Role of Sphingosine-1-Phosphate in Transplant Vasculopathy Evoked by Anti-HLA Antibody.

Transplant vasculopathy (TV) represents the main cause of late graft failure and limits the long-term success of organ transplantation. Cellular and humoral immune responses contribute to the pathogenesis of the concentric and diffuse intimal hyperplasia of arteries of the grafted organ. We recently reported that the mitogenic signaling, evoked in human vascular smooth muscle cells (hmSMC) by the anti-HLA class I monoclonal antibody W6/32, implicates neutral sphingomyelinase-2, suggesting a role for sphingolipids in intimal hyperplasia of TV. Here, we investigated whether the mitogenic sphingolipid, sphingosine-1-phosphate (S1P), is involved in intimal hyperplasia elicited by W6/32. Studies were done on cultured hmSMC and on an in vivo model of TV, consisting of human mesenteric arteries grafted into SCID/beige mice, injected weekly with W6/32. hmSMC migration and DNA synthesis elicited by W6/32 were inhibited by the sphingosine kinase-1 (SK1) inhibitor dimethylsphingosine, the anti-S1P antibody Sphingomab and the S1PR1/R3 inhibitor VPC23019. W6/32 stimulated SK1 activity, while siRNA silencing SK1, S1PR1 and S1PR3 inhibited hmSMC migration. In vivo, Sphingomab significantly reduced the intimal thickening induced by W6/32. These data emphasize the role of S1P in intimal hyperplasia elicited by the humoral immune response, and open perspectives for preventing TV with S1P inhibitors.

Aminothiols and allantoin in chronic dialysis patients: effects of hemodialysis sessions.

BACKGROUND: To investigate the possible relationship between homocysteine and allantoin levels in hemodialyzed patients, serum levels of thiols and purine compounds were analyzed before and after dialysis sessions. METHODS: 16 clinically stable non-diabetic patients hemodialyzed on polysulfone membranes were compared with 36 control subjects. Serum samples were collected before and after hemodialysis sessions. Total homocysteine, cysteine, glutathione, cysteinylglycine, uric acid, hypoxanthine, and allantoin were measured by capillary electrophoresis. RESULTS: Pre-dialysis homocysteine, allantoin, and uric acid were significantly elevated in dialysis patients as compared to controls. Cysteine, glutathione, and hypoxanthine levels were similar in both groups. Homocysteine significantly decreased, but did not normalize after dialysis sessions. Glutathione and cysteinylglycine levels remained unchanged after dialysis sessions, whereas cysteine decreased. Uric acid, hypoxanthine, and allantoin levels were significantly reduced by dialysis sessions. The allantoin/uric acid ratio was higher in dialyzed patients before hemodialysis (0.049 +/- 0.023 vs. 0.016 +/- 0.012 in controls; p < 0.001), and became elevated after a dialysis session (0.084 +/- 0.033; p = 0.002). CONCLUSIONS: Despite the use of biocompatible membranes, homeostasis of thiols and purine compounds is disturbed in hemodialysed patients. We suggest that allantoin could be used as a marker for oxidative stress in hemodialyzed patients.

Oxygen-regulated protein-150 prevents calcium homeostasis deregulation and apoptosis induced by oxidized LDL in vascular cells.

Oxidized LDLs (oxLDLs) induce apoptosis, which contributes to the pathogenesis of atherosclerosis. The 150 kDa oxygen-regulated protein (ORP150), an endoplasmic reticulum (ER)-resident chaperone, is upregulated by hypoxia and prevents ischemia-induced cell death. The aim of this work was to investigate whether and how ORP150 can prevent apoptosis induced by oxLDLs in vascular cells. OxLDLs induced ORP150 expression in the ER of human microvascular endothelial cell line (HMEC-1). ORP150 expression was blocked by antioxidants, by the permeant calcium chelator BAPTA-AM, and by inhibitors of the inositol-1,4,5 trisphosphate (IP3) receptors, 2-aminoethyl diphenylborinate (2-APB) and xestospongin C. ORP150 silencing by siRNA-enhanced oxLDL-induced apoptosis, while forced ORP150 expression increased the resistance of cells via an inhibition of the oxLDL-induced calcium rise, and of subsequent calpain activation, cytochrome c release, caspase 3 activation and apoptosis. A similar protective effect was achieved by BAPTA-AM, 2-APB and xestospongin C. Altogether, these data indicate that (i)ORP150 inhibits oxLDL-induced apoptosis by blocking calcium signaling and subsequent apoptosis, (ii)calcium released from ER stores through IP3 channels is involved in the oxLDL-induced calcium rise and apoptosis, and is inhibited by ORP150. Finally, ORP150 is expressed in advanced atherosclerotic lesions, where it may locally participate to reduce the apoptotic effect of oxLDLs and the subsequent risk of plaque rupture.

