Jun-ichi Ohsuga

Inhibition of diet-induced atheroma formation in transgenic mice expressing apolipoprotein E in the arterial wall.

Apolipoprotein E (apoE) plays a crucial role in lipoprotein metabolism both in plasma and in peripheral tissues. To test whether apoE in the vascular wall has a direct and local effect on atherogenesis, we established transgenic mice expressing human apoE under control of H2 Ld promoter. Studies on mRNA levels and immunohistochemistry demonstrated that this line was characterized by high expression of human apoE in the arterial wall while its expression was relatively low in other tissues as compared with the respective endogenous expression of mouse apoE. They showed no difference in plasma cholesterol levels and lipoprotein profile from controls when fed both normal and atherogenic diets. However, after 24 wk of an atherogenic diet, the formation of fatty streak lesions in proximal aorta was markedly inhibited in transgenic mice as compared with controls. Both lesion area and esterified cholesterol content were < 30% of those in controls. In a tissue cholesterol labeling study with 3H-cholesterol, the specific activity of aorta cholesterol was much less in transgenic mice, suggesting that apoE enhances cholesterol efflux from the aortic wall into plasma. Thus, apoE has anti-atherogenic action which is mediated via enhancing reverse cholesterol transport from arterial wall.

Secretion-recapture process of apolipoprotein E in hepatic uptake of chylomicron remnants in transgenic mice.

To investigate the role of apoE in hepatic uptake of chylomicron remnants, we studied chylomicron metabolism in transgenic mice overexpressing apoE in the liver. Plasma clearance of injected 125I-labeled human chylomicrons was fivefold faster in transgenic mice than in controls. Immunohistochemistry demonstrated that apoE was specifically localized at the basolateral surface of hepatocytes from fasted transgenic mice. After injection of a large amount of chylomicrons, the density of the cell surface apoE was markedly reduced and vesicular staining was observed in the cytoplasm, suggesting that the cell surface apoE was used for hepatic endocytosis of chylomicrons and remnants. Polyacrylamide gel analysis of chylomicrons and remnants that had been reisolated from plasma and from liver membrane after the injection of chylomicrons showed the particles to be enriched with apoE mainly after their influx into the liver rather than during their residence in plasma. These results provide strong evidence for the secretion-recapture process of apoE, whereby chylomicron remnants enter the sinusoidal space, acquire apoE molecules, and subsequently are endocytosed. Data from experiments with very low density lipoprotein and LDL showed that this system is specific for chylomicron remnants.

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.

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.

Transcription factor PU.1 mediates induction of c-fms in vascular smooth muscle cells: a mechanism for phenotypic change to phagocytic cells.

The macrophage colony-stimulating factor receptor encoded by the c-fms gene is expressed in vascular intimal smooth muscle cells isolated from atherosclerotic lesions. A combination of platelet-derived growth factor-BB and epidermal growth factor induces stable expression of c-fms in normal vascular medial smooth muscle cells. The mechanism by which these growth factors induce c-fms expression has now been investigated in an attempt to gain insight into the events that underlie the phenotypic conversion of vascular smooth muscle cells in atherosclerosis. Deletion analysis of the c-fms promoter revealed that the region including a binding site for transcription factor PU.1 was required for transcriptional activity in human aortic medial smooth muscle cells. Mutation in the PU.1 binding site markedly reduced promoter activity. Northern (RNA) blot analysis demonstrated that growth factors induced the expression of PU.1 mRNA in vascular medial smooth muscle cells and that PU.1 mRNA was expressed in vascular intimal smooth muscle cells. PU.1 antisense oligonucleotides inhibited growth factor-induced c-fms expression and foam cell formation. These results suggest that transcription factor PU.1 plays an essential role in the phenotypic conversion of vascular smooth muscle cells to macrophagelike cells by mediating the induction of c-fms.

Overexpression of Apolipoprotein E Prevents Development of Diabetic Hyperlipidemia in Transgenic Mice

To determine the role of apolipoprotein E (apoE) in diabetic hyperlipidemia, we induced diabetes in transgenic mice overexpressing apoE by intravenous injection of streptozotocin (STZ) and examined plasma lipoprotein metabolism in these mice. In STZ-induced diabetic mice, blood glucose levels were > 19 mmol/l (350 mg/dl) and plasma insulin levels were reduced to = 5 pmol/l (1 μU/ml). The diabetic nontransgenic mice developed hypercholesterolemia (plasma total cholesterol level: 4.55 ± 1.32 vs. 1.97 ± 0.13 mmol/l [176 ± 51 vs. 76 ± 5 mg/dl]) and hypertriglyceridemia (plasma triglyceride level: 0.82 ± 0.29 vx. 0.42 ± 0.11 mmol/l [73 ± 26 vs. 37 ± 10 mg/dl]) compared with values before induction of diabetes. In the diabetic nontransgenic mice, enhanced intestinal acylCoA:cholesterol acyltransferase activity was demonstrated, a factor that may contribute to the development of diabetic hyperlipidemia. Induction of apoE remarkably reduced the development of hyperlipidemia in diabetic transgenic mice compared with diabetic nontransgenic mice (plasma cholesterol level: 4.55 ± 1.32 vs. 3.31 ± 0.47 mmol/l [176 ± 51 vs. 128 ± 18 mg/dl], P < 0.01, and plasma triglyceride level: 0.82 ± 0.29 vs. 0.17 ± 0.11 mmol/l [73 ± 26 vs. 15 ± 10 mg/dl], P < 0.01). Plasma lipoprotein analysis by gel filtration chromatography showed that the reduction of plasma cholesterol and triglyceride levels was due to the disappearance of lipoproteins containing apoB. In these studies, we demonstrated the usefulness of STZ-induced diabetes in mice as an animal model for diabetic hyperlipidemia and demonstrated that endogenous induction of apoE in transgenic mice improved diabetic hyperlipidemia.

