Hiroyuki Tanigawa

Expression of Cholesteryl Ester Transfer Protein in Mice Promotes Macrophage Reverse Cholesterol Transport

Background— Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high-density lipoproteins to apolipoprotein (apo) B–containing lipoproteins and in humans plays an important role in lipoprotein metabolism. However, the role that CETP plays in mediation of reverse cholesterol transport (RCT) remains unclear. We used a validated in vivo assay of macrophage RCT to test the effect of CETP expression in mice (which naturally lack CETP) on macrophage RCT, including in mice that lack the low-density lipoprotein receptor or the scavenger receptor class B, type I. Method and Results— A vector based on adeno-associated virus serotype 8 (AAV8) with a liver-specific thyroglobulin promoter was used to stably express human CETP in livers of mice and was compared with an AAV8-lacZ control vector. The RCT assay was performed 4 weeks after vector injection and involved the intraperitoneal injection of acetylated low-density lipoprotein cholesterol–loaded and 3H-cholesterol–labeled J774 macrophages in mice with plasma sampling at several time points, liver and bile sampling at 48 hours, and continuous fecal collection to measure 3H-sterol as an integrated readout of macrophage RCT. In apobec-1–null mice, CETP expression reduced plasma high-density lipoprotein cholesterol levels but significantly increased fecal 3H-sterol excretion. In low-density lipoprotein receptor/apobec-1 double-null mice, CETP expression reduced high-density lipoprotein cholesterol levels and had no effect on fecal 3H-sterol excretion. Finally, in scavenger receptor class B, type I–null mice, CETP expression reduced high-density lipoprotein cholesterol levels and significantly increased fecal 3H-sterol excretion. Conclusion— The present results demonstrate that CETP expression promotes macrophage RCT in mice, that this effect is dependent on the low-density lipoprotein receptor, and that CETP expression restores to normal the impaired RCT in mice deficient in scavenger receptor class B, type I.

High Density Lipoprotein–Induced Angiogenesis Requires the Activation of Ras/MAP Kinase in Human Coronary Artery Endothelial Cells

Objective—Plasma high density lipoprotein (HDL) levels have been shown to be inversely correlated with coronary artery disease, but the mechanisms of the direct protective effect of HDL on endothelial cells (ECs) are not fully understood. In this study, we investigated the role of the HDL-mediated promotion of angiogenesis in human coronary artery ECs (HCECs). Methods and Results—We developed an in vitro model of HCEC tube formation on a matrix gel. We optimized the maximum dose of HDL required to induce tube formation in initial experiments, in which the dose response showed that the maximum effective dose of HDL was 100 &mgr;g/mL. PD98059, an inhibitor of p42/44 mitogen-activated protein kinase (MAPK) activity, but not SB203580, an inhibitor of p38 MAPK activity, suppressed HDL-induced tube formation. Dominant-negative Ras N17 inhibited HDL-induced tube formation. HDL activated Ras according to a ras pull-down assay, and this effect was inhibited by pertussis toxin. Moreover, HDL activated phospho(p)-p42/44 MAPK, whereas Ras N17 blocked HDL-induced pp42/44 MAPK. Conclusions—These results indicate that HDL induced a potent signal through a Ras/MAPK pathway mediated by a pertussis toxin–sensitive G-protein coupled receptor to the angiogenic phenotype in HCECs.

Lecithin: cholesterol acyltransferase expression has minimal effects on macrophage reverse cholesterol transport in vivo.

