Pitavastatin inhibits remnant lipoprotein-induced macrophage foam cell formation through ApoB48 receptor-dependent mechanism.

OBJECTIVE: Atherogenic remnant lipoproteins (RLPs) are known to induce foam cell formation in macrophages in vitro and in vivo. We examined the involvement of apoB48 receptor (apoB48R), a novel receptor for RLPs, in that process in vitro and its potential regulation by pitavastatin. METHODS AND RESULTS: THP-1 macrophages were incubated in the presence of RLPs (20 mg cholesterol/dL, 24 hours) isolated from hypertriglyceridemic subjects. RLPs significantly increased intracellular cholesterol ester (CE) and triglyceride (TG) contents (4.8-fold and 5.8-fold, respectively) in the macrophages. Transfection of THP-1 macrophages with short interfering RNA (siRNA) against apoB48R significantly inhibited RLP-induced TG accumulation by 44%. When THP-1 macrophages were pretreated with pitavastatin (5 micromol/L, 24 hours), the expression of apoB48R was significantly decreased and RLP-induced TG accumulation was reduced by 56%. ApoB48R siRNA also inhibited TG accumulation in THP-1 macrophage induced by beta-very-low-density lipoprotein derived from apoE-/- mice by 58%, supporting the notion that apoB48R recognizes and takes-up RLPs in an apoE-independent manner. CONCLUSIONS: RLPs induce macrophage foam cell formation via apoB48R. Pitavastatin inhibits RLP-induced macrophage foam cell formation. The underlying mechanism involves, at least in part, inhibition of apoB48R-dependent mechanism. Our findings indicate a potential role of apoB48R in atherosclerosis. RLPs induced macrophage foam cell formation via apoB48R. Pitavastatin inhibited RLP-induced macrophage foam cell formation, at least in part, via inhibition of apoB48R expression. Our findings indicate a potential role of apoB48R in atherosclerosis.

Danshen inhibits oxysterol-induced endothelial cell apoptosis in vivo.

Oxysterols induce apoptosis in vascular endothelial cells in vitro, but it is not clear whether they do so in vivo. We intravenously injected an oxysterol, cholestan-3beta, 5alpha, 6beta-triol, into rats and quantitatively analyzed endothelial cell apoptosis in the aorta. Oxysterol significantly promoted apoptosis in a time- and dose-dependent fashion. The apoptosis had increased 4.5-fold 6 hrs after injection, and returned to the background level at 48 hrs. An extract of the Chinese herb Danshen as well as probucol abolished triol-induced endothelial cell apoptosis in vitro and in vivo. Since apoptotic cells are quickly cleared, oxysterol-induced apoptosis could significantly affect endothelial integrity over a long period of time. Radical scavengers may be useful for the prevention of endothelial damage.

VLDL induces adipocyte differentiation in ApoE-dependent manner.

OBJECTIVE: To clarify the role of very low density lipoprotein (VLDL) and apolipoprotein E (apoE) in adipogenesis, we studied newly developed hyperlipidemic obese (ob/ob;apoE-/-) mice. Because hydrolysis of VLDL is believed to be the major source of adipogenic free fatty acids, a higher plasma level of VLDL in these mice should exaggerate obesity. METHODS AND RESULTS: When fed a high-fat, high-cholesterol diet, ob/ob;apoE-/- mice did not show increased body weight or an increased amount of adipose tissue in spite of increased plasma VLDL levels, whereas ob/ob mice showed an increased body weight and amount of adipose tissue, suggesting that there is a novel apoE-dependent pathway for adipogenesis. In vitro experiments using bone marrow stromal cells and 3T3-L1 cells confirmed this notion. ApoE-deficient VLDL did not induce adipogenesis, whereas normal VLDL induced adipogenesis in these cells. The incubation of apoE-deficient VLDL with recombinant human apoE restored its adipogenic activity. Tetrahydrolipstatin, a lipoprotein lipase inhibitor, did not affect the adipogenic activity of VLDL, suggesting that hydrolysis of VLDL did not play a major role in its effects. In fact, lipid components of VLDL or free fatty acids induced only partial adipogenesis. CONCLUSIONS: Our findings indicate that VLDL induces adipogenesis in an apoE-dependent manner both in vitro and in vivo.

Effect of troglitazone on serum gamma-glutamyltransferase activity and plasma t-PA/PAI-1 complex in type 2 diabetic patients.

BACKGROUND: This study was conducted to determine whether troglitazone (CAS 97322-87-7, Noscal), a thiazolidinedione with alpha-tocopherol in the side chain, had an antioxidant effect in patients with Type 2 diabetes. METHODS: In 46 Type 2 diabetic outpatients aged 64 +/- 7 years (male/female: 33/13), with a body mass index of 23.4 +/-2.8 kg/m2, a 2-year prospective study was performed. Patients were given troglitazone tablet (200 mg) twice a day. After treatment for 4 months, troglitazone was withheld for one month (washout period). Then troglitazone was resumed for a further 19 months. RESULTS: The study was completed in 28 patients. In 18 patients, it was not completed because of side effects in 4 female patients and because of withdrawal of troglitazone from the Japanese market in 14 patients. Fasting plasma glucose was decreased by troglitazone treatment from 170.2 +/- 38.2 mg/dl to 151.6 +/- 34.5 mg/dl (ANOVA: F = 8.054, p < 0.001). Washout caused it to return to the pre-treatment level (170.9 +/- 38.0 mg/dl), but it decreased again to 145.4 +/- 33.4 mg/dl with the resumption of treatment (F = 3.845, p < 0.001). Glycated hemoglobin was decreased by treatment from 8.2 +/- 1.6 % to 7.9 +/- 1.6 % (F = 7.558, D < 0.001). Washout caused it to return to the pre-treatment level (8.0 +/- 1.6 %), but it decreased again to 7.6 +/- 1.2 % with the resumption of treatment (F = 1.985, p = 0.041). Fasting serum immunoreactive insulin was decreased by treatment from 5.5 +/- 3.4 pU/ml to 4.0 +/- 2.2 pU/ml (F = 9.098, p < 0.001). Washout caused it to return to near the pre-treatment level (4.6 +/- 2.4 pU/ml). After resumption of treatment, it remained unchanged. Serum n-glutamyltransferase (gamma-GT) activity was decreased by treatment from 37.5 +/- 25.7 U/I to 25.6 +/- 12.3 U/I (F = 19.406, p < 0.001). Washout caused it to return to the pre-treatment level (36.5 +/- 25.1 U/I), but it decreased again to 24.2 +/- 10.1 U/l with the resumption of treatment (F = 7.931, p < 0.001). Plasma tissue plasminogen activator(t-PA)/plasminogen activator inhibitor-1 (PAI-1) complex was decreased by treatment from 21.7 + 8.6 ng/ml to 19.0 +/- 6.6 ng/ml (F = 8.784, p < 0.001). Washout caused it to return to the pretreatment level (21.9 +/- 7.4 ng/ml), but it decreased again to 14.3 +/- 4.3 ng/ml after the resumption of treatment (F = 12.341, p < 0.001). CONCLUSION: Decreases in serum gamma-GT activity and plasma t-PA/PAI-1 complex were closely related with antioxidant action of troglitazone.

The murine macrophage apoB-48 receptor gene (Apob-48r): homology to the human receptor.

Previously we cloned the human macrophage apolipoprotein B-48 receptor (ApoB-48R) and documented its expression in human atherosclerotic foam cells (1). Now we have identified and characterized the murine macrophage apob-48r cDNA gene sequence and its chromosomal location. The cDNA (3,615 bp) -deduced amino acid (aa) sequence (942 aa) is approximately 45% identical to the human macrophage APOB-48R, but not to other known gene families. The murine Apob-48r gene, like the human APOB-48R gene, consists of four exons interrupted by three small introns and is syntenically located on chromosome 7. Functionally significant conserved domains include an N-terminal hydrophobic domain, a glycosaminoglycan attachment site, an N-glycosylation site, and an ExxxLL internalization motif C-terminal to the putative internal transmembrane domain. Two conserved coiled-coil domains are likely involved in the spontaneous homodimerization that generates the active dimeric ligand binding species (mouse, approximately 190 kDa; human, approximately 200 kDa). Transfection of the murine apoB-48R into Chinese hamster ovary cells (CHOs) confers apoB-48R function: rapid, high-affinity, specific uptake of known triglyceride-rich lipoprotein ligands of the apoB-48R and, of note, uptake of the cholesteryl ester-rich apoB-48-containing very low density lipoproteins that accumulate in atherosclerosis-prone apoE-deficient mice. Uptake of these ligands by murine apoB-48R-transfected CHOs causes saturable, visible cellular triglyceride and cholesterol accumulation in vitro that resemble foam cells of atherosclerotic lesions. In aggregate, the data presented here and that previously published suggest that the apoE-independent murine apoB-48R pathway may contribute to the spontaneous development of atherosclerotic lesions rich in macrophage-derived foam cells observed in apoE-deficient mice, a murine model of human atherosclerosis.