Masakazu Sakai

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.

Lysophosphatidylcholine plays an essential role in the mitogenic effect of oxidized low density lipoprotein on murine macrophages

We previously demonstrated that the growth of starch-induced murine macrophages was stimulated by modified low density lipoproteins, such as oxidized low density lipoprotein (Ox-LDL) and acetylated low density lipoprotein (acetyl-LDL), and that the mitogenic effect of Ox-LDL was much greater than that of acetyl-LDL (Yui, S., Sasaki, T., Miyazaki, A., Horiuchi, S., and Yamazaki, M. (1993) Arterioscler. Thromb. 13, 331-337). The present study was undertaken to elucidate the factor(s) that are involved in this growth-stimulating effect of Ox-LDL. The growth-stimulating effect of acetyl-LDL on murine resident macrophages was negligibly weak compared with that of Ox-LDL. However, the treatment of acetyl-LDL with phospholipase A2 led to an increase in lysophosphatidylcholine (lyso-PC) (75% of total phospholipids) and a concomitant increase in the mitogenic activity of acetyl-LDL. In contrast, cell-free incubation of Ox-LDL with high density lipoprotein resulted in a decrease in lyso-PC content and a concomitant loss of growth-stimulating activity. These results suggest that lyso-PC may play an essential role in the mitogenic activity of Ox-LDL.

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.

The Scavenger Receptor Serves as a Route for Internalization of Lysophosphatidylcholine in Oxidized Low Density Lipoprotein-induced Macrophage Proliferation

We have recently demonstrated that the growth of murine macrophages is induced by oxidized low density lipoprotein (Ox-LDL) and that lysophosphatidylcholine (lyso-PC), a major phospholipid component of Ox-LDL, plays an essential role in its mitogenic effect. The present study was undertaken to further characterize the role of the macrophage scavenger receptor (MSR) in Ox-LDL-induced macrophage growth. The growth-stimulating effect of Ox-LDL on murine resident peritoneal macrophages was inhibited by maleylated bovine serum albumin (maleyl-BSA), a non-lipoprotein ligand for MSR but a poor carrier of lyso-PC, while maleyl-BSA itself failed to induce macrophage growth even in the presence of lyso-PC. Moreover, it competitively inhibited the endocytic uptake of 125I-Ox-LDL and the specific uptake of lyso-PC by MSR, whereas nonspecific lyso-PC transfer to cells was not affected. Furthermore, the Ox-LDL-induced cell growth of peritoneal macrophages obtained from MSR knockout mice was significantly weaker than that of macrophages obtained from their wild-type littermates. Our results suggest that the MSR is an important and efficient internalization pathway for lyso-PC in Ox-LDL-induced macrophage growth.

Two Intracellular Signaling Pathways for Activation of Protein Kinase C Are Involved in Oxidized Low-Density Lipoprotein–Induced Macrophage Growth

Recent studies demonstrated that oxidized LDL (Ox-LDL) induces macrophage growth in vitro. The present study was undertaken to elucidate the intracellular signaling pathways for macrophage growth. Ox-LDL initiated a rapid and transient rise in intracellular free calcium ion and induced activation of membrane protein kinase C (PKC). Pertussis toxin completely inhibited the Ox-LDL-induced rise in free calcium ion and significantly inhibited macrophage growth by 50%. Moreover, PKC inhibitors calphostin C and H-7 significantly inhibited Ox-LDL-induced macrophage growth by 80%. On the other hand, phospholipase A2-treated acetylated LDL did not induce a rise in calcium but significantly activated PKC and led to significant macrophage growth that was significantly inhibited by calphostin C by 90%. These results suggest the presence of two intracellular signaling pathways for activation of PKC, a rise in calcium that was mediated by pertussis toxin-sensitive G protein and the internalization of lysophosphatidylcholine through the scavenger receptors. These two pathways may play an important role in Ox-LDL-induced macrophage growth.

HMG-CoA reductase inhibitors suppress macrophage growth induced by oxidized low density lipoprotein.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors ameliorate atherosclerotic diseases in several models of vascular disease. This is largely due to their ability to reduce plasma cholesterol levels in vivo. Proliferation of cellular components is one of the major events in the development and progression of atherosclerotic lesions. We recently demonstrated that oxidized low density lipoprotein (Ox-LDL), a likely atherogenic lipoprotein present in vivo, is capable of inducing macrophage growth in vitro. In the present study, we investigated the effect of HMG-CoA reductase inhibitors, simvastatin and pravastatin, on Ox-LDL-induced macrophage growth. Our results demonstrated that these inhibitors effectively suppressed Ox-LDL-induced macrophage growth with concentrations required for 50% inhibition by simvastatin and pravastatin being 0.1 and 80 microM, respectively, and that this inhibitory effect was reversed by mevalonate but not by squalene. Under these conditions, simvastatin did not affect the endocytic degradation of Ox-LDL, nor subsequent accumulation of intracellular cholesteryl esters. Our results suggest that a non-cholesterol metabolites(s) of mevalonate pathway may play an important role in Ox-LDL-induced macrophage growth. Since it is well known that macrophage-derived foam cells are the key cellular element in the early stage of atherosclerosis, a significant inhibition of Ox-LDL-induced macrophage growth by HMG-CoA reductase inhibitors in vitro, particularly simvastatin, may also explain, at least in part, their anti-atherogenic action in vivo.

