Newly pressed extra-virgin olive oil contains oleocanthal--a compound whose pungency induces a strong stinging sensation in the throat, not unlike that caused by solutions of the non-steroidal anti-inflammatory drug ibuprofen. We show here that this similar perception seems to be an indicator of a shared pharmacological activity, with oleocanthal acting as a natural anti-inflammatory compound that has a potency and profile strikingly similar to that of ibuprofen. Although structurally dissimilar, both these molecules inhibit the same cyclooxygenase enzymes in the prostaglandin-biosynthesis pathway.

Our purpose was to determine whether the action of oxidative free radicals released by endothelial cells and vascular smooth muscle cells grown in culture could be responsible for certain modifications to low density lipoprotein (LDL). In these experiments we showed that after a 48-hour incubation with human umbilical vein endothelial cells or bovine aortic smooth muscle cells, human LDL: 1) became oxidized, as evidenced by reactivity to thiobarbituric acid; 2) lost variable amounts of sterol relative to protein (up to 20%); 3) had an increased relative electrophoretic mobility (by 30% to 70%); and 4) became toxic to proliferating fibroblasts. None of these changes occurred after a 48-hour incubation with confluent fibroblasts or bovine aortic endo- S everal investigators have reported that low density lipoprotein (LDL) is toxic to cultured vascular cells under certain conditions. 1 " 3 Our more recent results indicated that toxic LDL is formed by oxidation of a lipid component of the lipoprotein during its isolation from plasma if antioxidants are insufficient. 4 We have also demonstrated that this oxidation occurs by a free radical mechanism that involves superoxide anion and/or hydrogen peroxide. 5 Although oxygen-free radicals such as these have been implicated as effectors of tissue damage, 6 ' 7 the toxic action of oxidized LDL is effected by an oxidized lipid producad by free radicals rather than by free radicals generated during propagation of the oxidation reaction. 5 From Henriksen, et al. 8 " 10 have reported that cultured human umbilical vein endothelial cells (EC) and vascular smooth muscle cells (SMC), but not human fibroblasts or bovine aortic EC, can modify LDL. The changes in LDL produced by incubation with human umbilical vein EC and SMC include increased anodic electrophoretic mobility, a reduced ratio of total cholesterol to protein, and increased degradation by macrophages. 8 " 10 Bowman et al. 11 have reported that pulmonary artery EC, but not lung fibroblasts, produce superoxide anion in vitro. Since superoxide anion appeared to be involved in spontaneous oxidation of LDL during its isolation, 5 we chose to determine whether cultured EC and SMC could oxidize LDL and render it cytotoxic and to identify the relationship between oxidation and certain other EC modifications of LDL, including the changes in electrophoretic mobility and the decreased steroi content reported by Henriksen et al. Methods Cell PreparationThe methods for preparation of human umbilical vein EC, bovine aortic EC, and bovine aortic SMC have been described by DiCorleto and BowenPope. 12 Briefly, primary cultures of bovine aortic EC were isolated by previously used methods in 5% bovine cell-free plasma-derived serum, 13 which was 357 by guest on

Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte-derived free radicals. This is the first demonstration that short-lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.

The results of this study Indicate that when human VLDL or LDL Is prepared under conditions allowing oxidation, such oxidation renders the molecular complexes highly toxic to human skin flbroblasta growing In culture. The cytotoxicity can be predicted by assaying for the presence of thlobarbiturlc acld-reactlng substances on the lipoprotein. However, malondialdehyde, which reacts with thiobarblturlc acid and Is known to be Injurious to cells, was not cytotoxic In the same experimental system when dissolved In culture medium or covalently bound to non-toxic LDL. The toxic agent(s) on oxidized LDL Is(are) located In a llpld-extractable moiety. Since lipld peroxides and oxidized sterols can occur in vivo under various pathological conditions, the cytotoxicity of these llpoprotein-assoclated substances observed In vitro may be related to certain manifestations of these conditions. Under specific conditions, very low density lipoprotein (VLDL) has also been observed to be toxic to Received November 24, 1982; accepted February 14, 1983. cultured cells. Chan and Pollard 67 showed that VLDL fractions from rats in the late stage of pregnancy are toxic to tumorigenic and nontransformed cells. Gianturco et al. 8 showed decreased viability of bovine aortic endothelial cells exposed to VLDL from hypertriacylglycerolemic humans, and Arbogast et al. 9 found that porcine aortic endothelial cells died when exposed to VLDL from diabetic rat serum.In addition to the above reports of lipoprotein-related cell injury and death, there are a number of studies describing the inhibition of proliferation of various stimulated leukocyte populations by lipoproteins, principally VLDL and LDL. 10 " 14 Among these is the study by Schuh et al. 12 who found that LDL was inhibitory in stimulated lymphocyte populations only when there was evidence of LDL "autoxidation."The reports mentioned in the above paragraphs are evidence of a growing range of interests in the injurious effects of lipoproteins. Each of the above papers alludes to potential in vivo pathophysiological roles for these phenomena, since the lipoprotein concentrations involved are well within physiological limits.Our preliminary data 15 indicated that the cytotoxic phenomenon may be related to lipoprotein oxidation. The results of the present study indicate conclusively that oxidation renders LDL highly toxic to cultured skin fibroblasts in a concentration-dependent fashion. Our results also demonstrate that the toxic substance resides in the chloroform-methanol extractable fraction of the lipoprotein. Our measure of lipoprotein oxidation is in terms of the equivalents of