Background— Lecithin:cholesterol acyltransferase (LCAT) catalyzes the formation of plasma cholesteryl ester, plays a key role in high-density lipoprotein metabolism, and has been believed to be critical in the process of reverse cholesterol transport (RCT). Methods and Results— The role of LCAT in RCT from macrophages was quantified with a validated assay involving intraperitoneal injection in mice of 3H-cholesterol–labeled J774 macrophages and monitoring the appearance of tracer in plasma, liver, bile, and feces. Human LCAT overexpression in human apolipoprotein A-I transgenic mice substantially increased plasma high-density lipoprotein cholesterol levels but surprisingly did not increase macrophage RCT. Even in the setting of coexpression of scavenger receptor BI or cholesteryl ester transfer protein, both of which promoted the transfer of LCAT-derived high-density lipoprotein cholesterol ester to the liver, LCAT overexpression still had no effect on RCT. Serum from LCAT-overexpressing mice had reduced ability to promote cholesterol efflux from macrophages ex vivo via ABCA1. To determine the effect of LCAT deficiency on macrophage RCT, LCAT−/− and LCAT+/− mice were compared with wild-type mice. Despite extremely low plasma levels of high-density lipoprotein cholesterol, LCAT-deficient mice had only a 50% reduction in RCT. LCAT+/− mice had normal RCT despite a significant reduction in high-density lipoprotein cholesterol. Serum from LCAT-deficient mice had increased ability to promote ABCA1-mediated cholesterol efflux from macrophages ex vivo. Conclusions— These results demonstrate that LCAT overexpression does not promote an increased rate of macrophage RCT. Although LCAT activity does become rate limiting in the context of complete LCAT deficiency, RCT is reduced by only 50% even in the absence of LCAT. These data suggest that macrophage RCT may not be as dependent on LCAT activity as has previously been believed.

Pharmacologic Suppression of Hepatic ATP-Binding Cassette Transporter 1 Activity in Mice Reduces High-Density Lipoprotein Cholesterol Levels but Promotes Reverse Cholesterol Transport

Background— The role of hepatic ATP-binding cassette transporter 1 (ABCA1) in maintaining plasma high density lipoprotein cholesterol (HDL-C) levels is well established, but its role in reverse cholesterol transport (RCT) is unclear. Probucol is a compound that reduces HDL-C levels but also reduces atherosclerosis in animal models and xanthomas in humans. The aim of the present study was to test the hypothesis that probucol inhibits hepatic ABCA1 activity, thereby reducing HDL-C levels but promoting RCT from macrophages. Methods and Results— Wild-type (WT) C57BL/6 mice and scavenger receptor class B type I (SR-BI) knockout mice were fed a chow diet containing 0.5% probucol or normal chow for 2 weeks. In WT mice, probucol, despite decreasing HDL-C by >80%, effectively maintained macrophage RCT. In SR-BI knockout mice, probucol also substantially reduced HDL-C but significantly increased macrophage RCT. Furthermore, probucol significantly enhanced the excretion of HDL-derived cholesterol into feces in both WT and SR-BI knockout mice. Probucol inhibited ABCA1-dependent cholesterol efflux from mouse primary hepatocytes, and this effect was shown to be responsible for the effect of probucol on increasing the fecal excretion of HDL-derived cholesterol in vivo. Conclusions— We demonstrate that pharmacological inhibition of hepatic ABCA1 activity with probucol reduced HDL-C levels but promoted RCT through diversion of HDL-derived cholesterol from efflux back into plasma instead to excretion in the bile. These results explain the beneficial effects of probucol on atherosclerosis and xanthomas despite its HDL-lowering effects and suggest that inactivation of hepatic ABCA1 leads to increased RCT despite reducing plasma HDL-C levels.

Nifedipine-Induced Vascular Endothelial Growth Factor Secretion from Coronary Smooth Muscle Cells Promotes Endothelial Tube Formation via the Kinase Insert Domain-Containing Receptor/Fetal Liver Kinase-1/NO Pathway

Endothelial cells (ECs) are the critical cellular element responsible for postnatal angiogenesis. Since the calcium channel blocker (CCB) nifedipine indirectly upregulates endothelial superoxide dismutase expression by stimulating the production of vascular endothelial growth factor (VEGF) from smooth muscle cells (SMCs), we examined whether nifedipine would induce human coronary artery endothelial cell (HCEC) tube formation via an increase in VEGF production from human coronary artery SMCs (HCSMCs) in an in vitro model. Nifedipine stimulated VEGF production from HCSMCs, and this stimulation was abolished by protein kinase C (PKC) inhibitors and a bradykinin B2 receptor antagonist. In addition, supernatant derived from nifedipine-treated HCSMCs induced HCEC tube formation. This tube formation was inhibited by pretreatment with a specific inhibitor of kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1) tyrosine kinase and an inhibitor of nitric oxide (NO) synthase. In conclusion, nifedipine increases VEGF secretion through PKC activation via the B2 receptor. The VEGF secretion directly induces HCEC tube formation via the KDR/Flk-1/NO pathway. CCBs may thus have novel beneficial effects in improving coronary microvascular blood flow in addition to their main effect of reducing blood pressure.