HLA class I antibodies provoke graft arteriosclerosis in human arteries transplanted into SCID/beige mice.

Antibodies toward HLA class I and/or MICA are commonly observed in transplanted patients suffering from allograft arteriosclerosis, also called chronic vascular rejection (CVR). The relative importance of cellular versus humoral alloreactivity for CVR is still disputed. We demonstrate that antibodies toward HLA class I provoke lesions typical for CVR in human arteries in vivo in the absence of cellular immunity. To show this, we grafted segments of human mesenteric arteries from 8 deceased organ donors into 36 immunodeficient SCID/beige mice in the infrarenal aortic position. Three mice died postoperatively. The remaining 33 mice received weekly i.v. injections of either a monoclonal antibody toward HLA class I, toward MICA or an irrelevant monoclonal antibody. At sacrifice after 6 weeks, mice receiving the HLA antibody showed a significant neointimal thickening in the grafted artery due to smooth muscle cell (SMC) proliferation while control mice receiving anti-MICA or irrelevant antibody showed little or no thickening. Whereas antibodies toward HLA class I were mitogenic to SMC in vitro, those directed toward MICA did not have any effect. Humoral alloreactivity toward HLA may thus play a causal role for the development of CVR and this opens new possibilities for the treatment of CVR.

FTY720 Inhibits Tumor Necrosis Factor-alpha-Induced Proliferation and Extracellular Signal-Regulated Kinase Phosphorylation of Human Smooth Muscle Cells.

We investigated the effects of the sphingolipid FTY720 on tumor necrosis factor-alpha (TNF-alpha)-induced proliferation and signal transduction in human smooth muscle cells (SMC). We showed that clinically relevant concentrations of FTY720 inhibited TNF-alpha-induced SMC proliferation and extracellular signal-regulated kinase (ERK) phosphorylation. We concluded that FTY720 may be a useful drug to inhibit chronic vascular rejection.

Advanced lipid peroxidation end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for the inhibitors.

Reactive carbonyl compounds (RCCs) formed during lipid peroxidation and sugar glycoxidation, namely Advanced lipid peroxidation end products (ALEs) and Advanced Glycation end products (AGEs), accumulate with ageing and oxidative stress-related diseases, such as atherosclerosis, diabetes or neurodegenerative diseases. RCCs induce the 'carbonyl stress' characterized by the formation of adducts and cross-links on proteins, which progressively leads to impaired protein function and damages in all tissues, and pathological consequences including cell dysfunction, inflammatory response and apoptosis. The prevention of carbonyl stress involves the use of free radical scavengers and antioxidants that prevent the generation of lipid peroxidation products, but are inefficient on pre-formed RCCs. Conversely, carbonyl scavengers prevent carbonyl stress by inhibiting the formation of protein cross-links. While a large variety of AGE inhibitors has been developed, only few carbonyl scavengers have been tested on ALE-mediated effects. This review summarizes the signalling properties of ALEs and ALE-precursors, their role in the pathogenesis of oxidative stress-associated diseases, and the different agents efficient in neutralizing ALEs effects in vitro and in vivo. The generation of drugs sharing both antioxidant and carbonyl scavenger properties represents a new therapeutic challenge in the treatment of carbonyl stress-associated diseases.

T-cell function in maintenance renal transplant patients receiving mycophenolate mofetil and steroids with or without tacrolimus.

Nephrotoxicity-sparing protocols, using mycophenolate mofetil (MMF) and steroids without calcineurin inhibitors (CNIs) or mammalian target rapamycin (mTOR), could be used to treat maintenance renal transplant patients. However, the risk for acute rejection seems to be high. The aim of this pharmacodynamic study was to analyze T-cell function, T-cell activation, and T-cell proliferation among patients receiving MMF and steroids (n = 15) compared with patients receiving immunosuppression with CNI-based therapy including tacrolimus, MMF, and steroids. Our data suggested that among stable maintenance patients, dual therapy with MMF and steroids might provide a similar reduction in T-cell proliferation and T-cell activation as that observed among patients on standard immunosuppressive therapy. As expected, intralymphocytic interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) expressions were higher in patients not receiving CNIs.