Cost-effectiveness of intensive insulin therapy for type 2 diabetes: a 10-year follow-up of the Kumamoto study

To evaluate the cost and effectiveness of intensive insulin therapy for type 2 diabetes on the prevention of diabetes complications in Japan, we performed economic evaluation based on a randomized controlled trial. A total of 110 patients with type 2 diabetes were randomly assigned into two groups, a multiple insulin injection therapy (MIT) group or a conventional insulin injection therapy (CIT) group, and were followed-up for 10 years. Economic evaluation (cost-consequences analysis) was applied to evaluate both health and economic outcomes. As outcome measures for effectiveness of intensive insulin therapy, the frequency of complications, such as retinopathy, nephropathy, neuropathy, macrovascular event, and diabetes-related death, was used. For estimating costs, a viewpoint of the payer (the National Health Insurance) was adopted. Direct medical costs associated with diabetes care during 10 years were calculated and evaluated. In a base case analysis, all costs were discounted to the present value at an annual rate of 3%. Sensitivity analyses were carried out to assess the robustness of the results to changes in the values of important variables. MIT reduced the relative risk in the progression of retinopathy by 67%, photocoagulation by 77%, progression of nephropathy by 66%, albuminuria by 100% and clinical neuropathy by 64%, relative to CIT. Moreover, MIT prolonged the period in which patients were free of complications, including 2.0 years for progression of retinopathy (P<0.0001), 0.3 years for photocoagulation (P<0.05), 1.5 years for progression of nephropathy (P<0.01) and 2.2 years for clinical neuropathy (P<0.0001). The total cost (discounted at 3%) per patient during the 10-year period for each group was

Lysophosphatidylcholine Potentiates the Mitogenic Activity of Modified LDL for Human Monocyte–Derived Macrophages

30310 and 31525, respectively. The reduction of total costs in MIT over CIT was mainly due to reduced costs for management of diabetic complications. Our results show that MIT is more beneficial than CIT in both cost and effectiveness. Therefore, MIT is recommended for the treatment of type 2 diabetic patients who require insulin therapy as early as possible from the perspective of both patients and health policy.

Induction of Murine Macrophage Growth by Oxidized Low Density Lipoprotein Is Mediated by Granulocyte Macrophage Colony-stimulating Factor

The growth of murine peritoneal macrophages is induced by oxidized LDL (Ox-LDL), and lysophosphatidylcholine (lysoPC) plays an important role in its mitogenic activity. In the present++ study, Ox-LDL-induced macrophage growth was examined with human monocyte-derived macrophages. The cell growth of human macrophages was significantly induced by Ox-LDL but not by acetylated LDL (acetyl-LDL). The treatment of acetyl-LDL with phospholipase A2, however, led to a marked increase in its mitogenic activity, with a concomitant conversion of 75% of its phospholipids to lysoPC. The growth-stimulating activity became positive only when both acetyl-LDL and lysoPC were coincubated, although neither of them exhibited cell growth-promoting activity. These results suggest that Ox-LDL could stimulate the growth of human monocyte-derived macrophages, and lysoPC may play an essential role in the mitogenic activity of Ox-LDL.

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

We have examined whether certain secreted factor(s) is involved in oxidized low density lipoprotein (Ox-LDL)-induced murine macrophage growth. An antibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) effectively inhibited Ox-LDL-induced macrophage growth by >80%. Ox-LDL as well as phospholipase A2-treated acetylated LDL enhanced mRNA levels and protein release of GM-CSF from macrophages, while neither acetylated LDL nor lysophosphatidylcholine (lyso-PC) showed such effects. The maximal induction of GM-CSF by Ox-LDL was noted at 4 h, followed by a time-dependent decrease to a basal level within 24 h. Ox-LDL-induced macrophage growth was inhibited by 75% by replacement of the culture medium at 24 h by a fresh medium containing the same concentration of Ox-LDL, when GM-CSF had already returned to the basal level. Thus, a cytokine(s) other than GM-CSF is also expected to participate in Ox-LDL-induced macrophage growth in a later phase. The Ox-LDL-induced GM-CSF release was inhibited by calphostin C, a protein kinase C inhibitor, and was significantly reduced in macrophages from the knockout mice lacking class A, type I and type II macrophage scavenger receptors (MSR-AI/AII). These results taken together indicate that effective endocytosis of lyso-PC of Ox-LDL by macrophages through MSR-AI/AII and subsequent protein kinase C activation have led to GM-CSF release into the medium which may play a priming role in conjunction with other cytokines in Ox-LDL-induced macrophage growth.