Peroxisome Proliferator-Activated Receptor-α Activation Promotes Macrophage Reverse Cholesterol Transport Through a Liver X Receptor–Dependent Pathway

Objective—Peroxisome proliferator-activated receptor-&agr; (PPAR&agr;) activation has been shown in vitro to increase macrophage cholesterol efflux, the initial step in reverse cholesterol transport (RCT). However, it remains unclear whether PPAR&agr; activation promotes macrophage RCT in vivo. Methods and Results—We demonstrated that a specific potent PPAR&agr; agonist GW7647 inhibited atherosclerosis and promoted macrophage RCT in hypercholesterolemic mice expressing the human apolipoprotein A-I (apoA-I) gene. We compared the effect of GW7647 on RCT in human apoA-I transgenic (hA-ITg) mice with wild-type mice and showed that the PPAR&agr; agonist promoted RCT in hA-ITg mice to a much greater extent than in wild-type mice, indicating that human apoA-I expression is important for PPAR&agr;-induced RCT. We further investigated the dependence of the macrophage PPAR&agr;–liver X receptor (LXR) pathway on the promotion of RCT by GW7647. Primary murine macrophages lacking PPAR&agr; or LXR abolished the ability of GW7647 to promote RCT in hA-ITg mice. In concert, the PPAR&agr; agonist promoted cholesterol efflux and ATP binding cassette transporter A1/G1 expression in primary macrophages, and this was also by the PPAR&agr;-LXR pathway. Conclusion—Our observations demonstrate that a potent PPAR&agr; agonist promotes macrophage RCT in vivo in a manner that is enhanced by human apoA-I expression and dependent on both macrophage PPAR&agr; and LXR expression.

FAMP, a Novel ApoA‐I Mimetic Peptide, Suppresses Aortic Plaque Formation Through Promotion of Biological HDL Function in ApoE‐Deficient Mice

Background Apolipoprotein (apo) A‐I is a major high‐density lipoprotein (HDL) protein that causes cholesterol efflux from peripheral cells through the ATP‐binding cassette transporter A1 (ABCA1), thus generating HDL and reversing the macrophage foam cell phenotype. Pre‐β1 HDL is the smallest subfraction of HDL, which is believed to represent newly formed HDL, and it is the most active acceptor of free cholesterol. Furthermore it has a possible protective function against cardiovascular disease (CVD). We developed a novel apoA‐I mimetic peptide without phospholipids (Fukuoka University ApoA‐I Mimetic Peptide, FAMP). Methods and Results FAMP type 5 (FAMP5) had a high capacity for cholesterol efflux from A172 cells and mouse and human macrophages in vitro, and the efflux was mainly dependent on ABCA1 transporter. Incubation of FAMP5 with human HDL or whole plasma generated small HDL particles, and charged apoA‐I‐rich particles migrated as pre‐β HDL on agarose gel electrophoresis. Sixteen weeks of treatment with FAMP5 significantly suppressed aortic plaque formation (scrambled FAMP, 31.3±8.9% versus high‐dose FAMP5, 16.2±5.0%; P<0.01) and plasma C‐reactive protein and monocyte chemoattractant protein‐1 in apoE‐deficient mice fed a high‐fat diet. In addition, it significantly enhanced HDL‐mediated cholesterol efflux capacity from the mice. Conclusions A newly developed apoA‐I mimetic peptide, FAMP, has an antiatherosclerotic effect through the enhancement of the biological function of HDL. FAMP may have significant atheroprotective potential and prove to be a new therapeutic tool for CVD.

Dominant-Negative Lox-1 Blocks Homodimerization of Wild-Type Lox-1–Induced Cell Proliferation Through Extracellular Signal Regulated Kinase 1/2 Activation

