Akio Kawakami

Apolipoprotein CIII Induces Expression of Vascular Cell Adhesion Molecule-1 in Vascular Endothelial Cells and Increases Adhesion of Monocytic Cells

Background— Activation of vascular endothelial cells (ECs) plays an important role in atherogenesis and plaque instability. Lipoproteins containing apolipoprotein CIII (apoCIII) predict coronary heart disease (CHD). We recently reported that apoCIII has a proinflammatory effect on human monocytes. In this study, we looked for a direct effect of apoCIII on EC expression of adhesion molecules, leading to monocytic cell adhesion. Methods and Results— Treatment of ECs with apoCIII or apoCIII-rich VLDL caused human monocytic THP-1 cells to adhere to them under static condition or under laminar sheer stress (1.0 dyne/cm2). ApoCIII increased EC expression of vascular cell adhesion molecule-1 (VCAM-1) protein and intercellular cell adhesion molecule-1 (ICAM-1) protein (4.9±1.5-fold and 1.4±0.5-fold versus control, respectively). Furthermore, apoCIII remarkably increased membrane-bound protein kinase C (PKC) β in ECs, indicating activation. A selective inhibitor of PKCβ prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, exposure of ECs to apoCIII induced nuclear factor-&kgr;B (NF-&kgr;B) activation. PKCβ inhibition abolished apoCIII-induced NF-&kgr;B activation, and NF-&kgr;B inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. ApoCIII-rich VLDL also activated PKCβ and NF-&kgr;B in ECs and increased expression of VCAM-1. Pretreatment of ApoCIII-rich VLDL with anti-apoCIII neutralizing antibody abolished its effect on PKCβ activation. Conclusions— Our findings provide the first evidence that apoCIII increases VCAM-1 and ICAM-1 expression in ECs by activating PKCβ and NF-&kgr;B, suggesting a novel mechanism for EC activation induced by dyslipidemia. Therefore, apoCIII-rich VLDL may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them.

Apolipoprotein CIII in Apolipoprotein B Lipoproteins Enhances the Adhesion of Human Monocytic Cells to Endothelial Cells

Background— Lipoproteins containing apolipoprotein (apo) CIII predict coronary heart disease and associate with components of the metabolic syndrome. ApoCIII inhibits lipoprotein catabolism in plasma. However, it is unknown whether apoCIII itself, or in association with VLDL, LDL, or HDL, directly affects atherogenic mechanisms in vascular cells. Thus, we investigated the direct effect of lipoproteins that do or do not have apoCIII, and apoCIII itself, on adhesion of THP-1 cells, a human monocytic cell line, to vascular endothelial cells (ECs). Methods and Results— VLDL CIII+ and LDL CIII+ (100 &mgr;g apoB/mL) from fasting plasma of 18 normolipidemic volunteers increased THP-1 cell adhesion to ECs under static conditions by 2.4±0.3-fold and 1.8±0.7-fold, respectively (P<0.01), whereas VLDL or LDL without apoCIII did not affect THP-1 cell adhesion. ApoCIII (100 &mgr;g/mL), but not apoCI, apoCII or apoE, also increased THP-1 cell adhesion by 2.1±0.6-fold. Studies with human peripheral blood monocytes yielded similar results. ApoCIII also had strong proadhesive effects under shear flow conditions. VLDL CIII+, LDL CIII+, or apoCIII itself activated PKC&agr; and RhoA in THP-1 cells, which resulted in &bgr;1-integrin activation and enhancement of THP-1 cell adhesion. Interestingly, HDL CIII+ did not affect THP-1 cell adhesion, whereas HDL without apoCIII decreased their adhesion. Conclusions— ApoB lipoproteins that contain apoCIII increase THP-1 cell adhesion to ECs via PKC&agr; and RhoA-mediated &bgr;1-integrin activation. These results indicate that apoCIII not only modulates lipoprotein metabolism but also may directly contribute to the development of atherosclerosis.

