Troglitazone inhibits oxidized low-density lipoprotein-induced macrophage proliferation: impact of the suppression of nuclear translocation of ERK1/2.

Thiazolidinediones (TZDs), which were known as novel insulin-sensitizing antidiabetic agents, have been reported to inhibit the acceleration of atherosclerotic lesions. Macrophages play important roles in the development of atherosclerosis. We previously reported that oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation through ERK1/2-dependent GM-CSF production. In the present study, we investigated the effects of two TZDs, troglitazone and ciglitazone on Ox-LDL-induced macrophage proliferation. Troglitazone significantly inhibited Ox-LDL-induced increases in [(3)H]thymidine incorporation into and proliferation of mouse peritoneal macrophages, whereas ciglitazone had no effects. Troglitazone and ciglitazone both significantly induced PPARgamma activity, suggesting that the inhibitory effect of troglitazone was not mediated by PPARgamma. Ox-LDL-induced production of GM-CSF was significantly inhibited by troglitazone, but not by ciglitazone. Troglitazone inhibited Ox-LDL-induced production of intracellular reactive oxygen species, whereas ciglitazone had no effect. The antioxidant reagents NAC and NMPG each inhibited phosphorylation of ERK1/2, whereas troglitazone and ciglitazone had no effects. However, troglitazone, NAC and NMPG all inhibited nuclear translocation of ERK1/2. In conclusion, troglitazone inhibited Ox-LDL-induced GM-CSF production by suppressing nuclear translocation of ERK1/2, thereby inhibiting macrophage proliferation. This suppression of macrophage proliferation by troglitazone may, at least in part, explain its antiatherogenic effects.

Glycolaldehyde-modified bovine serum albumin downregulates leptin expression in mouse adipocytes via a CD36-mediated pathway.

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 gamma (PPAR-gamma) in macrophages, whereas PPAR-gamma 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.

CD36 is not involved in scavenger receptor-mediated endocytic uptake of glycolaldehyde- and methylglyoxal-modified proteins by liver endothelial cells.

Circulating proteins modified by advanced glycation end-products (AGE) are mainly taken up by liver endothelial cells (LECs) via scavenger receptor-mediated endocytosis. Endocytic uptake of chemically modified proteins by macrophages and macrophage-derived cells is mediated by class A scavenger receptor (SR-A) and CD36. In a previous study using SR-A knockout mice, we demonstrated that SR-A is not involved in endocytic uptake of AGE proteins by LECs [Matsumoto et al. (2000) Biochem. J. 352, 233-240]. The present study was conducted to determine the contribution of CD36 to this process. Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were used as AGE proteins. 125I-GA-BSA and 125I-MG-BSA underwent endocytic degradation by these cells at 37 degrees C, and this process was inhibited by several ligands for the scavenger receptors. However, this endocytic uptake of 125I-GA-BSA by LECs was not inhibited by a neutralizing anti-CD36 antibody. Similarly, hepatic uptake of (111)In-GA-BSA after its intravenous injection was not significantly attenuated by co-administration of the anti-CD36 antibody. These results clarify that CD36 does not play a significant role in elimination of GA-BSA and MG-BSA from the circulation, suggesting that the receptor involved in endocytic uptake of circulating AGE proteins by LEC is not SR-A or CD36.

Expression of class A scavenger receptor is enhanced by high glucose in vitro and under diabetic conditions in vivo: one mechanism for an increased rate of atherosclerosis in diabetes.

In the early stage of atherosclerosis, macrophages take up chemically modified low density lipoproteins (LDL) through the scavenger receptors, leading to foam cell formation in atherosclerotic lesions. To get insight into a role of the scavenger receptors in diabetes-enhanced atherosclerotic complications, the effects on class A scavenger receptor (SR-A) of high glucose exposure in vitro as well as the diabetic conditions in vivo were determined in the present study. The in vitro experiments demonstrated that high glucose exposure to human monocyte-derived macrophages led to an increased SR-A expression with a concomitant increase in the endocytic uptake of acetylated LDL and oxidized LDL. The endocytic process was significantly suppressed by an anti-SR-A neutralizing antibody. Stability analyses revealed a significant increased stability of SR-A at a mRNA level but not a protein level, indicating that high glucose-induced up-regulation of SR-A is due largely to increased stability of SR-A mRNA. High glucose-enhanced SR-A expression was prevented by protein kinase C and NAD(P)H oxidase inhibitors as well as antioxidants. High glucose-enhanced production of intracellular peroxides was visualized in these cells, which was attenuated by an antioxidant. The in vivo experiments demonstrated that peritoneal macrophages from streptozotocin-induced diabetic mice increased SR-A expression when compared with those from nondiabetic mice. Endocytic degradation of acetylated LDL and oxidized LDL were also increased with these macrophages but not with the corresponding macrophages from diabetic SR-A knock-out mice. These in vitro and in vivo results probably suggest that reactive oxygen species generated from a protein kinase C-dependent NAD(P)H oxidase pathway plays a role in the high glucose-induced up-regulation of SR-A, leading to the increased endocytic degradation of modified LDL for foam cell formation. This could be one mechanism for an increased rate of atherosclerosis in patients with diabetes.