C-type lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor-1 (Lox-1) belongs to the same family as natural killer cell receptors Ly49A and CD94 and functionally undergoes dimerization. Although Lys262 and Lys263 in the C terminus of bovine (b)Lox-1 play an important role in the uptake of Ox-LDL, mutation of these residues has not been suggested to be a potential source of the dominant-negative property. We hypothesize that dominant-negative human (h)Lox-1 forms a heterodimer with Lox-1–wild-type (WT) and blocks Lox-1–WT–induced cell signaling. Based on the use of molecular imaging techniques with laser scanning confocal microscopy and immunoprecipitation in an hLox-1–expressing Chinese hamster ovary cell system, homodimerization of hLox-1–WT was localized in the cell membrane, and Ox-LDL activated extracellular signal regulated kinase (ERK)1/2 without the translocation of hLox-1-WT. Lys266 and Lys267 of hLox-1, corresponding with Lys262 and Lys263 of bLox-1, were mutated (hLox1-K266A/K267A), and the mutant receptor inhibited hLox-1–WT–induced thymidine incorporation and ERK1/2 activation. Although Ox-LDL binds to the dominant-negative mutant receptor and is taken up by cytoplasm, ERK1/2 activation was blocked by heterodimerization with the mutant receptor and hLox-1–WT in the cell membrane. In addition, in human coronary artery smooth muscle cells, which express hLox-1–WT, we confirmed that the activation of ERK1/2 and [3H]-thymidine incorporation was caused by the addition of Ox-LDL, and these actions were blocked by hLox1-K266A/K267A. In conclusion, the present findings constitute the first evidence that strategies aimed at blocking cell-proliferative pathways at the receptor level could be useful for impairing Lox-1–induced cell proliferation.

Ras/Raf1-dependent signal in sphingosine-1-phosphate-induced tube formation in human coronary artery endothelial cells

Since we recently reported that high density lipoprotein, which contains the bioactive lipid sphingosine-1-phosphate (S1P) [Arterioscler. Thromb. Vasc. Biol. 23 (2003) 802], induced human coronary artery endothelial cell (HCEC) tube formation mediated by a Ras/Raf/ERK (extracellular signal-activated kinase) pathway, we thought that it would be very important to evaluate whether the signal in S1P-induced tube formation is Ras-dependent or -independent. In an in vitro model of HCEC tube formation on a matrix gel, S1P-induced tube formation. ERK1/2 inhibitor (PD98059) and pertussis toxin (PTX) suppressed S1P-induced tube formation. S1P activated phospho(p)-ERK1/2, while dominant-negative RasN17 blocked S1P-induced p-ERK1/2. Moreover, RasN17 inhibited S1P-induced tube formation. S1P activated Ras/Raf1 by Ras pull-down assay and this effect was inhibited by PTX. These results demonstrate that Ras/Raf1-dependent ERK activation mediated by PTX-sensitive G protein-coupled receptors may be a potent signal in S1P-induced HCEC tube formation.

Rosuvastatin Activates ATP-Binding Cassette Transporter A1–Dependent Efflux Ex Vivo and Promotes Reverse Cholesterol Transport in Macrophage Cells in Mice Fed a High-Fat Diet

Objective— It is controversial whether statins improve high-density lipoprotein (HDL) function, which plays an important role in reverse cholesterol transport in vivo. The aim of the present study was to clarify the effects of rosuvastatin and atorvastatin on reverse cholesterol transport in macrophage cells in vivo and their underlying mechanisms. Approach and Results— Male C57BL mice were divided into 3 groups (rosuvastatin, atorvastatin, and control groups) and orally administered rosuvastatin, atorvastatin, or placebo for 6 weeks under feeding with a 0.5% cholesterol+10% coconut oil diet. After administration, although there were no changes in plasma HDL cholesterol levels among the groups, plasma from the rosuvastatin group showed an increased ability to promote ATP-binding cassette transporter A1–mediated cholesterol efflux ex vivo. In addition, capillary electrophoresis revealed a shift in HDL toward the pre-&bgr; HDL fraction only in the rosuvastatin group. Mice in all 3 groups were intraperitoneally injected with 3H-cholesterol–labeled and cholesterol-loaded macrophages and then were monitored for the appearance of 3H-tracer in plasma and feces. The amount of 3H-tracer excreted into feces during 48 hours in the rosuvastatin group was greater than that in the control group. Finally, 3H-cholesteryl oleate-HDL was intravenously injected into all groups, blood samples were taken, and the count of 3H-cholesterol was analyzed. Plasma 3H-cholesteryl oleate-HDL changed similarly, and no differences in fractional catabolic rates were observed. Conclusions— Rosuvastatin enhanced the ATP-binding cassette transporter A1–dependent HDL efflux function of reverse cholesterol transport, and this finding highlights the potential of rosuvastatin for the regression of atherosclerosis.