⅢ in wild-type mice or in LDL receptor knockout mice not only induced hypertriglyceridemia, but also enhanced the development of atherosclerosis 17, 18). In these models, elevated apoCⅢ concentrations associated with the increased hepatic VLDL production rate and decreased catabolic rate of VLDL and their remnants. In contrast, apoCⅢ knockout mice showed the rapid catabolism of TRLs and hypotriglyceridemia 19). ApoCⅢ Predicts CHD Risk As apoCⅢ is elevated in metabolic syndrome and type 2 diabetes, major risk factors for CHD, and apoCⅢ modulates the metabolism of apoB lipoproteins, it is reasonable to hypothesize that apoCⅢ may affect the relationship between apoB lipoproteins and CHD risk in clinical studies. Alaupovic et al. reported that the concentration of apoB lipoproteins with apoCⅢ was the strongest lipoprotein predictor of the

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progression of coronary atherosclerosis, even in patients whose LDL cholesterol concentrations were aggressively lowered with lovastatin 20). Another coronary arteriographic study reported the relationship between apoCⅢ in apoB lipoproteins and CHD risk 21). The roles of apoCⅢ and apoE in predicting CHD events were compared in a large prospective study of patients with CHD (CARE trial), in which apoCⅢ concentration in VLDL＋LDL was associated with an increased risk of CHD regardless of the use of pravastatin 22, 23). Adjustment for other lipid risk factors did not affect the results for apoCⅢ, and apoCⅢ was a more specific marker of atherogenic particles than TG. Several studies reported that apoCⅢ in HDL was also associated with CHD in univariate analysis, and that apoCⅢ concentration in HDL had a positive rather than inverse correlation with other risk factors, such as VLDL and triglyceride 24). This suggests that apoCⅢ exerts atherogenic properties beyond its effect on apoB lipoprotein metabolism. Direct Effects of ApoCⅢ on Vascular Cells The causal association of apoCⅢ with CHD has been commonly attributed to its inhibitory effect on the catabolism and clearance of TRLs, prolonging the plasma residence time of atherogenic TLRs. While this is undoubtedly true in part, a recent study reported that apoCⅢ-rich VLDL do not have a longer residence time than their apoCⅢ-free counterparts 25), since slow clearance is balanced by rapid lipolytic conversion to LDL. Indeed, plasma concentrations of apoB lipoproteins with apoCⅢ are lower than those without apoCⅢ. Thus, apoB lipoproteins with apoCⅢ seem to augment CHD risk out of proportion to their concentration in plasma, suggesting that the correlation between apoCⅢ and CHD risk in population studies is in part attributable to apoCⅢ’s direct involvement in atherogenesis; however, the direct effects on vascular cells of apoB lipoproteins with apoCⅢ specifically or apoCⅢ itself have been untested. We hypothesized that apoCⅢ-containing lipoproteins have enhanced atherogenicity relative to their apoCⅢ-free counterparts, and examined the direct effects of apoCⅢ alone on peripheral blood monocytes and vascular endothelial cells (ECs). ApoCⅢ Activates Human Peripheral Monocytes The adhesion of circulating monocytes to vascular ECs contributes importantly to the inflammatory aspects of atherogenesis. We found that apoCⅢ-rich

Kawakami and Yoshida

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Fig. 1. ApoCⅢ-rich lipoproteins modulate monocyte adhesion to endothelial cells. (A) Human peripheral monocytes were incubated in the presence of the indicated lipoproteins (100 μg apoB/mL), apoCⅢ (100 μg/ mL) or PBS (Control) for 8 hours, and static adhesion assays to human umbilical vein endothelial cells (HUVECs) were carried out. ＊p ＜ 0.05 vs. Control, #p ＜ 0.05 vs. VLDL CⅢ− or LDL CⅢ−. (B) Human peripheral monocytes were incubated with PBS (Control), HDL CⅢ＋ or HDL CⅢ− (500 μg chol/mL) for 8 hours, and static adhesion assays to HUVECs were carried out. ＊p ＜ 0.05 vs. Control.

