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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...

The objective of the study was to analyze the intracellular antioxidative response of macrophages (Mphi) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit Mphi, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human Mphi with oxidized-LDL (ox-LDL) resulted in an induction of MnSOD mRNA production as shown by RT-PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of Mphi. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P<0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=-0.67, P<0.001) and GSH concentration (r=-0.57, P<0.01) with plaque size. Immunohistology of the atherosclerotic intima revealed MnSOD-IR in Mac-1 (CD 11b/CD 18)-immunoreactive (ir) Mphi of human arteries and, similarly, in RAM-11-ir Mphi of rabbit ones. The relation of MnSOD-ir Mphi decreased with plaque advancement, which is consistent with biochemical findings. Most MnSOD-ir Mphi in atherosclerotic plaques revealed TUNEL-positive nuclei, indicating DNA strand breaks, and p53-IR. We conclude that mitochondrial antioxidants such as MnSOD are induced in Mphi in vitro and in atherosclerotic arteries as a reply to increased mitochondrial oxidation. As normal consequences of an increased oxidative stress due to the exposure to ox-LDL nuclear DNA strand breaks occur, which are suggested to be a signal to increase p53 protein levels. Reactive oxygen species-mediated mitochondrial-dependent pathways are suggested as major contributing pathomechanisms to nuclear damage, which eventually may result in apoptosis. A common response to increased oxidative stress due to modified LDL is presumed in rabbit and human atherosclerotic plaques.

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The study aimed to 1) quantify oxidative stress in spinal cord after crush injury at T6, 2) determine whether the administration of the procysteine compound L-2-oxothiazolidine-4-carboxylate (OTC) would up-regulate glutathione (GSH) synthesis and decrease oxidative stress, and 3) determine whether decreased oxidative stress results in better tissue and function retention. We demonstrate that spinal cord compression (5 s with a 50 g aneurysm clip) at T6 in rats results in oxidative stress that is extensive (significant increases in oxidative stress seen at C3 and L4) and rapid in onset. Indices of oxidative stress used were GSH content, protein carbonyl content, and inactivation of glutathione reductase. Administration of OTC resulted in a marked decrease in oxidative stress associated with a sparing of white matter at T6 (16+/-1.9% retained in OTC-treated animals vs. less than 1% in saline-treated). Behavioral indices in control, saline-treated, and OTC-treated animals after 6 wk were respectively: angle board scores (59 degrees, 32 degrees, and 42 degrees ), modified Tarlov score (7, 2.4, and 4.1), and Basso-Beattie-Bresnahan score (21, 5.3, and 12.9). We conclude that administration of OTC after spinal cord trauma greatly decreases oxidative stress and allows tissue preservation, thereby enabling otherwise paraplegic animals to locomote.

SUMMARYTumour antigen presentation by dendritic cells (DCs) to T cells in lymphoid organs is crucial for induction of anti-tumour immune responses. It has been previously reported that tumour necrosis factor-a (TNF-a) is required for DC activation and subsequent induction of optimal immune responses, and thus DCs for anti-tumour vaccination are often generated by culture in exogenous TNF-a. In the present study, we investigated the effect on anti-tumour immunity of vaccination with Mut1 tumour peptide-pulsed DCs engineered to express a TNF-a transgene. Our data shows that transfection of DCs with recombinant adenovirus AdV-TNFa resulted in greater maturation of the DCs than occurred with control DCs cultured in exogenous TNF-a, as determined by up-regulated expression of pro-inflammatory cytokines (e.g. interleukins 1b and 18), chemokines [e.g. interferon-c-inducible protein-10 and macrophage inflammatory protein-1b (MIP-1b)], the CC chemokine receptor CCR7, and immunologically important cell surface molecules (CD40, CD86 and intercellular adhesion molecule-1). These transgenic DCs stimulated stronger allogeneic T-cell responses in vitro and T-cell activation in vivo; displayed 2 . 4-fold enhanced chemotactic responses to the MIP-3b in vitro (P<0 . 05); and, perhaps most importantly, trafficked into the draining lymph nodes dramatically (seven-fold, P<0 . 01) more efficiently than the control DCs. Our data also demonstrate that vaccination of mice with Mut1 peptide-pulsed, AdV-TNF-a-transfected DCs stimulated more efficient in vitro Mut1-specific CD8+ cytotoxic T-cell responses and solid tumour immunity in vivo, when compared to the in vitro TNF-a-cultivated DCs. Thus, DCs engineered to secrete TNF-a may offer a new strategy in DC cancer vaccines.

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