Toshimori Inaba

Adiponectin Reduces Atherosclerosis in Apolipoprotein E-Deficient Mice

Background—Dysregulation of adipocyte-derived bioactive molecules plays an important role in the development of atherosclerosis. We previously reported that adiponectin, an adipocyte-specific plasma protein, accumulated in the injured artery from the plasma and suppressed endothelial inflammatory response and vascular smooth muscle cell proliferation, as well as macrophage-to-foam cell transformation in vitro. The current study investigated whether the increased plasma adiponectin could actually reduce atherosclerosis in vivo. Methods and Results—Apolipoprotein E-deficient mice were treated with recombinant adenovirus expressing human adiponectin (Ad-APN) or &bgr;-galactosidase (Ad-&bgr;gal). The plasma adiponectin levels in Ad-APN–treated mice increased 48 times as much as those in Ad-&bgr;gal treated mice. On the 14th day after injection, the lesion formation in aortic sinus was inhibited in Ad-APN–treated mice by 30% compared with Ad-&bgr;gal–treated mice (P <0.05). In the lesions of Ad-APN–treated mice, the lipid droplets became smaller compared with Ad-&bgr;gal–treated mice (P <0.01). Immunohistochemical analyses demonstrated that the adenovirus-mediated adiponectin migrate to foam cells in the fatty streak lesions. The real-time quantitative polymerase chain reaction revealed that Ad-APN treatment significantly suppressed the mRNA levels of vascular cell adhesion molecule-1 by 29% and class A scavenger receptor by 34%, and tended to reduce levels of tumor necrosis factor-&agr; without affecting those of CD36 in the aortic tissue. Conclusions—These findings documented for the first time that elevated plasma adiponectin suppresses the development of atherosclerosis in vivo.

T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice.

Liver X receptors (LXRα and LXRβ) are nuclear receptors, which are important regulators of cholesterol and lipid metabolism. LXRs control genes involved in cholesterol efflux in macrophages, bile acid synthesis in liver and intestinal cholesterol absorption. LXRs also regulate genes participating in lipogenesis. To determine whether the activation of LXR promotes or inhibits development of atherosclerosis, T‐0901317, a synthetic LXR ligand, was administered to low density lipoprotein receptor (LDLR)−/− mice. T‐0901317 significantly reduced the atherosclerotic lesions in LDLR−/− mice without affecting plasma total cholesterol levels. This anti‐atherogenic effect correlated with the plasma concentration of T‐0901317, but not with high density lipoprotein cholesterol, which was increased by T‐0901317. In addition, we observed that T‐0901317 increased expression of ATP binding cassette A1 in the lesions in LDLR−/− mice as well as in mouse peritoneal macrophages. T‐0901317 also significantly induced cholesterol efflux activity in peritoneal macrophages. These results suggest that LXR ligands may be useful therapeutic agents for the treatment of atherosclerosis.

Enhanced expression of platelet-derived growth factor-β receptor by high glucose. Involvement of platelet-derived growth factor in diabetic angiopathy

Coronary heart disease is a major complication of diabetic subjects, and platelet-derived growth factor (PDGF) has been implicated in the development of atherosclerosis. We investigated the effects of high glucose on expression of PDGF-β receptor. In a binding assay with 125I-labeled PDGF-BB homodimer, high concentrations of glucose increased high-affinity binding of PDGF-BB on human monocyte-derived macrophages and rabbit aortic medial smooth muscle cells. Northern blot analysis confirmed the enhanced effect of glucose on expression of PDGF-β receptor mRNA in human monocyte-derived macrophages. The protein kinase C inhibitor, staurosporin, completely suppressed an increase in PDGF-BB binding by high glucose, and high glucose significantly activated protein kinase C. These results indicated that PDGF-β receptor expression was enhanced by high glucose through the activation of protein kinase C. Furthermore, we observed similar effects of high glucose on both PDGF-β receptor expression and protein kinase C activation in rat mesangial cells and human capillary endothelial cells. Our results suggest that stimulation of the PDGF system is significantly involved in the development not only of diabetic atherosclerosis but also of microangiopathy.