Statins suppress oxidized low density lipoprotein-induced macrophage proliferation by inactivation of the small G protein-p38 MAPK pathway.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) ameliorate atherosclerotic diseases. Macrophages play an important role in the development and subsequent stability of atherosclerotic plaques. We reported previously that oxidized low density lipoprotein (Ox-LDL) induced macrophage proliferation through the secretion of granulocyte/macrophage colony-stimulating factor (GM-CSF) and the consequent activation of p38 MAPK. The present study was designed to elucidate the mechanism of the inhibitory effect of statins on macrophage proliferation. Mouse peritoneal macrophages were used in our study. Cerivastatin and simvastatin each inhibited Ox-LDL-induced [(3)H]thymidine incorporation into macrophages. Statins did not inhibit Ox-LDL-induced GM-CSF production, but inhibited GM-CSF-induced p38 MAPK activation. Farnesyl transferase inhibitor and geranylgeranyl transferase inhibitor inhibited GM-CSF-induced macrophage proliferation, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the effect of statins. GM-CSF-induced p38 MAPK phosphorylation was also inhibited by farnesyl transferase inhibitor or geranylgeranyl transferase inhibitor, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the suppression of GM-CSF-induced p38 MAPK phosphorylation by statins. Furthermore, we found that statin significantly inhibited the membrane translocation of the small G protein family members Ras and Rho. GM-CSF-induced p38 MAPK activation and macrophage proliferation was partially inhibited by overexpression of dominant negative Ras and completely by that of RhoA. In conclusion, statins inhibited GM-CSF-induced Ras- or RhoA-p38 MAPK signal cascades, thereby suppressing Ox-LDL-induced macrophage proliferation. The significant inhibition of macrophage proliferation by statins may also explain, at least in part, their anti-atherogenic action.

Advanced glycation end products-modified proteins and oxidized LDL mediate down-regulation of leptin in mouse adipocytes via CD36.

Advanced glycation end products (AGE)-modified proteins as well as oxidized-LDL (Ox-LDL) undergo receptor-mediated endocytosis by CHO cells overexpressing CD36, a member of class B scavenger receptor family. The purpose of the present study was to examine the effects of glycolaldehyde-modified BSA (GA-BSA) as an AGE-ligand and Ox-LDL on leptin expression in adipocytes. GA-BSA decreased leptin expression at both protein and mRNA levels in 3T3-L1 adipocytes and mouse epididymal adipocytes. Ox-LDL showed a similar inhibitory effect on leptin expression in 3T3-L1 adipocytes, which effect was protected by N-acetylcysteine, a reactive oxygen species (ROS) inhibitor. Binding of (125)I-GA-BSA or (125)I-Ox-LDL to 3T3-L1 adipocytes and subsequent endocytic degradation were inhibited by a neutralizing anti-CD36 antibody. Furthermore, this antibody also suppressed Ox-LDL-induced leptin down-regulation. These results clarify that the interaction of GA-BSA and Ox-LDL with CD36 leads to down-regulation of leptin expression via ROS system(s) in 3T3-L1 adipocytes, suggesting that a potential link of AGE- and/or Ox-LDL-induced leptin down-regulation might be linked to insulin-sensitivity in metabolic syndrome.

Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase mediate macrophage proliferation induced by oxidized low-density lipoprotein.