Ezetimibe improves liver steatosis and insulin resistance in obese rat model of metabolic syndrome

Non‐alcoholic fatty liver disease (NAFLD) is associated with the metabolic syndrome characterized by dislipidemia and insulin resistance. We hypothesized that ezetimibe, an inhibitor of NPC1L1, improves these metabolic disorders in Zucker obese fatty rats (ZOF). Ezetimibe significantly lowered total cholesterol and triglycerides in ZOF with prominent reduction in the remnant lipoprotein fraction and small dense low density lipoprotein fraction. Moreover, lipid deposition and fibrosis of liver were decreased by ezetimibe. Interestingly, ezetimibe improved insulin and plasma glucose response after intraperitoneal glucose injection. Further, ezetimibe enhanced insulin signaling in cultured hepatocytes. Our results indicate the potential of ezetimibe in treating the metabolic syndrome and NAFLD.

Apolipoprotein CIII Links Hyperlipidemia With Vascular Endothelial Cell Dysfunction

Background— Apolipoprotein CIII (apoCIII) is a component of some triglyceride-rich very-low-density and low-density lipoprotein and is elevated in dyslipidemia with insulin resistance and the metabolic syndrome. We previously reported that apoCIII directly activates proinflammatory and atherogenic signaling in vascular endothelial cells through protein kinase C-&bgr; (PKC&bgr;). Because PKC&bgr; impairs the response of vascular endothelial cells to insulin, we tested the hypothesis that apoCIII affects insulin signaling in vascular endothelial cells and its function in vitro and in vivo. Methods and Results— ApoCIII inhibited insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), decreasing phosphatidylinositol 3-kinase (PI3K)/Akt activation in human umbilical vein endothelial cells. These effects of apoCIII led to reduced endothelial nitric oxide synthase (eNOS) activation and NO release into the media. ApoCIII activated PKC&bgr; in human umbilical vein endothelial cells, resulting in IRS-1 dysfunction via serine phosphorylation. ApoCIII also activated mitogen-activated protein kinase through PKC&bgr;. The impaired insulin signaling was restored by PKC&bgr; inhibitor or MEK1 inhibitor. ApoCIII-rich very-low-density lipoprotein and apoCIII impaired insulin signaling in the aorta of C57BL/6J mice and in human umbilical vein endothelial cells, which was recovered by PKC&bgr; inhibitor. They also inhibited endothelium-dependent relaxation of the aortas of C57BL/6J mice. In summary, apoCIII in very-low-density lipoprotein impaired insulin stimulation of NO production by vascular endothelium and induced endothelial dysfunction in vivo. This adverse effect of apoCIII was mediated by its activation of PKC&bgr;, which inhibits the IRS-1/PI3K/Akt/eNOS pathway. Conclusion— Our results suggest that apoCIII is a crucial link between dyslipidemia and insulin resistance in vascular endothelial cells with consequential deleterious effects on their atheroprotective functions.

Atorvastatin Attenuates Remnant Lipoprotein-Induced Monocyte Adhesion to Vascular Endothelium Under Flow Conditions

Abstract— Remnant lipoproteins have been reported to play a causative role in atherogenesis. We investigated the effect of remnant-like lipoprotein particles (RLPs) on monocyte-endothelial interaction and their potential regulation by atorvastatin. Monocytic U937 cells were incubated with RLPs isolated from hypertriglyceridemia subjects and their adhesion to human umbilical vein endothelial cells (HUVECs) was examined under flow conditions. Incubation of U937 cells with 15 &mgr;g protein/mL RLPs increased their adhesion to HUVECs activated with IL-1&bgr; (untreated: 6.8±1.6 cells/HPF versus RLPs: 16.2±3.3 cells/HPF, P <0.05). Flow cytometric analysis revealed that incubation with RLPs increased expression levels of CD11a, CD18, and CD49d in U937 cells. Moreover, RLP-induced RhoA activation as well as FAK activation was seen in U937 cells, and RLP-induced RhoA activation seemed to be involved with PKC-dependent signaling. To explore the effect of atorvastatin on RLP-induced U937 cell adhesion to HUVECs, U937 cells were incubated with RLPs in the presence of atorvastatin. Pretreatment of U937 cells with 10 &mgr;mol/L atorvastatin significantly decreased RLP-induced U937 cell adhesion to activated HUVECs (RLP 15.2±1.5 cells/HPF versus atorvastatin+RLP 10.2±1.0 cells/HPF;P <0.05) and decreased the enhanced integrin expression in RLP-treated U937 cells. Atorvastatin also inhibited RLP-induced RhoA activation and FAK activation in U937 cells. In summary, RLPs induced monocyte adhesion to vascular endothelium by sequential activation of PKC, RhoA, FAK, and integrins, indicating a role of remnant lipoproteins in vascular inflammation during atherogenesis. Atorvastatin attenuated this enhanced monocyte adhesion to HUVECs, suggesting an antiinflammatory role for this compound.

