Oxysterols are common components of oxidized low-density lipoprotein and accumulate in the core of fibrotic plaques as a mixture of cholesterol and cholesteryl ester oxidation products. The proapoptotic effects of a biologically representative mixture of oxysterols was compared with equimolar amounts of 7-ketocholesterol and unoxidized cholesterol. The oxysterol mixture in a concentration range actually detectable in hypercholesterolemic patients did not stimulate programmed cell death in cultivated murine macrophages. Unoxidized cholesterol also produced no effect. By contrast, when given alone, 7-ketocholesterol strongly stimulated the mitochondrial pathway of apoptosis with cytochrome c release, caspase-9 activation, and eventually caspase-3 activation. Subsequent experiments showed that when 7-ketocholesterol was administered to cells together with another oxysterol, namely 7betaOH-cholesterol, the strong proapoptotic effect of 7-ketocholesterol was markedly attenuated. As regards the mechanism underlying this quenching, we found that the combined oxysterol treatment counteracted the ability of 7-ketocholesterol, when administered alone, to strongly up-regulate the steady-state levels of reactive oxygen species (ROS) without interfering with sterol uptake. Furthermore, this increase in intracellular ROS appeared to be responsible for the up-regulation of proapoptotic factor, p21, after treatment with 7-ketocholesterol but not in cells challenged with the oxysterol mixture. Competition among oxysterols, apparently at the level of NADPH oxidase, diminishes the ROS induction and direct toxicity that is evoked by specific oxysterols. As a consequence, a more subtle gene modulation by oxysterols becomes facilitated in vascular cells.

Background: During neoplastic progression, alterations in transforming growth factor β1 (TGF-β1) dependent control of cell growth may be an important mechanism of selective proliferation of transformed cellular clones. Defective regulation of TGF-β1 receptors has been reported to occur in a number of human malignant tumours while little is known of the actual levels of this growth inhibitory cytokine in cancer. On the basis of the demonstrated ability of major lipid peroxidation products such as 4-hydroxynonenal to modulate TGF-β1 expression and synthesis, we speculated that decreased lipid oxidation, as frequently observed in neoplastic tissues, would contribute to the selective promotion of tumour growth through decreased expression of the cytokine within the tumour mass. Aims: To seek a possible association between steady state levels of major aldehydic end products of lipid peroxidation and TGF-β1 content in human colon cancer at different stages of growth. Patients and methods: Tissue biopsies from 15 adult patients with colon adenocarcinoma of different TNM and G stagings were compared with regard to lipid peroxidation aldehydes and net TGF-β1 levels. For a more comprehensive analysis, cytokine type I and II receptors were measured in tumour biopsies. In one set of experiments, to support the conclusions, the apoptotic effect of TGF-β1 was evaluated in a human colon cancer cell line, CaCo-2, retaining receptor changes consistent with those observed in cancer patients. Results: With the exception of two extremely advanced cases (T4/G3) in which tissue levels of lipid peroxidation were within the normal range, 4-hydroxynonenal was significantly decreased in all other cancer specimens. Consistent with lipid peroxidation levels, TGF-β1 protein was markedly decreased or even negligible compared with the corresponding normal tissue surrounding the tumour in all tested biopsies except for the two T4/G3 colon cancers in which cytokine content was again within the normal range. As regards TGF-β1 receptors, both in tumour sections and CaCo-2 cells, downregulation was greater for TGF-β1 receptor I than for receptor II. Of note, in CaCo-2 cells, incubation with appropriate doses of TGF-β1 led to marked nuclear fragmentation and apoptosis. Conclusions: Evasion of human colon cancer cells from TGF-β1 mediated growth inhibition appears to be due not only to downregulation of TGF-β1 receptors, which is inconsistent and unrelated to cancer development, but also to the constant low concentration of this cytokine in the tumour mass. The associated levels of lipid peroxidation aldehydes, much lower than in control tissue, probably represent a lower stimulus for TGF-β1 production in the neoplastic area and thus a favourable condition for neoplastic progression.

Liver ischemia-reperfusion is characterized by an increased oxygen-dependent free radical chain-reaction rate and an increased steady-state concentration of reactive oxygen species. The aim of this study was to evaluate the in situ generation of reactive oxygen species and its relationship with phagocyte activation and recruitment in reperfused rat liver. Rat livers were subjected to 2 hours of selective lobular ischemia and reperfusion for up to 12 hours. The following parameters were determined: in situ liver chemiluminescence, understood to reflect the tissue steady-state concentration of singlet oxygen ( 1 O 2 ); myeloperoxidase tissue activity; the number of neutrophils; and the degree of necrosis. An early chemiluminescence burst was measured after 30 minutes of blood reflow (early phase of oxidative stress), followed by a relapse and a further increase after 4 to 12 hours of reperfusion (late phase of oxidative stress). Both early and late phases were modified by pretreatment with gadolinium chloride (GdCl 3 ), pointing to a key role of the Kupffer cells. Neutrophils infiltrated into the liver, myeloperoxidase activity, in situ chemiluminescence, and necrosis were found to be strongly correlated over the 4-to 12-hour reperfusion period (r ‫؍‬ .960; average of the 4 correlation coefficients). Together with resident phagocytes, neutrophil recruitment and activation appear to provide a major contribution to the increase of oxygendependent free-radical reactions and amplification of liver reperfusion damage. Surface chemiluminescence appears to properly describe the in situ and in vivo progressive organization of the acute inflammatory response with phagocytemediated liver injury. (HEPATOLOGY 2000;31:622-632.)

The liver is particularly susceptible to Fas-mediated cytotoxicity. Mice given an adequate parenteral dose of agonistic antiFas antibody (aFas) or of FasL are known to develop a devastating liver injury and to die in a few hours. The present work shows that mice lacking TNFR1 and TNFR2 (R À ) both survive a single dose of aFas, otherwise rapidly lethal, and develop a mild form of hepatic damage, compared to the much more severe liver injury that in a few hours strikes wildtype mice (R þ ), eventually involving increased activity of proteases of different families (caspase 3-, 8-, and 9-like, calpains, cathepsin B). Neither the overall tissue levels of Fas and FasL nor Fas expression at the hepatocyte surface are altered in the liver of R À animals. The DNA-binding activity of the NF-jB transcription factor is enhanced after aFas treatment, but much more markedly in R À than in R þ mice. Bcl2, while unchanged in untreated animals, is markedly upregulated in R À but not in R þ mice challenged with aFas. The requirement of a normal TNFR1/TNFR2 phenotype for full deployment of the general and liver-specific aFas toxicity in mice most likely implies that treatment with aFas in some way results in activation of the TNFa-TNFRs system and that this activation synergizes with Fas-mediated signals in causing the fulminant liver injury and the animal death. The precise cellular and molecular details underlying this interplay between Fas-and TNFRs-mediated signaling systems in the general and liver-specific aFas toxicity largely remain to be clarified.

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