Yoshichika Anami

Glucocorticoid Inhibits Oxidized LDL-Induced Macrophage Growth by Suppressing the Expression of Granulocyte/Macrophage Colony-Stimulating Factor

Glucocorticoid, an anti-inflammatory agent, inhibits the development of atherosclerosis in various experimental animal models. This is partially explained by its ability to inhibit smooth muscle cell migration and proliferation in the intima and to reduce chemotaxis of circulating monocytes and leukocytes into the subendothelial spaces. We have recently demonstrated that oxidized LDL (Ox-LDL) has a mitogenic activity for macrophages in vitro in which Ox-LDL-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) production plays an important role. Proliferation of cellular components is one of the characteristic events in the development and progression of atherosclerotic lesions. In the present study, we investigated the effects of glucocorticoids on Ox-LDL-induced macrophage growth. Dexamethasone, prednisolone, and cortisol inhibited Ox-LDL-induced thymidine incorporation into macrophages by 85%, 70%, and 50%, respectively. Ox-LDL induced a significant production of GM-CSF by macrophages, which was effectively inhibited by dexamethasone, prednisolone, and cortisol by 80%, 65%, and 50%, respectively. Dexamethasone-mediated inhibition of Ox-LDL-induced GM-CSF mRNA expression and macrophage growth was significantly abrogated by RU-486, a glucocorticoid receptor antagonist. Our results suggest that the inhibitory effects of glucocorticoids on macrophage growth may be due to the inhibition of Ox-LDL-induced GM-CSF production through transactivation of the glucocorticoid receptor.

Human β-migrating very low density lipoprotein induces foam cell formation in human mesangial cells

Abstract To elucidate the mechanism of foam cell formation in the mesangial region of a kidney observed in a familial type III hyperlipoproteinemic patient presenting with diabetes mellitus and nephrotic syndrome, we have examined, in the present study, the effect of human β -VLDL (apo E2/E2) on foam cell formation in human mesangial cells, since an increase in β -VLDL is a characteristic feature of this patient. Human β -VLDL (apo E2/E2) induced foam cell formation in human mesangial cells. The binding of [ 125 I]LDL to human mesangial cells was inhibited completely by both LDL and β -VLDL. On the other hand, the binding of [ 125 I] β -VLDL was completely inhibited by β -VLDL, but partially by LDL. The LDL receptor, but not the VLDL receptor was down-regulated by accumulation of cholesteryl esters. These results suggest that human β -VLDL (apo E2/E2)-induced foam cell formation in mesangial cells is mediated through both the LDL receptor pathway and the β -VLDL specific pathway, in which the VLDL receptor is one of the candidates.

β-very low density lipoprotein induces triglyceride accumulation through receptor mediated endocytotic pathway in 3T3-L1 adipocytes

Abstract To elucidate the mechanism of triglyceride (TG) accumulation in adipocytes induced by TG-rich lipoproteins, we examined the effect of β -very low density lipoprotein ( β -VLDL) on TG accumulation in 3T3-L1 adipocytes. β -VLDL did not induce TG accumulation in 3T3-L1 preadipocytes but in 3T3-L1 adipocytes. TG accumulation was significantly inhibited by cytochalasin B, an inhibitor of receptor mediated endocytosis. In contrast, cytochalasin B did not inhibit free fatty acid induced TG accumulation in adipocytes. The binding of [ 125 I] β -VLDL to preadipocytes was inhibited completely by both β -VLDL and LDL. In sharp contrast, the binding of [ 125 I] β -VLDL to adipocytes was inhibited completely by β -VLDL, but partially by LDL. The VLDL receptor mRNA was only expressed in adipocytes. These results suggest that β -VLDL induced TG accumulation in adipocytes may be mediated through the VLDL receptor pathway.

Effects of probucol on cholesterol metabolism in mouse peritoneal macrophages: Inhibition of HDL-mediated cholesterol efflux

Macrophage-derived foam cells are known to play an essential role in the development and progression of atherosclerotic lesions. Probucol prevents oxidative modification of low-density lipoprotein (LDL) and lowers plasma contents of LDL and high-density lipoprotein (HDL). A recent report using apoE -/- mice demonstrated that probucol treatment enhanced atherosclerosis in apoE -/- mice more rapidly than that in untreated apoE -/- mice, and a reduction in plasma cholesterol by probucol was not the cause of enhancement of atherosclerotic lesions in probucol-treated apoE -/- mice. Moreover, probucol was reported to inhibit apoA-I mediated cholesterol efflux from mouse macrophages. These reports suggested that probucol might directly affect cholesterol metabolism in mouse macrophages. Thus, we investigated the effects of probucol on cholesterol metabolism in mouse resident peritoneal macrophages. Probucol did not affect degradation of acetylated LDL (Ac-LDL), degradation of LDL and endogenous cholesterol synthesis in mouse macrophages. However, it significantly inhibited HDL-mediated cholesterol efflux. Moreover, probucol partially (30%) inhibited the binding of HDL to mouse macrophages, and significantly activated acyl-coenzyme A:cholesterol acyltransferase (ACAT). Our results suggested that probucol inhibited HDL-mediated cholesterol efflux by inhibiting the binding of HDL to mouse macrophages and reducing HDL-accessible free cholesterol content by ACAT activation, thereby worsening atherosclerotic lesions in apoE -/- mice. However, it remains unclear whether probucol inhibits HDL-mediated cholesterol efflux from human macrophages.