In presence of oleate and taurocholate, differentiated CaCo-2 cell monolayers on membranes were able to assemble and secrete chylomicrons. Under these conditions, both cellular uptake and secretion into chylomicrons of ␤ -carotene ( ␤ -C) were curvilinear, time-dependent (2-16 h), saturable, and concentration-dependent (apparent K m of 7-10 M) processes. Under linear concentration conditions at 16 h incubation, the extent of absorption of all-trans ␤ -C was 11% (80% in chylomicrons), while those of 9-cis-and 13-cis-␤ -C were significantly lower (2-3%). The preferential uptake of the all-trans isomer was also shown in hepatic stellate HSC-T6 cells and in a cell-free system from rat liver (microsomes), but not in endothelial EAHY cells or U937 monocyte-macrophages. Moreover, extents of absorption of ␣ -carotene ( ␣ -C), lutein (LUT), and lycopene (LYC) in CaCo-2 cells were 10%, 7%, and 2.5%, respectively. Marked carotenoid interactions were observed between LYC/ ␤ -C and ␤ -C/ ␣ -C. The present results indicate that ␤ -C conformation plays a major role in its intestinal absorption and that cis isomer discrimination is at the levels of cellular uptake and incorporation into chylomicrons. Moreover, the kinetics of cellular uptake and secretion of ␤ -C, the inhibition of the intestinal absorption of one carotenoid by another, and the cellular specificity of isomer discrimination all suggest that carotenoid uptake by intestinal cells is a facilitated process. -During, A., M. M. Hussain, D. W. Morel, and E. H. Harrison. Carotenoid uptake and secretion by CaCo-2 cells: ␤ -carotene isomer selectivity and carotenoid interactions. J.

Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2 Ϯ 0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL 3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 M, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 M), with increasing cholate concentration there was an increase in the hydrolysis of LDL-and HDL 3 -CE; at 100 M cholate, the percent hydrolysis per hour was 32 Ϯ 2 and 1.6 Ϯ 0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL 3 -TG hydrolysis was also observed, but was only ‫ف‬ 5-10% of that for HDL 3 -CE at either 10 or 100 M cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content.

Modification of low density lipoprotein (LDL) by free radical oidat renders this molecular complex cytotoxic. Oxidized lipoproteins exist in vivo In atherosclerotic lesions and in the plasma of diabetic i , suggesting that Upoprotein-induced tissue damage may occur in cen diseases. We undertook purifiation and Identicati of the major cytotoxin In oxidized LDL. The lipid extract from oxidized LDL was subjected to multiple HPLC separations, and the fractions were assayed for cytotoxicity. Mass spectrometry and nuclear magnetic resonance Identified the purified toxin as 7p-hydroperoxycholest-5-en-3I3-ol Vascular cells are susceptible to the toxic effects of low density lipoprotein (LDL) or very low density lipoprotein (VLDL) after modification of the lipoproteins by free radical oxidation (1-4). Oxidation of LDL leads to cytotoxin formation whether oxidation is mediated by lipoxygenases (5), metal ions (6), or ultraviolet irradiation (7) in cell-free systems or by the action of free radicals from cultured endothelial cells (8), vascular smooth muscle cells (8), neutrophils (9), or stimulated human monocytes (10). The cytotoxic moiety of oxidized LDL (oxLDL) is extractable with organic solvents (4), and numerous candidate substances could be proposed to explain its toxic action (11).Oxidized lipoproteins occur in vivo in vascular lesions (12)(13)(14) and in plasma of certain diabetic subjects (15). OxLDL has been proposed to play a causal role in atherosclerosis (11,(16)(17)(18) is an important step toward testing evolving theories of atherogenesis that include lipoprotein oxidation (11,(16)(17)(18) and vascular injury (20,21) as putative early events.by dialysis against 2-6 /uM cupric sulfate for various times (23). OxLDL preparations were dialyzed against 0.15 M NaCl/0.5 mM EDTA, pH 8.5, to remove cupric ions. Relative oxidation, measured as thiobarbituric acid reactivity (6,24), was equivalent to 4-6 nmol of malondialdehyde (MDA) per mg of LDL cholesterol.Human foreskin fibroblasts were plated in a 1:1 (vol/vol) mixture of Dulbecco's minimal essential medium and Ham's F-12 medium (DME/F-12) supplemented with 5% (vol/vol) fetal bovine serum (6) Aliquots of5 mg ofnative LDL or oxLDL were lyophilized overnight, and the lipid was extracted with 5 ml of acetone. The mixture was sonicated for 10-20 sec, mixed for 1 min, and allowed to stand for 20 min. After centrifugation at 1000x g for 10-20 min, the residue was reextracted twice, as above. Preliminary experiments revealed that the recovery of the cytotoxic activity by this extraction procedure was equivalent or superior to other means, including chloroform/ methanol extraction ofaqueous lipoprotein solutions. Pooled extracts were dried under nitrogen and redissolved in isopropanol/acetonitrile, 1:1 (vol/vol). After centrifugation for 5 min at 1000 x g, the supernatant was analyzed by reversephase HPLC (Waters 1LBondapak C1s preparative column).The solvent gradient for elution consisted of water/ acetonitrile, 1:1 (vol/vol), which was increased over 5 ...

Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (greater than 30,000 Daltons) and in a lipoprotein-free, low molecular weight fraction (less than 30,000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury.