Yu-Ichiro Sakamoto

Intravenous Injection of Rabbit Apolipoprotein A-I Inhibits the Progression of Atherosclerosis in Cholesterol-Fed Rabbits

The effects of intravenous injection of purified rabbit apoA-I on the progression of aortic atherosclerosis in cholesterol-fed rabbits were examined. In experiment 1, 28 rabbits were equally divided into groups A and B and fed a 0.5% cholesterol diet for 90 days. For the last 30 days, group B received 40 mg apoA-I every week. The fatty streak lesions in group B (23.9 +/- 15.6%) were significantly suppressed compared with those in group A (46.0 +/- 24.9%) (P < .05). In experiment 2, 33 rabbits were divided into four groups (8 or 9 rabbits per group) and fed a 0.5% cholesterol diet. Group A was killed on day 105, while groups B, C, and D were maintained for an additional 60 days on a normal diet, during which time groups C and D received 1 mg apoA-I every other day or 40 mg apoA-I every week, respectively. The lesions in group C (70.2 +/- 15.4%) and group D (65.7 +/- 20.0%) were significantly suppressed compared with those in group B (86.2 +/- 13.7%) (P < .05) but were not reduced to the level of group A (50.0 +/- 22.9%). Although apparent regression was not observed under these conditions, the present study provided the first evidence for the antiatherogenic effect of homologous and apoA-I on the progression of atherosclerosis in cholesterol-fed rabbits.

Scavenger receptors for oxidized and glycated proteins

Summary. Our present knowledge on chemically modified proteins and their receptor systems is originated from a proposal by Goldstein and Brown in 1979 for the receptor for acetylated LDL which is involved in foam cell formation, one of critical steps in atherogenesis. Subsequent extensive studies using oxidized LDL (OxLDL) as a representative ligand disclosed at least 11 different scavenger receptors which are collectively categorized as “scavenger receptor family”. Advanced glycation endproducts (AGE) and their receptor systems have been studied independently until recent findings that AGE-proteins are also recognized as active ligands by scavenger receptors including class A scavenger receptor (SR-A), class B scavenger receptors such as CD36 and SR-BI, type D scavenger receptor (LOX-1) and FEEL-1/FEEL-2. Three messages can be summarized from these experiments; (i) endocytic uptake of OxLDL and AGE-proteins by macrophages or macrophage-derived cells is mainly mediated by SR-A and CD36, which is an important step for foam cell formation in the early stage of atherosclerosis, (ii) selective uptake of cholesteryl esters of high density lipoprotein (HDL) mediated by SR-BI is inhibited by AGE-proteins, suggesting a potential pathological role of AGE in a HDL-mediated reverse cholesterol transport system, (iii) a novel scavenger receptor is involved in hepatic clearance of plasma OxLDL and AGE-proteins.

Human Urotensin II Accelerates Foam Cell Formation in Human Monocyte-Derived Macrophages

Human urotensin II (U-II), the most potent vasoconstrictor peptide identified to date, and its receptor (UT) are involved in hypertension and atherosclerosis. Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) converts intracellular free cholesterol into cholesterol ester (CE) for storage in lipid droplets and plays an important role in the formation of macrophage-derived foam cells in atherosclerotic lesions. We examined the effects of U-II on ACAT-1 expression and CE accumulation in human monocyte-derived macrophages. U-II increased ACAT activity in a concentration-dependent manner after 7 days in monocyte primary culture. Immunoblotting analysis showed that U-II at 25 nmol/L increased ACAT-1 protein expression level by 2.5-fold, which was completely abolished by anti–U-II antibody, selective UT receptor antagonists (urantide and 4-aminoquinoline), a G-protein inactivator (GDP-&bgr;-S), a c-Src protein tyrosine kinase inhibitor (PP2), a protein kinase C (PKC) inhibitor (rottlerin), a mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059), or a Rho kinase (ROCK) inhibitor (Y27632). Northern blotting analysis indicated that among the 4 ACAT-1 mRNA transcripts (2.8-, 3.6-, 4.3-, and 7.0-kb), the 2.8- and 3.6-kb transcript levels were selectively upregulated by ≈1.7-fold by U-II (25 nmol/L). Further, U-II (25 nmol/L) significantly increased acetylated LDL (acetyl-LDL)–induced CE accumulation in monocyte-derived macrophages but not scavenger receptor class A (SR-A) function as assessed by endocytic uptake of [125I]acetyl-LDL. Our results suggest that U-II may play a novel role in the formation of macrophage-derived foam cells by upregulating ACAT-1 expression via the UT receptor/G-protein/c-Src/PKC/MEK and ROCK pathways but not by SR-A, thus contributing to the relatively rapid development of atherosclerosis in hypertension.