We previously reported that oxidized low-density lipoprotein (Ox-LDL)-induced expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) via PKC, leading to activation of phosphatidylinositol-3 kinase (PI-3K), was important for macrophage proliferation [J Biol Chem 275 (2000) 5810]. The aim of the present study was to elucidate the role of extracellular-signal regulated kinase 1/2 (ERK1/2) and of p38 MAPK in Ox-LDL-induced macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages assessed by [3H]thymidine incorporation and cell counting assays was significantly inhibited by MEK1/2 inhibitors, PD98059 or U0126, and p38 MAPK inhibitors, SB203580 or SB202190, respectively. Ox-LDL-induced GM-CSF production was inhibited by MEK1/2 inhibitors but not by p38 MAPK inhibitors in mRNA and protein levels, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by p38 MAPK inhibitors but enhanced by MEK1/2 inhibitors. Recombinant GM-CSF-induced PI-3K activation and Akt phosphorylation were significantly inhibited by SB203580 but enhanced by PD98059. Our results suggest that ERK1/2 is involved in Ox-LDL-induced macrophage proliferation in the signaling pathway before GM-CSF production, whereas p38 MAPK is involved after GM-CSF release. Thus, the importance of MAPKs in Ox-LDL-induced macrophage proliferation was confirmed and the control of MAPK cascade could be targeted as a potential treatment of atherosclerosis.

Statins downregulate ATP-binding-cassette transporter A1 gene expression in macrophages.

The ATP-binding-cassette transporter A1 (ABCA1) plays an essential role in cellular cholesterol efflux and helps prevent macrophages from becoming foam cells. The statins are widely used as cholesterol-lowering agents and have other anti-atherogenic actions. We tested the effects of four different statins (fluvastatin, atorvastatin, simvastatin, and lovastatin) on ABCA1 expression in macrophages in vitro. The statins suppressed ABCA1 mRNA expression in RAW246.7 and THP-1 macrophage cell lines and in mouse peritoneal macrophages. The effect was time- and dose-dependent and was abolished by the addition of the post-reductase product, mevalonate. These findings imply that there is a possible modulation of the well-known beneficial effects of the statins on the reverse cholesterol transport pathway.

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB.

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.

Acyl-coenzyme A:cholesterol acyltransferase inhibitors for controlling hypercholesterolemia and atherosclerosis.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT; Sterol O-acyltransferase/SOAT) is an intracellular enzyme that catalyzes the formation of cholesteryl esters from cholesterol and fatty acyl-coenzyme A. ACAT inhibitors reduce plasma cholesterol levels by suppressing absorption of dietary cholesterol and by suppressing the assembly and secretion of apolipoprotein B-containing lipoproteins such as very low density lipoprotein in liver and chylomicron in intestine. Moreover, ACAT inhibitors prevent the conversion of macrophages into foam cells in the arterial walls. Thus, ACAT inhibitors are under investigation for controlling hypercholesterolemia and the development of atherosclerosis. Some potent ACAT inhibitors have been tested for their efficacy and safety in humans.

Advanced glycation end products (AGE) inhibit scavenger receptor class B type I-mediated reverse cholesterol transport: a new crossroad of AGE to cholesterol metabolism.

Advanced glycation end products (AGE) -modified proteins behave as active ligands for several receptors belonging to the scavenger receptor family. Scavenger receptor class B type I (SR-BI) was identified as the first high density lipoprotein (HDL) receptor that mediates selective uptake of HDL-cholesteryl esters (HDL-CE). This study investigated whether AGE proteins serve as ligands for SR-BI and affect SR-BI-mediated cholesterol transport using Chinese hamster ovary (CHO) cells overexpressing hamster SR-BI (CHO-SR-BI cells). [125I] AGE-bovine serum albumin (AGE-BSA) underwent active endocytosis and subsequent lysosomal degradation by CHO-SR-BI cells, indicating that SR-BI serves as an AGE receptor. SR-BI-mediated selective uptake of HDL-CE by CHO-SR-BI cells was efficiently inhibited by AGE-BSA although AGE-BSA had no effect on HDL binding to CHO-SR-BI cells. In addition, AGE-BSA significantly inhibited the efflux of [3H] cholesterol from CHO-SR-BI cells to HDL. These findings suggest the possibility that AGE proteins in the circulation interfere with the functions of SR-BI in reverse cholesterol transport by inhibiting the selective uptake of HDL-CE, as well as cholesterol efflux from peripheral cells to HDL, thereby accelerating diabetes-induced atherosclerosis.

[The mechanisms of the development and progression of diabetic macrovascular complications].

Patients with diabetes mellitus increases in number in recent years and atherosclerosis-related vascular complications are the major cause of death in diabetic patients. A massive cluster of macrophage-derived foam cells in the subendothelial spaces is one of the characteristic features of the early stages of atherosclerotic lesions. In the present work, we mainly focused on the possible links of glycated-proteins and AGE-modified proteins to the development and progression of diabetic macrovascular complications.