β-Migrating very low density lipoproteins induce foam cell formation in mouse mesangial cells

To elucidate whether beta-migrating very low density lipoproteins (beta-VLDL) induce foam cell formation in mesangial cells or not, surface binding and foam cell formation with beta-VLDL were studied in mouse mesangial cells. Specific binding kinetics for beta-VLDL and low density lipoproteins (LDL) on the mesangial cells were observed with Kd = 3.8 and 13.7 micrograms/ml, and Bmax = 65.9 and 71.9 ng/ml cell protein at 4 degrees C, respectively. The binding of beta-VLDL was inhibited by excess amounts of LDL or beta-VLDL, but not by acetyl-low density lipoproteins. Ligand blotting using beta-VLDL or LDL and immunoblotting using anti-human LDL receptor monoclonal antibody detected the same apparent single protein (approx. 130 kDa). Incorporation of [14C]oleate into cholesteryl ester in mouse mesangial cells was enhanced by beta-VLDL to 3-fold higher than that by LDL, and it was inhibited by chloroquine or anti-human LDL receptor monoclonal antibody. The light microscopic findings also demonstrated that cholesteryl ester deposition increased in these cells incubated with beta-VLDL, but not with LDL. In conclusion, beta-VLDL was specifically taken up by receptor-mediated endocytosis in mouse mesangial cells through LDL receptors, resulting in foam cell formation.

Release of fructose and hexose phosphates from perivascular cells induced by low density lipoprotein and acceleration of protein glycation in vitro

We investigated whether low density lipoprotein (LDL) under oxidative stress might induce the release of fructose, glucose-6-phosphate and fructose-6-phosphate from perivascular cells, and also whether these substances might accelerate the formation of advanced glycation end products (AGE) from proteins in vitro. When vascular smooth muscle cells were incubated with LDL in Hams F10 at 37 degrees C for 48 h. release of all these substances was increased dose-dependently by oxidized LDL. Fructose release was increased in a dose-dependent manner by glucose. Indomethacin (20 microM) significantly (P < 0.01) suppressed the release of fructose (25.4 +/- 15.7% of control) and hexose phosphates (29.4 +/- 4.0) with the inhibition of release of lactate dehydrogenase (35.5 +/- 4.9) as well as probucol, whereas an aldose reductase inhibitor, epalrestat, significantly (P < 0.001) inhibited only the fructose release (0.9 +/- 0.8). Release of fructose and hexose phosphates from vascular endothelial cells was also induced by oxidized LDL. AGE immunoreactivities and AGE-related fluorescence formed from proteins and glucose were significantly increased (P < 0.001) in the presence of small amounts of the cellular glucose metabolites (6.6%) with glucose (93.4%). These data suggest that release of potent AGE initiators, fructose and hexose phosphates, from perivascular cells induced by oxidized LDL may be an important phenomenon for vascular complications.

Group-II phospholipase A2 enhances oxidized low density lipoprotein-induced macrophage growth through enhancement of GM-CSF release

Inflammatory process plays an important role in the development and progression of atherosclerotic lesions. Recently, group-II phospholipase A(2) (PLA(2)), an inflammatory mediator, was reported to exist in human atherosclerotic lesions and to enhance the development of murine atherosclerotic lesions. Oxidized low density lipoprotein (Ox-LDL) stimulates the growth of several types of macrophages in vitro. Since proliferation of macrophages occurs in atherosclerotic lesions, it is possible to assume that the Ox-LDL-induced macrophage proliferation might be involved in the progression of atherosclerosis. In this study, the role of group-II PLA(2) in the Ox-LDL-induced macrophage growth was investigated using thioglycollate-elicited mouse peritoneal macrophages. Thioglycollate-elicited macrophages significantly expressed group-II PLA(2) and released it into the culture medium. The Ox-LDL-induced thymidine incorporation into thioglycollate-elicited macrophages was three times higher than that into resident macrophages, whereas under the same conditions, granulocyte/macrophage colony-stimulating factor (GM-CSF) equally induced thymidine incorporation into both types of macrophages. Moreover, the Ox-LDL-induced GM-CSF release from thioglycollate-elicited macrophages was significantly higher than that from resident macrophages. In addition, the Ox-LDL-induced thymidine incorporation into macrophages obtained from human group-II PLA(2) transgenic mice and the GM-CSF release from these cells were significantly higher than those from their negative littermates, and the Ox-LDL-induced thymidine incorporation into human group-II PLA(2) transgenic macrophages was significantly inhibited by a polyclonal anti-human group-II PLA(2) antibody. These results suggest that the expression of group-II PLA(2) in thioglycollate-elicited macrophages may play an enhancing role in the Ox-LDL-induced macrophage growth through the enhancement of the GM-CSF release.