Species difference in cholesteryl ester cycle and HDL-induced cholesterol efflux from macrophage foam cells.

The species difference in the turnover rates of the cholesteryl ester (CE) cycle in macrophage foam cells (MFC) was examined in mice and rats. MFC were induced by acetyl-LDL and pulsed with [3H]oleate, followed by a chase with [14C]oleate. The replacement of the initial amount of cholesteryl [3H]oleate by cholesteryl [14C]oleate within 24 hours was 63% in mouse MFC, whereas it was 33% in rat MFC. The corresponding replacement in rabbit MFC was < 10%. In addition, HDL removed 41% of the CE mass from mouse MFC but only 22% from rat MFC. HDL-induced CE reduction from mouse MFC was enhanced by 40% by the inhibitor for acyl-coenzyme A:cholesterol acyltransferase (58-035), whereas the enhancing effect was not observed with rat MFC. These results indicate that the rate of CE turnover may serve as a critical factor to determine the capacity of MFC to respond to HDL-induced CE reduction, suggesting the possibility that the species difference in the turnover rates of the CE cycle in MFC might explain, in part, the species difference in susceptibility to experimental atherosclerosis.

Cross-linking of apolipoproteins is involved in a loss of the ligand activity of high density lipoprotein upon Cu2+-mediated oxidation

A recent study demonstrated that Cu2+‐mediated oxidation of high density lipoprotein (HDL) resulted in a loss of the capacity to reduce cholesterol from macrophage foam cells [(1991) Proc. Natl. Acad. Sci. USA 88, 6457–6461]. In the present study we characterized the physicochemical properties of oxidized HDL and correlated them with the ligand activity toward the HDL receptor. Among them, the cross‐linking of apolipoproteins and an increase in lipid peroxides were characteristic and closely similar to those of tetranitromethane‐treated HDL, an abortive ligand for the HDL receptor. Cellular experiments with murine peritoneal macrophages revealed that both the cellular binding activity of HDL and its capacity to enhance cholesterol efflux from macrophage foam cells were markedly reduced upon oxidation. These results suggest that cross‐linking of HDL apolipoproteins is involved in the loss of the ligand activity of oxidized HDL.

Differential effects of an acyl-coenzyme A: cholesterol acyltransferase inhibitor on HDL-induced cholesterol efflux from rat macrophage foam cells

When rat macrophages were converted to foam cells with acetylated low density lipoprotein (acetyl‐LDL) and then reacted with high density lipoprotein (HDL) and an inhibitor of acyl‐coenzyme A:cholesterol acyltransferase (58‐035) (sequential incubation system), 58‐035 did not enhance HDL‐induced cholesterol efflux. In contrast, when macrophages were exposed to acetyl‐LDL in the presence of both HDL and 58‐035 (simultaneous incubation system), HDL‐induced cholesterol efflux was enhanced 1.6‐fold by 58‐035. Cholesterol efflux with HDL alone was 2‐fold greater in simultaneous incubation than in sequential incubation. These results suggest the presence of an efficient cholesterol efflux pathway in simultaneous incubation which is not available in sequential incubation. This pathway, which we refer to as the neutral cholesterol ester hydrolase‐independent pathway, is characterized by the efflux of lysosome‐derived cholesterol without re‐esterification.

Reconstituted high density lipoprotein reduces the capacity of oxidatively modified low density lipoprotein to accumulate cholesteryl esters in mouse peritoneal macrophages

Oxidized low density lipoprotein (ox-LDL) was incubated with discoidal complexes of apolipoprotein A-I (apo A-I) and dimyristoylphosphatidylcholine (DMPC) (DMPC/apo A-I) in a cell-free system and re-isolated on Sephacryl S-400 gel filtration chromatography. Analyses of re-isolated ox-LDL showed that apo A-I was transferred from DMPC/apo A-I to ox-LDL, which accounted for 10% of the total protein of ox-LDL. Re-isolated ox-LDL also showed a 2.2-fold increase in phospholipid and a 14% decrease in cholesterol content on an apo B basis. The electrophoretic mobility of re-isolated ox-LDL was markedly reduced almost to that of native LDL. Moreover, the amounts of re-isolated ox-LDL to be degraded by mouse peritoneal macrophages as well as the capacity of re-isolated ox-LDL to accumulate cholesteryl esters (CE) in these cells were markedly reduced (60% and 80% reduction, respectively), suggesting that the ligand activity of ox-LDL for the scavenger receptor was significantly reduced upon treatment with DMPC/apo A-I. Parallel incubation of ox-LDL with free apo A-I led to a similar incorporation of apo A-I into ox-LDL. However, it had no effects on the ligand activity of ox-LDL. Thus, it is likely that the reduction in the ligand activity of ox-LDL by DMPC/apo A-I is explained by the change in the lipid moiety (mainly phospholipid) of ox-LDL. Since discoidal high density lipoprotein (HDL) is known to occur in vivo, this phenomenon might explain one of the anti-atherogenic functions of HDL.