Effects of Platelet-Derived Growth Factor on the Synthesis of Lipoprotein Lipase in Human Monocyte–Derived Macrophages

Lipoprotein lipase (LPL), which is secreted by the two predominant cell types in atherosclerotic plaque, macrophages and smooth muscle cells, may be involved in atherosclerosis by generating atherogenic remnant lipoproteins. We investigated the effects of platelet-derived growth factor (PDGF)-BB on the synthesis of LPL by human monocyte-derived macrophages. These cells were cultured in the presence of PDGF-BB for 8 days, after which the enzyme activity, mass, and mRNA levels of LPL were determined. The effect of PDGF-BB was time-dependent and dose-dependent at concentrations of 1 to 10 ng/mL. At 10 ng/mL PDGF-BB enhanced twofold to 2.3-fold the secretion of LPL, and a pulse-labeling study with [35S]methionine revealed that 10 ng/mL PDGF-BB significantly increased the synthesis of LPL. Northern blotting analysis showed that the LPL mRNA level increased dose dependently in macrophages treated with PDGF-BB, and 10 ng/mL PDGF-BB enhanced twofold the expression of LPL mRNA. The protein kinase C inhibitor staurosporine suppressed the effect of PDGF-BB on LPL activity. These results indicate that PDGF-BB stimulated transcription of the LPL gene in human monocyte-derived macrophages through protein kinase C activation and resulted in an increased synthesis of LPL. Therefore, we hypothesize that the augmented synthesis of LPL by PDGF-BB modulates atherosclerosis by influencing lipoprotein metabolism in the vascular wall.

Overexpression of human lipoprotein lipase enhances uptake of lipoproteins containing apolipoprotein B-100 in transfected cells.

To investigate the role in lipoprotein metabolism of lipoprotein lipase (LPL) secreted by tissues, we established two cell lines. Fusion plasmids containing either human LPL cDNA or antisense LPL cDNA under control of the cytomegalovirus promoter were transfected into Chinese hamster ovary (CHO) cells, designated as CHO-LPL and CHO-anti-LPL, respectively. CHO-LPL constitutively produced a high level of LPL, whereas CHO-anti-LPL produced a minimal level. When very-low-density lipoprotein (VLDL) was incubated with CHO-LPL, VLDL triglycerides were hydrolyzed, intermediate-density lipoprotein (IDL) was produced, and apolipoprotein E contents increased. CHO-LPL took up and degraded 125I-VLDL at 37 degrees C four times more strongly than did CHO-anti-LPL. Whereas the degradation of apolipoprotein E-deficient VLDL was only 12% that of normal VLDL in CHO-LPL, structural changes of the lipoprotein, including apolipoprotein E expression on the lipoprotein surface, may be important for the cellular uptake of VLDL. Furthermore, we found that binding at 4 degrees C of VLDL and LDL to CHO-LPL was greater than to CHO-anti-LPL, and this binding difference was abolished by washing the cells with heparin. This suggests that cell surface LPL plays a role in the binding of lipoproteins to the cells. We conclude that both the composition of VLDL particles and their cellular binding are influenced by LPL secreted by cells, both of which may enhance the cellular uptake of VLDL.

Role of macrophage colony-stimulating factor in the initial process of atherosclerosis.

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 role of M-CSF in the atherogenic process in vitro and in vivo, 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), and 300 micrograms of M-CSF was intravenously injected into WHHL rabbits aged 2.5 months for 8.5 months. M-CSF (100 ng/ml) enhanced acidic and neutral CE hydrolase, and ACAT activities by 3.2-fold, 4-fold, and 2.3-fold, respectively, in the presence of acetyl LDL, and M-CSF increased ratios of both acidic and neutral CE hydrolase activities to ACAT activity. After M-CSF injection into WHHL rabbits, 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 (4.21 +/- 0.65 mg/g tissue). The results suggest that M-CSF prevents the progression of atherosclerosis in WHHL rabbits by increasing net hydrolysis of cholesteryl ester in macrophages.