Toll-Like Receptor 2 Mediates Apolipoprotein CIII–Induced Monocyte Activation: Retracted

Apolipoprotein (apo)CIII predicts risk for coronary heart disease. We recently reported that apoCIII directly activates human monocytes. Recent evidence indicates that toll-like receptor (TLR)2 can contribute to atherogenesis through transduction of inflammatory signals. Here, we tested the hypothesis that apoCIII activates human monocytoid THP-1 cells through TLR2. ApoCIII induced the association of TLR2 with myeloid differentiation factor 88, activated nuclear factor (NF)-&kgr;B in THP-1 cells, and increased their adhesion to human umbilical vein endothelial cells (HUVECs). Anti-TLR2 blocking antibody, but not anti-TLR4 blocking antibody or isotype-matched IgG, inhibited these processes (P<0.05). ApoCIII bound with high affinity to human recombinant TLR2 protein and showed a significantly higher (P<0.05) and saturable binding to 293 cells overexpressing human TLR2 than to parental 293 cells with no endogenous TLR2. Overexpression of TLR2 in 293 cells augmented apoCIII-induced NF-&kgr;B activation and &bgr;1 integrin expression, processes inhibited by anti-apoCIII antibody as well as anti-TLR2 antibody. Exposure of peripheral blood monocytes isolated from C57BL/6 (wild-type) mice to apoCIII activated their NF-&kgr;B and increased their adhesiveness to HUVECs. In contrast, apoCIII did not activate monocytes from TLR2-deficient mice. Finally, intravenous administration to C57BL/6 mice of apoCIII-rich very-low-density lipoprotein (VLDL), but not of apoCIII-deficient VLDL, activated monocytes and increased their adhesiveness to HUVECs, processes attenuated by anti-TLR2 or anti-apoCIII antibody. ApoCIII-rich VLDL did not activate monocytes from TLR2-deficient mice. In conclusion, apoCIII activated monocytes at least partly through a TLR2-dependent pathway. The present study identifies a novel mechanism for proinflammatory and proatherogenic effects of apoCIII and a role for TLR2 in atherosclerosis induced by atherogenic lipoproteins.

Apolipoprotein CIII-Induced THP-1 Cell Adhesion to Endothelial Cells Involves Pertussis Toxin-Sensitive G Protein- and Protein Kinase Cα-Mediated Nuclear Factor-κB Activation

Objective—Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC&agr; as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC&agr; in human monocytic THP-1 cells, leading to NF-&kgr;B activation. Methods and Results—Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC&agr; activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC&agr; activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC&agr; activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-&kgr;B (NF-&kgr;B) through PKC&agr; in THP-1 cells and augmented &bgr;1-integrin expression. The NF-&kgr;B inhibitor peptide SN50 partially inhibited apoCIII-induced &bgr;1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. Conclusions—PTX-sensitive G protein pathway participates critically in PKC&agr; stimulation in THP-1 cells exposed to apoCIII, activating NF-&kgr;B, and increasing &bgr;1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-&kgr;B activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.

