Alpha-tocopherol decreases CD36 expression in human monocyte-derived macrophages.

Cholesterol-laden macrophages are the hallmark of atherogenesis. The class B scavenger receptor, CD36, binds oxidized low density lipoprotein (OxLDL), is found in atherosclerotic lesions, and is upregulated by OxLDL. We tested the effects of alpha-tocopherol (AT) enrichment of human monocyte-derived macrophages on CD36 expression and cholesteryl ester accumulation. Monocytes isolated from normal volunteers were cultured into macrophages. Macrophages were enriched overnight with various doses of AT (25, 50, and 100 microM). LDL from normal volunteers was oxidized or acetylated (AcLDL) and incubated with macrophages for 48 h at a concentration of 50 or 100 microg/ml. CD36 expression was assessed by flow cytometry. Quantitative analysis of scavenger receptor class A (SR-A) activity was performed with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled LDL. CD36 expression was maximal after 8;-10 days of culture. AT (> or =50 microM) significantly decreased CD36 expression upregulated by OxLDL and AcLDL (P < 0.01). Other antioxidants (beta- or gamma-tocopherol) or protein kinase C inhibitors failed to decrease CD36 expression. Concomitantly, DiI-AcLDL and DiI-OxLDL uptake was significantly decreased after AT treatment (P < 0.001). Cholesteryl ester accumulation was significantly decreased after AT enrichment (AcLDL + AT, 77% inhibition; OxLDL + AT, 42% inhibition). In conclusion, AT decreases both CD36 and SR-A expression and cholesteryl ester accumulation in human macrophages. This provides additional scientific support for the antiatherogenic properties of AT.

The promoter of human tissue factor pathway inhibitor gene: identification of potential regulatory elements.

Tissue factor pathway inhibitor is the major potent physiologic inhibitor of tissue factor-induced coagulation. Several potential binding sites for transcription factors have been described in the 750 bp of the 5' flanking region of the human tissue factor pathway inhibitor gene reported earlier. To identify elements that regulate the expression of tissue factor pathway inhibitor in endothelial, hepatocyte, and monocyte cells, the sequence of an additional 770 bp of tissue factor pathway inhibitor was determined. Comparison of this new sequence as well as that reported earlier with consensus sequences for transcription factor binding sites provided matches for GATA-2, SP1, and c-Myc sequences. Moreover, plasmids containing deletion mutants of the 5' tissue factor pathway inhibitor promoter region and the luciferase reporter gene were transfected into HepG2, ECV304, and THP1 cells. Three negative regulatory elements were localized between -548 to -390, - 390 to -75, and -1158 to -796 relative to the transcriptional start, respectively, in HepG2, ECV304 and THP-1 cells.

In vivo action of 15-lipoxygenase in early stages of human atherogenesis.

Oxidative modification of low density lipoprotein has been suggested as patho-physiologically relevant process in atherogenesis and the lipid peroxidizing enzyme 15-lipoxygenase may be involved. For experimental evidence on the in vivo action of this enzyme in the time course of plaque formation we analyzed the lipid extracts of lesional areas representing various stages of human atherogenesis for the occurrence of specific 15-lipoxygenase products. In advanced human lesions the degree of oxygenation of the lesion lipids measured as hydroxy linoleic acid/linoleic acid ratio varied between 0.2 and 3.2%. Here an unspecific pattern of oxygenated lipids that did not differ from the pattern formed during copper-catalyzed LDL oxidation was detected. In both cases an enantiomer ratio (S/R-ratio) of 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) of approximately 1:1 was found. In young human lesions which were obtained from the collection of the pathological determinants of atherosclerosis in youth (PDAY) program the hydroxy linoleic acid/linoleic acid ratio was much smaller (variation between 0.05 and 0.6%), and a significant share of specific 15-lipoxygenase products was detected (S/R-ratio of 13-hydroxy linoleic acid of 54 +/- 3.1/46 +/- 3.1 [mean +/- SD]). These data suggest that the 15-lipoxygenase is enzymatically active on endogenous substrates in young human lesions and thus, may be of patho-physiological importance for early atherogenesis. In advanced human plaques the 15-lipoxygenase may be functionally silent and specific lipoxygenase products formed in earlier stages may be decomposed or superimposed by large amounts of nonenzymatic lipid peroxidation products.

15-Lipoxygenase expression in smooth muscle cells from atherosclerotic rabbit aortas.

To determine the extent and origin of the stimulation of 15-lipoxygenase activity in atherosclerotic aortas, formation of hydroxy-derivatives from arachidonic acid was measured by HPLC-analysis and 15-lipoxygenase mRNA expression was investigated by RNA blot and in situ hybridization in atherosclerotic and normal rabbit aortic tissues. The synthesis of hydroxy-eicosatetraenoic acids (HETE) from exogenously added [14C]arachidonic acid was unchanged in atherosclerotic aortas in comparison with healthy aortas, but pretreatment with indomethacin demonstrated that 15-HETE production resulted essentially (75%) from cyclooxygenase activity in healthy aorta and from lipoxygenase activity in atherosclerotic aorta. The RNA blot and in situ hybridization with radiolabelled oligonucleotide probe demonstrated that 15-lipoxygenase mRNA was strictly localized in intimal thickening of atherosclerotic aortas. The immunostaining using anti-alpha smooth muscle actin, revealed that smooth muscle cell rich areas of the intimal thickening expressed 15-lipoxygenase mRNA. In addition, RNA blot hybridization indicated that cultured smooth muscle cells from atherosclerotic aortas expressed strongly 15-lipoxygenase mRNA. These results demonstrate that augmentation of 15-lipoxygenase activity in atherosclerotic aortas is correlated with 15-lipoxygenase mRNA expression in atherosclerotic plaque, and that intimal smooth muscle cells were involved, in addition to macrophages, in the expression of 15-lipoxygenase.

A new apheresis system using a copolymer (polyvinyl alcohol triethylene glycol diacrylate) for removal of LDL from dog whole blood.

Studies were undertaken to determine the effectiveness of a copolymer composed of PVA-TEGDA (Poly Vinyl Alcohol TriEthylene Glycol) as a plasma-cholesterol lowering procedure. For a comparative study, five dogs underwent plasmapheresis including the transfusion bag containing gel in the plasma line, while three control dogs underwent the same plasmapheresis without gel. Numerous biological determinations were performed firstly in whole blood circulation before and after treatment over 10 days, and secondly in plasma before and after LDL binding on the gel. In the whole blood circulation, the average significant depletion of cholesterol levels was 31-51% for treated, 0-16% for control dogs and the average significant depletion of LDL cholesterol was 26-75% for treated and 0-3% for control dogs. Gel was therefore able to bind 121-217 mg of total cholesterol and 34-70 mg of LDL cholesterol per gram of gel. Lipid and lipoprotein levels rebounded 3-4 days after treatment. Adverse effects were not observed during all plasmapheresis. No significant differences between control plasmapheresis and gel-plasmapheresis were obtained for blood cell counts while lengthened coagulation times were observed during 24 h. Complement was not significantly activated by the copolymer as shown by a same decreased activity in the blood stream of all dogs: in fact, CH 50 depletion in the gel incubated plasma was due to a protein adsorption on the hydrogel. This new approach for LDL apheresis appears to be a promising new technique.