Growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) is a new member of the transforming growth factor beta (TGF-beta) superfamily, which has most recently been found in activated macrophages (MPhi). We have now investigated GDF-15/MIC-1 in human MPhi after exposure to oxidized low-density lipoproteins (oxLDL) related mediators in vitro and in arteriosclerotic carotid arteries. Using RT-PCR and Western blotting a pronounced induction of GDF-15/MIC-1 expression by oxLDL, C6-ceramide, tumor necrosis factor (TNFalpha) and hydrogen peroxide (H2O2) was found in cultured human MPhi. In 11 human arteriosclerotic carotid arteries, immunohistochemical analyses supported by computer-assisted morphometry and regression analyses demonstrated a significant colocalization of GDF-15/MIC-1 immunoreactivity (IR) with oxLDL IR and manganese superoxide dismutase (MnSOD) IR in CD68 immunoreactive (ir) MPhi, which were also expressing AIF-IR (apoptosis-inducing factor), caspase-3-IR (CPP32), PARP-IR, c-Jun/AP-1-IR and p53-IR. Our data suggest that GDF-15/MIC-1 is inducible in human MPhi by oxLDL and its mediators in vitro and is supposed to contribute to oxidative stress dependent consequences in arteriosclerotic plaques, e.g. modulating apoptosis and inflammatory processes in activated MPhi.

Oxidized low density lipoprotein (oxLDL) induces apoptosis in human macrophages (Mphi), a significant feature in atherogenesis. We found that induction of apoptosis in Mphi by oxLDL, C2-ceramide, tumor necrosis factor alpha (TNF-alpha), and hydrogen peroxide (H2O2) was associated with enhanced expression of manganese superoxide dismutase (MnSOD) and p53. Treatment of cells with p53 or MnSOD antisense oligonucleotides prior to stimulation with oxLDL, C2-ceramide, TNF-alpha, or H2O2 caused an inhibition of the expression of the respective protein together with a marked reduction of apoptosis. Exposure to N-acetylcysteine before treatment with oxLDL, C2-ceramide, TNF-alpha, or H2O2 reversed a decrease in cellular glutathione concentrations as well as the enhanced production of p53 and MnSOD mRNA and protein. In apoptotic macrophages of human atherosclerotic plaques, colocalization of MnSOD and p53 immunoreactivity was found. These results indicate that in oxLDL-induced apoptosis, a concomitant induction of p53 and MnSOD is critical, and suggest that it is at least in part due to an enhancement of the sphingomyelin/ceramide pathway.

Abstract-Low-density lipoprotein (LDL) can be transformed to an atherogenic moiety by nonoxidative, enzymatic degradation. Enzymatically degraded LDL induces macrophage foam cell formation, provokes release of cytokines, and also activates complement. To determine whether complement activation may contribute to atherogenesis, 6 pairs of homozygous C6-deficient rabbits and their non-C6-deficient heterozygous siblings were fed a cholesterol-rich diet for 14 weeks. Cholesterol levels and plasma lipoprotein profiles of the animals in the C6-competent and C6-deficient groups did not significantly differ, and the high density lipoprotein and LDL cholesterol ratios at the end of the experiment were 0.07Ϯ0.01 and 0.08Ϯ0.01 (SEM), respectively. However, differences in atherosclerotic plaque formation were discernible macroscopically, with extensive aortic lesions being visible in all C6-competent animals and absent in all C6-deficient animals. Aortas were sectioned from thorax to abdomen, and 10 sections were stained from each aorta. Quantification of atherosclerotic lesions and lumen stenosis with the use of computer-based morphometry documented a dramatic protective effect of C6 deficiency on the development of diet-induced atherosclerosis. We conclude that the terminal complement sequence is centrally involved in atherosclerotic lesion progression. (Arterioscler Thromb Vasc Biol. 1998;18:1790-1795.)Key Words: complement activation Ⅲ atherosclerosis T he possible relevance of complement activation in atherogenesis has not received much attention to date, and only a few reviews are available on the topic. 1,2 The first immunohistochemical studies on complement deposition in atherosclerotic lesions appeared in 1985 to 1987, 3-5 and C5b-9 complexes were subsequently quantified by ELISA in detergent extracts of lesion homogenates. 6 In those studies, the stages of lesion development were not defined, so it could not be excluded that complement activation might have occurred subsequent to tissue damage. An experimental study was then performed in rabbits, leading to the clear demonstration that diet-induced deposition of lipids in the subendothelium was temporally associated with complement activation, which occurred before lesion infiltration by monocytes. 7 A directed search led to tentative identification of the complement-activating entity. Heterogeneously sized lipid droplets containing high amounts of free cholesterol were isolated from early lesions and were shown to be capable of spontaneously activating the alternative complement pathway. 8 The origin of this lipid, termed the lesion complement activator (LCA), was unknown, but the possibility that it represented an LDL derivative was obvious. To corroborate this assumption, attempts were undertaken to transform LDL in vitro into a complement-activating moiety. This was found to be possible by combined treatment of the lipoprotein with a protease, cholesterol-esterase, and neuraminidase, 9 enzymes that occur ubiquitously in lysosomes of mammalian cells and that are...

Background-Enzymatic, nonoxidative modification transforms LDL to an atherogenic molecule (E-LDL) that activates complement and macrophages and is present in early atherosclerotic lesions. Methods and Results-We report on the atherogenic effects of E-LDL on human vascular smooth muscle cells (SMC).E-LDL accumulated in these cells, and this was accompanied by selective induction of monocyte chemotactic protein-1 in the absence of effects on the expression of interleukin (IL)-8, RANTES, or monocyte inflammatory proteins-1␣ and -␤). Furthermore, E-LDL stimulated the expression of gp130, the signal-transducing chain of the IL-6 receptor (IL-6R) family, and the secretion of IL-6. E-LDL invoked mitogenic effects on SMC through 2 mechanisms. First, an autocrine mitogenic circuit involving platelet-derived growth factor and fibroblast growth factor-␤ was induced. Second, upregulation of gp130 rendered SMC sensitive to transsignaling through the IL-6/sIL-6R activation pathway. Because E-LDL promoted release of both IL-6 and sIL-6R from macrophages, application of macrophage cell supernatants to prestimulated SMC provoked a pronounced and sustained proliferation of the cells. Conclusions-E-LDL can invoke alterations in SMC that are characteristic of the evolving atherosclerotic lesion.