Macrophage colony stimulating factor prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits

The early atherosclerotic lesion is characterized by the presence of macrophage-derived foam cells. Macrophage colony stimulating factor (M-CSF) specifically stimulates the functions of the monocyte-macrophages. To elucidate the effects of M-CSF in the atherogenic process in vivo, we administered human recombinant M-CSF into Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia. Three hundred micrograms of M-CSF were intravenously injected into WHHL rabbits aged 2.5 months, three times a week for 8.5 months. After the M-CSF treatment, we found very retarded progression of atherosclerosis. The accumulation of cholesterol ester was remarkably decreased in the aortae of M-CSF-treated animals (0.60 +/- 0.32 mg/g tissue), as compared to those of controls (4.32 +/- 0.61 mg/g tissue). Furthermore, the percentage of the surface area of the aorta with macroscopic plaque in animals treated with M-CSF was 14.3 +/- 6.2%, much less than that in controls receiving saline injection (38.8 +/- 8.0%). Thus, M-CSF definitely prevented the progression of atherosclerosis in WHHL rabbits by influencing macrophage functions.

Liver X receptor agonists inhibit tissue factor expression in macrophages

Exposure of blood to tissue factor (TF) rapidly initiates the coagulation serine protease cascades. TF is expressed by macrophages and other types of cell within atherosclerotic lesions and plays an important role in thrombus formation after plaque rupture. Macrophage TF expression is induced by pro‐inflammatory stimuli including lipopolysaccharide (LPS), interleukin‐1β and tumor necrosis factor‐α. Here we demonstrate that activation of liver X receptors (LXRs) LXRα and LXRβ suppresses TF expression. Treatment of mouse peritoneal macrophages with synthetic LXR agonist T0901317 or GW3965 reduced TF expression induced by pro‐inflammatory stimuli. LXR agonists also suppressed TF expression and its activity in human monocytes. Human and mouse TF promoters contain binding sites for the transcription factors AP‐1, NFκB, Egr‐1 and Sp1, but no LXR‐binding sites could be found. Cotransfection assays with LXR and TF promoter constructs in RAW 264.7 cells revealed that LXR agonists suppressed LPS‐induced TF promoter activity. Analysis of TF promoter also showed that inhibition of TF promoter activity by LXR was at least in part through inhibition of the NFκB signaling pathway. In addition, in vivo, LXR agonists reduced TF expression within aortic lesions in an atherosclerosis mouse model as well as in kidney and lung in mice stimulated with LPS. These findings indicate that activation of LXR results in reduction of TF expression, which may influence atherothrombosis in patients with vascular disease.

Induction of LDL receptor-related protein during the differentiation of monocyte-macrophages. Possible involvement in the atherosclerotic process.

The low-density lipoprotein receptor-related protein (LRP) is a multifunctional receptor that binds to apolipoprotein E-rich lipoproteins, lipoprotein lipase, alpha 2-macroglobulin, lactoferrin, and tissue plasminogen activator. We studied the mRNA expression of LRP in human monocyte-derived macrophages and THP-1 cells. mRNA expression of LRP was induced during cell differentiation from human monocytes to macrophages or after incubation with phorbol ester (tetradecanoylphorbol acetate 100 ng/mL) in THP-1 cells, and the addition of 30 ng/mL macrophage colony-stimulating factor further enhanced LRP expression. These results indicated that the expression of LRP depended on the stage of differentiation and maturation of monocytic cells. mRNA expression of LRP was also enhanced in human monocyte-derived macrophages in the presence of acetylated low-density lipoprotein and in aorta of rabbits fed a high-cholesterol diet. We hypothesize that the LRP induced in monocyte-derived macrophages is involved in the initial process of atherosclerosis by interacting with its multiple ligands.

Overexpression of Human Lipoprotein Lipase Protects Diabetic Transgenic Mice From Diabetic Hypertriglyceridemia and Hypercholesterolemia

We investigated the role of the overexpression of lipoprotein lipase (LPL) in lipoprotein abnormalities in transgenic mice with streptozotocin-induced diabetes mellitus. Before the induction of diabetes, LPL activity was 4.6-fold in skeletal muscle and 2.0-fold higher in the heart in transgenic mice than in their nontransgenic littermates. LPL activity in skeletal muscles in diabetic nontransgenic mice and cardiac LPL activity in diabetic nontransgenic and transgenic mice were decreased. Body weights were similarly reduced, and no appreciable amount of adipose tissue was observed in diabetes in both groups. The plasma triglyceride level was lower in diabetic transgenic mice than in diabetic nontransgenic mice (33.2 +/- 22.5 versus 185.3 +/- 57.4 mg/dL). Induction of diabetes was associated with a significant increase in the plasma cholesterol level in nontransgenic mice (90.0 +/- 11.1 versus 163.9 +/- 39.3 mg/dL) but much less in transgenic mice. Our results indicate that overexpression of LPL in transgenic mice inhibited diabetes-associated hypertriglyceridemia and hypercholesterolemia but did not affect the loss of body weight induced by diabetes.