Inhibition of hepatic Niemann-Pick C1-like 1 improves hepatic insulin resistance

The present study attempted to define the role of hepatic Niemann-Pick C1-like 1 (NPC1L1), a cholesterol transporter, in hepatic insulin resistance as well as hepatic steatosis. The inhibition of NPC1L1 and its molecular consequences were examined in Zucker obese fatty (ZOF) rats and cultured steatotic hepatocytes using ezetimibe, a pharmacoloigcal inhibitor of NPC1L1, and short hairpin RNA (shRNA) of NPC1L1. Ezetimibe improved hepatic insulin signaling as well as hepatic steatosis in ZOF rats. It also restored insulin sensitivity in steatotic hepatocytes in vitro through a reduction in hepatic reactive oxygen species (ROS) generation, JNK activation, and ER stress. In addition, ezetimibe recovered insulin-induced Akt activation and reduced gluconeogenic genes in the liver of ZOF rats and cultured steatotic hepatocytes. Transfection of NPC1L1 shRNA into hepatocytes also reduced ROS generation and ER stress. These results indicate that NPC1L1 contributes to hepatic insulin resistance through cholesterol accumulation, and its inhibition could be a potential therapeutic target of hepatic insulin resistance.

Pitavastatin Inhibits Remnant Lipoprotein-Induced Macrophage Foam Cell Formation Through ApoB48 Receptor–Dependent Mechanism

Objective— Atherogenic remnant lipoproteins (RLPs) are known to induce foam cell formation in macrophages in vitro and in vivo. We examined the involvement of apoB48 receptor (apoB48R), a novel receptor for RLPs, in that process in vitro and its potential regulation by pitavastatin. Methods and Results— THP-1 macrophages were incubated in the presence of RLPs (20 mg cholesterol/dL, 24 hours) isolated from hypertriglyceridemic subjects. RLPs significantly increased intracellular cholesterol ester (CE) and triglyceride (TG) contents (4.8-fold and 5.8-fold, respectively) in the macrophages. Transfection of THP-1 macrophages with short interfering RNA (siRNA) against apoB48R significantly inhibited RLP-induced TG accumulation by 44%. When THP-1 macrophages were pretreated with pitavastatin (5 &mgr;mol/L, 24 hours), the expression of apoB48R was significantly decreased and RLP-induced TG accumulation was reduced by 56%. ApoB48R siRNA also inhibited TG accumulation in THP-1 macrophage induced by &bgr;–very-low-density lipoprotein derived from apoE−/− mice by 58%, supporting the notion that apoB48R recognizes and takes-up RLPs in an apoE-independent manner. Conclusions— RLPs induce macrophage foam cell formation via apoB48R. Pitavastatin inhibits RLP-induced macrophage foam cell formation. The underlying mechanism involves, at least in part, inhibition of apoB48R-dependent mechanism. Our findings indicate a potential role of apoB48R in atherosclerosis.

Remnant Lipoprotein-Induced Smooth Muscle Cell Proliferation Involves Epidermal Growth Factor Receptor Transactivation

Background—Remnant lipoproteins (RLPs) have been shown to play a causative role during atherosclerosis. Furthermore, it is known that vascular smooth muscle cell (SMC) proliferation is crucial for the development of atherosclerosis and restenosis after percutaneous coronary intervention. We examined the direct effect of RLPs on the proliferation and signal transduction of SMCs. Methods and Results—Incubation in the presence of RLPs (20 mg cholesterol per dL) for 48 hours induced rat aortic SMC proliferation (2.3-fold over medium alone). RLPs also induced the phosphorylation of epidermal growth factor (EGF) receptor in SMCs, which was followed by the activation of mitogen-activated protein kinases. Moreover, the activation of protein kinase C (PKC) as well as the shedding of membrane-bound soluble heparin-binding EGF-like growth factor (HB-EGF) was observed after RLP treatment of SMCs, whereas PKC inhibitors and metalloprotease inhibitors inhibited RLP-induced EGF receptor transactivation and HB-EGF shedding in SMCs. Furthermore, anti-HB-EGF neutralizing antibody inhibited RLP-induced EGF receptor transactivation. Phosphorylation of EGF receptor and HB-EGF shedding were also observed in the aortas of apolipoprotein E–knockout mice but not in those of C57BL6 mice. Conclusions—These results suggest that RLPs transactivate EGF receptor via PKC and HB-EGF shedding from SMCs, resulting in SMC proliferation.