Glycolaldehyde-Modified Bovine Serum Albumin Downregulates Leptin Expression in Mouse Adipocytes via a CD36-Mediated Pathway

Abstract: Previous observations by us have clarified that proteins modified by advanced glycation end products (AGEs) are recognized as effective ligands by CD36‐overexpressed CHO cells and undergo receptor‐mediated endocytosis. CD36, a member of the class B scavenger receptor family, also acts as a fatty acid transporter in adipocytes. Oxidized low‐density lipoprotein (Ox‐LDL), a ligand for CD36, is known to upregulate CD36 by activating peroxisome proliferator‐activated receptor γ (PPAR‐γ) in macrophages, whereas PPAR‐γ ligands such as troglitazone and 15‐deoxy‐delta12,14‐prostaglandin J2 decrease leptin secretion from adipocytes. The purpose of this study was to examine effects of AGE ligands on leptin expression in adipocytes. Glycolaldehyde‐modified bovine serum albumin (GA‐BSA) decreased leptin expression at both the protein and mRNA levels in 3T3‐L1 adipocytes and mouse epididymal adipocytes. The binding to and subsequent endocytic degradation of GA‐BSA by 3T3‐L1 adipocytes were effectively inhibited by a neutralizing anti‐CD36 antibody. These results indicate that the ligand interaction of GA‐BSA with CD36 leads to downregulation of leptin expression in 3T3‐L1 adipocytes, suggesting that AGE‐induced leptin downregulation is linked to reduction of the insulin sensitivity in metabolic syndrome.

The heparin-bound fraction of human lipoprotein-deficient serum inhibits endocytic uptake of oxidized low density lipoprotein by macrophages

We recently demonstrated that bovine lactoferrin, a cationic whey protein from bovine milk, interacts with the negative charges of modified low density lipoproteins (modified LDL) such as acetylated LDL (acLDL) and oxidized LDL (oxLDL), which markedly interferes with their endocytic uptake by rat peritoneal macrophages (Kajikawa M, Ohta T, Takase M, Kawase K, Shimamura S, Matsuda I. Biochim Biophys Acta 1994;1213:82-90). In the present study, we examined whether human lipoprotein-deficient serum (LPDS) might contain protein(s) that could inhibit the endocytic uptake of oxLDL by mouse macrophages. We fractionated LPDS by heparin affinity chromatography and found that the cellular binding of oxLDL to mouse macrophages and subsequent endocytic uptake were inhibited by 50%-60% with the heparin-bound fraction, whereas the heparin-unbound fraction had no effect. Similar results were obtained in the experiments with acetylated LDL. Sephacryl S-300 gel-filtration chromatography of a mixture of oxLDL and the heparin-bound fraction revealed that a 150-kDa protein was associated with oxLDL. These results indicate that the electrostatic interaction of oxLDL with some component(s) of the heparin-bound fraction might interfere with the endocytic uptake of oxLDL by the macrophage scavenger receptor.

Granulocyte macrophage colony-stimulating factor plays a priming role in murine macrophage growth induced by oxidized low density lipoprotein.

One of the characteristic events in the atherosclerotic lesion is the proliferation of cellular components. It is generally accepted that smooth muscle cells migrated from media into intima proliferate in the atherosclerotic lesions. It was recently demonstrated, however, that macrophages also proliferate in the early stage of atherosclerotic lesions.1 Using an in vitro culture system, we have shown that macrophages obtained from mouse,2–5 rat,6 and human7 are able to proliferate upon incubation with oxidized LDL (Ox-LDL). It then becomes clear that the specific uptake of lysophosphatidylcholine (lyso-PC) of Ox-LDL through the macrophage scavenger receptor type A-I/ A-II (MSR-AI/AII) is essential for Ox-LDL–induced macrophage proliferation.3,5,7 The activation of protein kinase C (PKC) was also shown to be involved in this phenomenon.8 These in vitro observations strongly suggest that Ox-LDL acts as a growth factor for macrophage in vivo. The present study was undertaken to elucidate the molecular cascade(s) leading to Ox-LDL–induced macrophage proliferation. The results indicate that Ox-LDL–mediated release of granulocyte macrophage colony-stimulating factor (GM-CSF) from macrophages may play an important role in macrophage proliferation.