VLDL particles (VLDL CⅢ＋), but not VLDL particles without apoCⅢ increase the adhesion of monocytes to vascular ECs; apoCⅢ protein itself caused this effect (Fig. 1A) 26), which was observed in a concentration-dependent manner. Proadhesive concentrations of apoCⅢ-containing lipoproteins, 50−100 μg apoB/ mL, are well within the range found in fasting plasma, e.g. 50 μg apoB/mL in normolipidemic individuals, and ＞100 μg apoB/mL in hypertriglyceridemic individuals or those with CHD, supporting their clinical relevance. Some HDL preparations can inhibit the expression of integrins and adhesion molecules in leukocytes and vascular ECs, and reduce their adhesive interaction 27), which is supposed to be one of antiatherogenic properties of HDL. We examined the effect of apoCⅢ-rich HDL particles (HDL CⅢ＋) on monocyte adhesion. HDL particles without apoCⅢ (HDL CⅢ−) reduced monocyte adhesion to vascular ECs in a concentration-dependent manner 26), while HDL CⅢ＋ did not (Fig . 1B). ApoCⅢ might have counteracted potentially atheroprotective actions of other HDL components. Our current data suggest a novel mechanism for HDL dysfunction induced by apoCⅢ. ApoCⅢ-containing lipoproteins also contain other apolipoproteins, such as apoCⅠ, apoCⅡ, and apoE, as well as various lipids; however, other apolipoproteins or lipids extracted from apoCⅢ-containing VLDL did not increase monocyte adhesion. Antibodies against apoCⅢ but not against apoCⅠ, apoCⅡ or

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Fig. 2. Schema depicting the mechanisms by which apoCⅢ activates monocytes and endothelial cells. ApoCⅢ in TRLs activates PKC families and NF-κB, and increases the expression of integrins and adhesion molecules in monocytes and endothelial cells, causing their interaction. PC-PLC, phosphatidylcholine-specific phospholipase C.

apoE decreased the proadhesive effect. Thus, apoCⅢ itself but not other apolipoproteins that commonly cluster with apoCⅢ on apoB lipoproteins or lipoprotein lipids mediate the enhanced monocyte adhesion to vascular ECs; however, further study is needed to determine whether concomitant apoCⅠ, apoCⅡ, or apoE modifies the effects of apoCⅢ on monocyte adhesion. We then examined the mechanisms of apoCⅢinduced monocyte activation. ApoCⅢ alone as well as VLDL CⅢ＋ activated β1-integrin in monocytes. Protein kinase C (PKC) plays an important role in several mechanisms that promote atherosclerosis, including monocyte-endothelial interaction 28). We showed that among PKCs, PKCα plays an important role in monocyte β1-integrin activation by VLDL CⅢ＋ or apoCⅢ itself. We further identified pertussis toxin (PTX)sensitive G protein-coupled receptors and phosphatidylcholine-specific phospholipase C (PC-PLC) as key molecules that activate PKCα, NF-κB and β1-integrin in monocytes (Fig. 2) 29). Our observations may provide a role for apoCⅢ as a distinct contributor to inflammation and atherosclerosis through monocyte activation. ApoCⅢ Activates Vascular Endothelial Cells The induction of adhesion molecules in vascular ECs and the subsequent recruitment of circulating monocytes are proinflammatory events that promote

ApoCⅢ Induces Atherosclerosis

atherogenesis and plaque instability. We demonstrated that apoCⅢ activates PKCβ rather than PKCα, and increases the expression of VCAM-1 in non-activated ECs, thus recruiting the adhesion of monocytes 30). Moreover, VLDL CⅢ＋, but not VLDL CⅢ−, activated PKCβ in ECs, and anti-apoCⅢ antibody inhibited PKCβ activation induced by VLDL CⅢ＋, suggesting that apoCⅢ in VLDL plays a pivotal role in this process (Fig. 1). This study identified NF-κB as the molecular link between apoCⅢ-induced PKCβ activation and increased expression of VCAM-1. Since a portion of TLRs ordinarily contains apoCⅢ31), our results provide a novel mechanism for vascular EC activation by TRLs that is independent of lipid moieties and their oxidation in these particles. ApoCⅢ Induces Insulin Resistance in Vascular Endothelial Cells and Causes Endothelial Dysfunction Endothelial dysfunction importantly contributes to CHD and is characterized by the reduced bioavailability of nitric oxide (NO), which has potent vasodilatory and anti-atherosclerotic properties. Insulin activates endothelial NO synthase (eNOS) in endothelial cells and stimulates the production of NO, and insulin resistance in vascular ECs leads to its dysfunction. Insulin resistance and subsequent endothelial dysfunction are often seen in diabetes, obesity, and dyslipidemia, and major risk factors for CHD, and plasma apoCⅢ level is high in these conditions. As PKCβ inhibits insulin signaling in endothelial cells, and apoCⅢ activates PKCβ in the same cells, we tested the effect of apoCⅢ on endothelial insulin signaling 32). ApoCⅢ in VLDL inhibited insulin activation of the eNOS pathway and the production of NO in vascular ECs. ApoCⅢ also impaired endothelium-dependent relaxation of mouse aortas. This adverse effect of apoCⅢ was mediated by PKCβⅡ, which inhibits the function of IRS-1 (Fig. 3). Insulin resistance and endothelial dysfunction associated with hypertriglyceridemia have been attributed to free fatty acids and other lipid moieties in TRLs; however, our findings may add a new mechanism that apoCⅢ in TRLs impairs insulin signaling in vascular ECs, and suggest that apoCⅢ could link dyslipidemia with endothelial dysfunction. Remnant Lipoproteins and ApoCⅢ ApoCⅢ-rich VLDL or LDL particles are also rich in apoE as well as TG and cholesterol, which have similar properties to atherogenic remnant lipoprotein

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Fig. 3. Schema depicting the mechanisms by which apoCⅢ causes insulin resistance in vascular endothelial cells. Insulin resistance in hepatocytes increases apoCⅢ production and secretion into blood of apoCⅢ. ApoCⅢ in TRLs in turn impairs insulin-induced NO production by inhibiting IRS-1 function in vascular endothelial cells. The inhibitory effects of apoCⅢ on NO production are likely to be mediated by the activation of PKCβⅡ and partly by ERK, which induce serine phosphorylation of IRS-1.

particles (RLPs) 33). Plasma TRL concentrations, especially those of RLPs, independently correlate with atherosclerosis 34, 35). RLPs consist of heterogeneous TRL particles, and some RLPs have a high content of apoCⅢ as well as apoE 36). ApoCⅢ-rich VLDL or LDL particles may be constituents of RLP particles or their precursors. We reported that several mechanisms of RLPs directly promoted atherosclerosis 37), in which RLPs stimulated monocyte adhesion 38), induced the proliferation of smooth muscle cells 39), and facilitated foam cell formation 40). ApoCⅢ may not only be involved in the formation and accumulation of RLPs, but may also importantly contribute to these atherogenic processes by inducing inflammatory processes. Conclusion This review described the role of apoCⅢ in the metabolism of TRLs, and the correlation between apoCⅢ and CHD risk. We also focused on the direct effect of apoCⅢ on vascular cells. Many points remain to be elucidated regarding the precise mechanisms by which apoCⅢ-rich lipoproteins activate PKCs and

Kawakami and Yoshida

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NF-κB in vascular cells, although our data suggest receptors for apoCⅢ other than apoB/E receptor. Further studies are also needed to elucidate the kinetics of apoCⅢ-containing lipoproteins, and their atherogenicity on other types of cells, which will help to understand the overall atherogenicity of TRLs. Many previous studies have focused on the roles of lipid moieties, such as oxidized lipids in atherogenic lipoproteins; however, our observations may provide novel insights into the role of apoCⅢ as a direct and distinct contributor to inflammation and atherosclerosis. ApoCⅢ may be a new target for interventions, particularly in subjects with insulin resistance, metabolic syndrome and type 2 diabetes, because lowering apoCⅢ not only improved the impaired metabolism of TRLs in these conditions, but also may directly contribute to the prevention of atherosclerosis. Acknowledgements We thank Makoto Harada for technical assistance. Grant Support This study was supported by grants from the Ministry of Education, Science and Technology (10178102), ONO Medical Research Foundation, Takeda Science Foundation, Mitsukoshi Health and Welfare Foundation, Uehara Memorial Foundation, and a Sakakibara Memorial Research Grant from the Japan Research Promotion Society for Cardiovascular Diseases. References 1) Karathanasis SK: Apolipoprotein multigene family: tandem organization of human apolipoprotein AⅠ, CⅢ, and AⅣ genes. Proc Natl Acad Sci USA, 1985; 82:6374-6378 2) Jong MC, Hofker MH, Havekes LM: Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3. Arterioscler Thromb Vasc Biol, 1999; 19:472-484 3) Ooi EM, Barrett PH, Chan DC, Watts GF: Apolipoprotein C-Ⅲ: understanding an emerging cardiovascular risk factor. Clin Sci, 2008; 114:611-624 4) Chen M, Breslow JL, Li W, Leff T: Transcriptional regulation of the apoC-Ⅲ gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels. J Lipid Res, 1994; 35:1918-1924 5) Altomonte J, Cong L, Harbaran S, Richter A, Xu J, Meseck M, Dong HH: Foxo1 mediates insulin action on apoC-Ⅲ and triglyceride metabolism. J Clin Invest, 2004; 114:1493-1503 6) Olivieri O, Bassi A, Stranieri C, Trabetti E, Martinelli N, Pizzolo F, Girelli D, Friso S, Pignatti PF, Corrocher R:

Apolipoprotein C-Ⅲ, metabolic syndrome, and risk of coronary artery disease. J Lipid Res, 2003; 44:2374-2381 7) Cohn JS, Patterson BW, Uffelman KD, Davignon J, Steiner G: Rate of production of plasma and very-lowdensity lipoprotein (VLDL) apolipoprotein C-Ⅲ is strongly related to the concentration and level of production of VLDL triglyceride in male subjects with different body weights and levels of insulin sensitivity. J Clin Endocrinol Metab, 2004; 89:3949-3955 8) Staels B, Vu-Dac N, Kosykh VA, Saladin R, Fruchart JC, Dallongeville J, Auwerx J: Fibrates downregulate apolipoprotein C-Ⅲ expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates. J Clin Invest, 1995; 95:705-712 9) Lee SJ, Moye LA, Campos H, Williams GH, Sacks FM: Hypertriglyceridemia but not diabetes status is associated with VLDL containing apolipoprotein CⅢ in patients with coronary heart disease. Atherosclerosis, 2003; 167:293302 10) Windler E, Havel RJ: Inhibitory effects of C apolipoproteins from rats and humans on the uptake of triglyceriderich lipoproteins and their remnants by the perfused rat liver. J Lipid Res, 1985; 26:556-565 11) Quarfordt SH, Michalopoulos G, Schirmer B: The effect of human C apolipoproteins on the in vitro hepatic metabolism of triglyceride emulsions in the rat. J Biol Chem, 1982; 257:14642-14647 12) Clavey V, Lestavel-Delattre S, Copin C, Bard JM, Fruchart JC: Modulation of lipoprotein B binding to the LDL receptor by exogenous lipids and apolipoproteins CⅠ, CⅡ, CⅢ, and E. Arterioscler Thromb Vasc Biol, 1995; 15:963971 13) Sehayek E, Lewin-Velvert U, Chajek-Shaul T, Eisenberg S: Lipolysis exposes unreactive endogenous apolipoprotein E-3 in human and rat plasma very low density lipoprotein. J Clin Invest, 1991; 88:553-560 14) Wang CS, McConathy WJ, Kloer HU, Alaupovic P: Modulation of lipoprotein lipase activity by apolipoproteins. Effect of apolipoprotein C-Ⅲ. J Clin Invest, 1985; 75:384-390 15) Ginsberg HN, Le NA, Goldberg IJ, Gibson JC, Rubinstein A, Wang-Iverson P, Norum R, Brown WV: Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CⅢ and AⅠ. Evidence that apolipoprotein CⅢ inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo. J Clin Invest, 1986; 78:12871295 16) Batal R, Tremblay M, Barrett PH, Jacques H, Fredenrich A, Mamer O, Davignon J, Cohn JS: Plasma kinetics of apoC-Ⅲ and apoE in normolipidemic and hypertriglyceridemic subjects. J Lipid Res, 2000; 41:706-718 17) Ito Y, Azrolan N, O’Connell A, Walsh A, Breslow JL: Hypertriglyceridemia as a result of human apo CⅢ gene expression in transgenic mice. Science, 1990; 249:790-793 18) Masucci-Magoulas L, Goldberg IJ, Bisgaier CL, Serajuddin H, Francone OL, Breslow JL, Tall AR: A mouse model with features of familial combined hyperlipidemia. Science, 1997; 275:391-394 19) Maeda N, Li H, Lee D, Oliver P, Quarfordt SH, Osada J:

ApoCⅢ Induces Atherosclerosis

Targeted disruption of the apolipoprotein C-Ⅲ gene in mice results in hypotriglyceridemia and protection from postprandial hypertriglyceridemia. J Biol Chem, 1994; 269:23610-23616 20) Alaupovic P, Mack WJ, Knight-Gibson C, Hodis HN: The role of triglyceride-rich lipoprotein families in the progression of atherosclerotic lesions as determined by sequential coronary angiography from a controlled clinical trial. Arterioscler Thromb Vasc Biol, 1997; 17:715-722 21) Hodis HN: Triglyceride-rich lipoprotein remnant particles and risk of atherosclerosis. Circulation, 1999; 99:28522854 22) Sacks FM, Alaupovic P, Moye LA, Cole TG, Sussex B, Stampfer MJ, Pfeffer MA, Braunwald E: VLDL, apolipoproteins B, CⅢ, and E, and risk of recurrent coronary events in the Cholesterol and Recurrent Events (CARE) trial. Circulation, 2000; 102:1886-1892 23) Lee SJ, Campos H, Moye LA, Sacks FM: LDL containing apolipoprotein CⅢ is an independent risk factor for coronary events in diabetic patients. Arterioscler Thromb Vasc Biol, 2003; 23:853-858 24) Onat A, Hergenç G, Sansoy V, Fobker M, Ceyhan K, Toprak S, Assmann G: Apolipoprotein C-Ⅲ, a strong discriminant of coronary risk in men and a determinant of the metabolic syndrome in both genders. Atherosclerosis, 2003; 168:81-89 25) Zheng C, Khoo C, Ikewaki K, Sacks FM: Rapid turnover of apolipoprotein C-Ⅲ-containing triglyceride-rich lipoproteins contributing to the formation of LDL subfractions. J Lipid Res, 2007; 48:1190-1203 26) Kawakami A, Aikawa M, Libby P, Alcaide P, Luscinskas FW, Sacks FM: Apolipoprotein CⅢ in apolipoprotein B lipoproteins enhances the adhesion of human monocytic cells to endothelial cells. Circulation, 2006; 113:691-700 27) Barter PJ, Nicholls S, Rye KA, Anantharamaiah GM, Navab M, Fogelman AM: Antiinflammatory properties of HDL. Circ Res, 2004; 95:764-772 28) Rask-Madsen C, King GL: Proatherosclerotic mechanisms involving protein kinase C in diabetes and insulin resistance. Arterioscler Thromb Vasc Biol, 2005; 25:487-496 29) Kawakami A, Aikawa M, Nitta N, Yoshida M, Libby P, Sacks FM: Apolipoprotein CⅢ-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G-protein- and protein kinase C alpha-mediated nuclear factor-kappaB activation. Arterioscler Thromb Vasc Biol, 2007; 27:219-225 30) Kawakami A, Aikawa M, Alcaide P, Luscinskas FW, Libby P, Sacks FM: Apolipoprotein CⅢ induces expression of

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vascular cell adhesion molecule-1 in vascular endothelial cells and increases adhesion of monocytic cells. Circulation, 2006; 114:681-687 31) Campos H, Perlov D, Khoo C, Sacks FM: Distinct patterns of lipoproteins with apoB defined by presence of apoE or apoC-Ⅲ in hypercholesterolemia and hypertriglyceridemia. J Lipid Res, 2001; 42:1239-1249 32) Kawakami A, Osaka M, Tani M, Azuma H, Sacks FM, Shimokado K, Yoshida M: Apolipoprotein CⅢ links hyperlipidemia with vascular endothelial cell dysfunction. Circulation, 2008; 118:731-742 33) Nakajima K, Saito T, Tamura A, Suzuki M, Nakano T, Adachi M, Tanaka A, Tada N, Nakamura H, Campos E, et al: Cholesterol in remnant-like lipoproteins in human serum using monoclonal anti apo B-100 and anti apo A-Ⅰ immunoaffinity mixed gels. Clin Chim Acta, 1993; 223:5371 34) Karpe F, Boquist S, Tang R, Bond GM, de Faire U, Hamsten A: Remnant lipoproteins are related to intima-media thickness of the carotid artery independently of LDL cholesterol and plasma triglycerides. J Lipid Res, 2001; 42:17-21 35) Nakada Y, Kurosawa H, Tohyama J, Inoue Y, Ikewaki K: Increased remnant lipoprotein in patients with coronary artery disease-evaluation utilizing a newly developed remnant assay, remnant lipoproteins cholesterol homogenous assay (RemL-C). J Atheroscler Thromb, 2007; 14:56-64 36) Marcoux C, Tremblay M, Nakajima K, Davignon J, Cohn JS: Characterization of remnant-like particles isolated by immunoaffinity gel from the plasma of type Ⅲ and type Ⅳ hyperlipoproteinemic patients. J Lipid Res, 1999; 40:636-647 37) Kawakami A, Yoshida M: Remnant lipoproteins and atherogenesis. J Atheroscler Thromb, 2005; 12:73-76 38) Kawakami A, Tanaka A, Nakajima K, Shimokado K, Yoshida M: Atorvastatin attenuates remnant lipoprotein-induced monocyte adhesion to vascular endothelium under flow conditions. Circ Res, 2002; 91:263-271 39) Kawakami A, Tanaka A, Chiba T, Nakajima K, Shimokado K, Yoshida M: Remnant lipoprotein-induced smooth muscle cell proliferation involves epidermal growth factor receptor transactivation. Circulation, 2003; 108:2679-2688 40) Kawakami A, Tani M, Chiba T, Yui K, Shinozaki S, Nakajima K, Tanaka A, Shimokado K, Yoshida M: Pitavastatin inhibits remnant lipoprotein-induced macrophage foam cell formation through ApoB48 receptor-dependent mechanism. Arterioscler Thromb Vasc Biol, 2005; 25:424-429