Involvement of FAN in TNF-induced apoptosis.

TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.

Effect of FTY720 on apoptosis of smooth muscle cells.

BACKGROUND: Hyperproliferation of smooth muscle cells (SMCs) plays a key role in allograft arteriosclerosis. This prompted us to investigate the effect of the novel immune modulator and synthetic sphingolipid FTY720 on apoptosis of SMCs. METHODS: Rabbit SMC cultures were treated with FTY720 and apoptosis and necrosis were detected by fluorescence microscopy. RESULTS: We investigated dose- and time-dependent effects of FTY720 and found that clinically relevant low doses of FTY720 (<1 micromol/L) did not induce apoptosis, whereas 10 micromol/L FTY720 induced apoptosis after 48 hours incubation. CONCLUSION: At doses of FTY720 used in clinics for treatment of renal allografts and multiple sclerosis. FTY720 did not induce SMC apoptosis.

Sphingolipid mediators in cardiovascular cell biology and pathology.

Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications.

Activation of epithelial growth factor receptor pathway by unsaturated fatty acids.

Nonesterified fatty acids (NEFAs) are acutely liberated during lipolysis and are chronically elevated in pathological conditions, such as insulin resistance, hypertension, and obesity, which are known risk factors for atherosclerosis. The purpose of this study was to investigate the effect and mechanism of action of NEFAs on the epithelial growth factor (EGF) receptor (EGFR). In the ECV-304 endothelial cell line, unsaturated fatty acids triggered a time- and dose-dependent tyrosine phosphorylation of EGFR (polyunsaturated fatty acids [PUFAs] were the most active), whereas saturated FAs were inactive. Although less potent than PUFAs, oleic acid (OA) was used because it is prominent in the South European diet and is only slightly oxidizable (thus excluding oxidation derivatives). EGFR is activated by OA independent of any autocrine secretion of EGF or other related mediators. OA-induced EGFR autophosphorylation triggered EGFR signaling pathway activation (as assessed through coimmunoprecipitation of SH2 proteins such as SHC, GRB2, and SHP-2) and subsequent p42/p44 mitogen-activated protein kinase (as shown by the use of EGFR- deficient B82L and EGFR- transduced B82LK(+) cell lines). OA induced in vitro both autophosphorylation and activation of intrinsic tyrosine kinase of immunopurified EGFR, thus suggesting that EGFR is a primary target of OA. EGFR was also activated by mild surfactants, Tween-20 and Triton X-100, both in vitro (on immunopurified EGFR) and in intact living cells, thus indicating that EGFR is sensitive to amphiphilic molecules. These data suggest that EGFR is activated by OA and PUFAs, acts as a sensor for unsaturated fatty acids (and amphiphilic molecules), and is a potential transducer by which diet composition may influence vascular wall biology.

Sphingomyelin metabolites in vascular cell signaling and atherogenesis.

The atherosclerotic lesion most probably develops through a number of cellular events which implicate all vascular cell types and include synthesis of extracellular proteins, cell proliferation, differentiation and death. Sphingolipids and sphingolipid metabolizing enzymes may play important roles in atherogenesis, not only because of lipoprotein alterations but also by mediating a number of cellular events which are believed to be crucial in the development of the vascular lesions such as proliferation or cell death. Exogenous sphingolipids may mediate various biological effects such as apoptosis, mitogenesis or differentiation depending on the cell type. Moreover, several molecules present in the atherogenic lesion, such as oxidized LDL, growth factors or cytokines, which activate intracellular signaling pathways leading to vascular cell modifications, can stimulate sphingomyelin hydrolysis and generation of ceramide (and other metabolites as sphingosine-1-phosphate). Here we review the potential implication of the sphingomyelin/ceramide cycle in vascular cell signaling related to atherosclerosis, and more generally the role of sphingolipids in the events observed during the atherosclerotic process as cell differentiation, migration, adhesion, retraction, proliferation and death.

Oxidative theory of atherosclerosis and antioxidants.