Macrophage colony-stimulating factor regulates both activities of neutral and acidic cholesteryl ester hydrolases in human monocyte-derived macrophages.

Macrophage colony-stimulating factor (M-CSF) regulates cholesterol metabolism in vivo and in vitro. We studied the effects of M-CSF on enzyme activities of acidic cholesteryl ester (CE) hydrolase, neutral CE hydrolase, and acyl-coenzyme A:cholesterol acyltransferase (ACAT), all of which are involved in cellular cholesterol metabolism in macrophages. During the differentiation of monocytes to macrophages, these enzyme activities were induced and further enhanced in response to M-CSF. M-CSF (100 ng/ml) enhanced acidic and neutral CE hydrolase and ACAT activities by 3.2-, 4-, and 2.3-fold, respectively, in the presence of acetyl LDL. The presence of acetyl LDL influenced these enzyme activities. ACAT and acidic CE hydrolase activities were increased and neutral CE hydrolase activity was decreased, indicating that these enzymes are regulated by intracellular cholesterol enrichment. M-CSF increased the ratios of acidic CE hydrolase to ACAT activity and of neutral CE hydrolase to ACAT activity. The results suggest that M-CSF enhances net hydrolysis of CE by stimulating the two CE hydrolases to a greater extent than ACAT, and M-CSF may reduce the rate of atherogenesis.

Induction of sustained expression of proto-oncogene c-fms by platelet-derived growth factor, epidermal growth factor, and basic fibroblast growth factor, and its suppression by interferon-gamma and macrophage colony-stimulating factor in human aortic medial smooth muscle cells

Vascular medial smooth muscle cells migrate, proliferate and transform to foam cells in the process of atherosclerosis. We have reported that the intimal smooth muscle cells express proto-oncogene c-fms, a characteristic gene of monocyte-macrophages, which is not normally expressed in medial smooth muscle cells. In the present study, we demonstrated that combinations of platelet-derived growth factor (PDGF)-BB and either epidermal growth factor (EGF) or fibroblast growth factor (FGF) induced high expression of c-fms in normal human medial smooth muscle cells to the level of intimal smooth muscle cells or monocyte-derived macrophages, whereas c-fms expression by PDGF-BB alone was 1/10 and both EGF and FGF had no independent effect on c-fms expression. By contrast, interferon (IFN)-gamma and macrophage colony-stimulating factor (M-CSF) suppressed the induction of c-fms expression. These results indicate that multiple growth factors and cytokines may play a role in the phenotypic transformation of medial smooth muscle cells to intimal smooth muscle cells in atherosclerotic lesions by altering c-fms expression.

Induction of macrophage colony-stimulating factor receptor (c-fms) expression in vascular medial smooth muscle cells treated with heparin binding epidermal growth factor-like growth factor.

Vascular smooth muscle cells migrate, proliferate, and transform to foam cells during the atherosclerotic process. We have reported that smooth muscle cells derived from the intima of atherosclerotic lesions express the proto-oncogene c-fms and a scavenger receptor, which are not normally expressed in normal medial smooth muscle cells. In the present study, we demonstrated that heparin binding epidermal growth factor-like growth factor (HB-EGF) induced the expression of c-fms and the scavenger receptor in normal human medial smooth muscle cells to the level observed in the intima. The expression of c-fms was partially inhibited by a protein kinase C inhibitor, suggesting that HB-EGF induces c-fms via pathways that are both dependent on and independent of protein kinase C. By contrast, most of the scavenger receptor induction by HB-EGF was suppressed by protein kinase C inhibitors. These results indicate that two characteristic genes of monocyte-derived macrophages were induced by HB-EGF via different mechanisms. The alteration of gene expression in response to HB-EGF may play an important role in the phenotypic change of smooth muscle cells to macrophage-like foam cells during the atherosclerotic process.