Atherosclerosis is a multifactorial process that begins early in infancy and affects all the humans. Early steps of atherogenesis and the evolution towards complex atherosclerotic plaques are briefly described. After a brief history of the 'Lipid theory of atherosclerosis', we report the most prominent discoveries on lipoproteins, their receptors and metabolism, and their role in atherogenesis. The main focus is the 'oxidative theory of atherosclerosis', with emphasis on free radicals and reactive oxygen species, lipid peroxidation and LDL oxidation, biological properties of oxidized LDL and their potential role in atherogenesis. Then, we report the properties of antioxidants and antioxidant systems and their effects in vitro, on cultured cells, in animal models and in humans. The surprising discrepancy between the efficacy of antioxidants in vitro and in animal models of atherosclerosis and the lack of protective effect against cardiovascular events and death in epidemiological study and clinical trials are discussed. In contrast, epidemiological studies seem to indicate that the Mediterranean diet may protect (in part) against atherosclerosis complications (myocardial infarction and cardiovascular death).

UV-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cells. 4. Calcium is involved in the cytotoxicity of UV-treated LDL on lymphoid cell lines.

In lymphoid cells pulsed with 'cytotoxic' concentrations of UV-treated LDL, the study of the variations of free cytosolic calcium concentration, of the influence of extracellular calcium and of the protective effect of calcium chelators suggests that both intra- and extracellular calcium could play a major role in the genesis of cell injury leading to cell death. (1) A dramatic sustained rise of cytosolic free calcium (the level of free cytosolic calcium was higher than 500 nmol/l for 6 h or more) occurred several hours after the beginning of the pulse with UV-treated LDL (lag period between 6 and 12 h). (2) The rise of the free cytosolic calcium and the 'cytotoxicity' induced by UV-treated LDL were largely dependent on the concentration of extracellular calcium which has an effect on the uptake of UV-treated LDL and on the expression of the 'cytotoxicity' at the cellular level. (3) The study of the sequence of intracellular events showed that the cellular oxidative stress generated by oxidized LDL was followed by the rise of free cytosolic calcium and later by the rise of 'cytotoxicity' indexes. (4) The intracellular calcium chelators, BAPTA/AM and EGTA/AM, were able to partially protect lymphoid cells against the 'cytotoxicity' of oxidized LDL. The supposed mechanisms of the free cytosolic calcium rise and the respective role of calcium or/and other factors (for instance direct lesions of the plasma membrane by the oxidative stress due to oxidized LDL) in the genesis of cellular lesions leading to cell death are discussed.

Pyrenemethyl laurate, a new fluorescent substrate for continuous kinetic determination of lipase activity.

A fluorescent acyl derivative of pyrenemethanol, pyrenemethyl laurate, was synthesized and used for the determination of several lipases by a continuous kinetic assay. The influence of the physical parameters of the substrate (pyrenemethyl laurate) and its hydrolysis product (pyrenemethanol), on the fluorescence emission was studied. The hydrolysis of pyrenemethyl laurate could be monitored directly in a spectrofluorometer because of the very high monomeric emission of pyrenemethanol at about 375 nm, whereas an aqueous dispersion of pyrenemethyl laurate emitted at 475 nm ('excimeric'). Pyrenemethyl laurate was hydrolyzed by gastric lipase, cellular lipases of haemopoietic cells, and the bacterial lipase of Rhizopus arrhizus.

Degradation of fluorescent and radiolabelled sphingomyelins in intact cells by a non-lysosomal pathway.

The aim of the present study was to investigate the role of the entitled neutral, sphingomyelinase in the non-lysosomal pathway of sphingomyelin degradation by intact cells (Spence et al. (1983) J. Biol. Chem. 258, 8595-8600; Levade et al. (1991) J. Biol. Chem. 266, 13519-13529). The uptake and degradation of sphingomyelin by intact living cells was studied using cell lines exhibiting a wide range of activity levels of acid, lysosomal and neutral sphingomyelinases as determined in vitro on cell homogenates by their respective standard assays. For this purpose, neuroblastoma, skin fibroblasts, lymphoid and leukemic cell lines, some of them derived from patients with Niemann-Pick disease (deficient in the acid, lysosomal sphingomyelinase) were incubated with radioactive, [oleoyl-3H]sphingomyelin or fluorescent, pyrene-sulfonylaminoundecanoyl-sphingomyelin. Either compound was taken up by a pathway which was not receptor-mediated and hydrolyzed by all intact cells, including those derived from Niemann-Pick disease patients. Moreover, their degradation by the intact cells was not inhibited by treatment with chloroquine, indicating hydrolysis by a non-lysosomal sphingomyelinase. The intracellular sphingomyelin degradation rates showed no correlation with the activity of the 'classical' neutral sphingomyelinase as determined in vitro. In particular, fibroblasts derived from Niemann-Pick patients lacking the lysosomal sphingomyelinase, and having no detectable in vitro activity of the 'classical' neutral sphingomyelinase, were able to degrade the exogenously supplied sphingomyelins. Indeed, in vitro these cells were shown to exhibit neutral, magnesium- and dithiothreitol-dependent sphingomyelinase activities, that might contribute to the non-lysosomal pathway for sphingomyelin degradation to ceramide in intact cells.

Cholesterol sulfate is not degraded but does not accumulate in Epstein-Barr virus-transformed lymphoid cells from patients with X-linked ichthyosis.

The metabolism of cholesterol sulfate (CS) was investigated in immortalized, Epstein-Barr virus-transformed lymphoid cell lines derived from normal individuals and patients affected with recessive X-linked ichthyosis (XLI). Normal lymphoid cells expressed arylsulfatase C and steroid sulfatase (including cholesterol sulfatase) activities, and these two sulfohydrolases showed the same enzyme properties as in other human cells, e.g., leukocytes or skin fibroblasts. XLI-derived lymphoid cell lines exhibited extremely deficient activity of both arylsulfatase C and steroid sulfatase. While normal and XLI intact, living lymphoid cells could take up exogenous radiolabelled CS through a non-receptor-mediated process. XLI cells were completely unable to degrade CS to cholesterol. However, despite their defect in CS degradation, steroid sulfatase-deficient cells did not accumulate CS because of outflux of this sterol. The potential implications of these findings to the pathogenesis of increased CS content in plasma and epidermis of XLI patients are discussed. This study also demonstrates that immortalized lymphoid cell lines may represent a useful experimental model system for the study of XLI.

Phospholipid hydrolysis of mildly oxidized LDL reduces their cytotoxicity to cultured endothelial cells. Potential protective role against atherogenesis.

Oxidized low density lipoproteins (LDL) are cytotoxic to cultured endothelial cells and thereby are potentially involved in endothelial cell injury and atherogenesis. Oxidized phospholipids of oxLDL undergo spontaneous hydrolysis (PL-hydrolysis) by LDL-associated phospholipase A2 (PLA2) activities. The present study aimed to investigate whether hydrolysis of oxidized phospholipids contained in mildly oxLDL could influence their cytotoxicity to cultured endothelial cells. PL-hydrolysis (spontaneous or mediated by exogenous PLA2) of mildly oxLDL elicited a significant reduction of their cytotoxicity to cultured endothelial cells. The reduced cytotoxicity of PL-hydrolysed oxLDL was not due to their reduced uptake by cells, but rather to their reduced content of oxidation products which are liberated by PL-hydrolysis and released (at least the more polar compounds) in the aqueous phase, as shown by ultrafiltration experiments. Oxidation products released in the aqueous phase were not or only slightly cytotoxic to endothelial cells, probably because a selective uptake of non oxidized fatty acids as shown by studies of uptake of oxidized and non oxidized [1-14C]linoleic acid. These data suggest that during PL-hydrolysis of mildly oxLDL, (i) oxidized phospholipids are hydrolysed; (ii) oxidation products liberated from oxLDL particles are released (at least in part) to the aqueous phase; (iii) the cytotoxicity of oxLDL to endothelial cells is reduced, probably because oxidized free fatty acids (released by PL-hydrolysis towards the aqueous phase) are not taken up by the cells. Finally, the possibility of a favourable role of PL-hydrolysis of oxLDL against atherogenesis is discussed.

Significance of two point mutations present in each HEXB allele of patients with adult GM2 gangliosidosis (Sandhoff disease) homozygosity for the Ile207-->Val substitution is not associated with a clinical or biochemical phenotype.

The molecular defects in the HEXB gene encoding the common beta-subunit of lysosomal beta-hexosaminidase A (beta-Hex A, alpha beta) and beta-Hex B (beta beta) were investigated in a Portuguese family affected with late onset Sandhoff disease (GM2-gangliosidosis variant 0). This family comprised two unaffected daughters and three affected sibs who developed at about age 17 cerebellar ataxia and mental deficiency. Their parents were consanguineous and clinically asymptomatic. There was no detectable beta-Hex B activity and a profound reduction in the activity of beta-Hex A in the leukocytes and transformed lymphoid cell lines from the affected sibs. The expected intermediate values were observed in the parents as well as in one daughter and her children. Western analysis revealed the presence of reduced, but detectable amounts of mature beta-chain protein in cell lysates from the probands and intermediate levels in the parents. Nucleotide sequencing of amplified, reverse-transcribed beta-chain mRNA demonstrated the presence of two single point mutations: an A619 to G transition in exon 5 (Ile207-->Val), and a G1514 to A transition in exon 13 (Arg505-->Gln). Both of these two mutations have been previously linked to the adult form of Sandhoff disease in compound heterozygote patients. All three affected sibs were found to be homoallelic for both mutations. Interestingly, while the mother was heterozygous for each mutation, the father was homozygote for the A619-->G substitution and heterozygote for the G1514-->A transition. Since the father is homozygote for the A619-->G mutation but expresses a biochemical phenotype consistent with a carrier of Sandhoff disease and is clinically asymptomatic, this substitution is likely a neutral mutation. We confirmed this hypothesis by finding this transition present in 4 of 30 alleles from normal individuals. We conclude that homozygosity for the G1514-->A mutation is exclusively responsible for the adult form of Sandhoff disease in this family, and that the A619-->G substitution is not a deleterious mutation but rather a common HEXB polymorphism.

Ultraviolet-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cells. III. The protective effect of antioxidants (probucol, catechin, vitamin E) against the cytotoxicity of oxidized LDL occurs in two different ways.

Comparison of the protective effect of three antioxidants (from three different chemical classes) against cell injury due to LDL oxidation, allowed us to clearly discriminate between two different lines of defence. The ultraviolet-induced lipid peroxidation of LDL was strongly inhibited by 10 mumol/l catechin and 25 mumol/l probucol, but only poorly by 100 mumol/l vitamin E. The ultraviolet-treated LDL protected by catechin or probucol (i.e. LDL irradiated by ultraviolet in the presence of effective concentrations of antioxidants inhibiting the lipid peroxidation) were much less 'cytotoxic' than unprotected ultraviolet-treated LDL. In contrast, LDL treated by ultraviolet in the presence of 100 mumol/l vitamin E were 'cytotoxic' similarly to unprotected LDL. The level of 'cytotoxicity' of LDL treated by ultraviolet in the presence of antioxidants (protected ultraviolet-treated LDL) was well correlated with their content in lipid peroxidation markers. Therefore these markers can be useful for predicting the 'cytotoxicity' of oxidized LDL and subsequently the protective effect of the tested antioxidants. The 'cytotoxicity' of unprotected ultraviolet-treated LDL (i.e. LDL irradiated by ultraviolet in the absence of exogenous antioxidant) can be effectively blocked by preincubation of the cells with antioxidants. Catechin (10 mumol/l) and vitamin E (100 mumol/l) are very effective cytoprotective agents, whereas probucol (up to 50 mumol/l) was completely ineffective under these experimental conditions. The cytoprotective effect of vitamin E was associated to a complete inhibition of the cellular TBARS formation induced by ultraviolet-treated LDL, whereas the cytoprotective effect of catechin was relatively independent on the TBARS inhibition. All these results showed that: (1) probucol (25 mumol/l) is very effective to prevent lipid peroxidation of LDL and their subsequent 'cytotoxicity', but it cannot protect cells against the 'cytotoxicity' of previously oxidized LDL; (2) vitamin E (100 mumol/l) prevents poorly the ultraviolet-induced lipid peroxidation of LDL, but is able to block simultaneously the cellular oxidative stress and the 'cytotoxicity' induced by previously oxidized LDL; and (3) catechin (10 mumol/l) exhibited two types of protective effects: it inhibits the lipid peroxidation of LDL (and their subsequent 'cytotoxicity') and very effectively protects the cells against 'toxicity' of previously oxidized LDL (with only little inhibition of the cellular oxidative stress).(ABSTRACT TRUNCATED AT 400 WORDS)