Search

When Congress enacted Section 230 of the Communications Decency Act, it made an implicit deal with every Interactive Computer Service (ICS): at least attempt to clean your website of defamatory or otherwise illegal third-party content in exchange for immunity from vicarious liability. However, the majority of courts applying Section 230 have since construed this aptly-titled "good Samaritan" law as a grant of blanket ICS immunity, offering protection regardless of whether an ICS actually regulates or edits its website. This piece analyzes an aparent split among the circuit courts, and explains that blanket ICS immunity does not square with Congress' underlying intent of encouraging ICS self-regulation. In the end, this article highlights four potential scenarios in which an ICS could lose its Section 230 "good Samaritan" immunity status when it does not act like a "good Samaritan."

Electronegative low density lipoprotein (LDL−) formation that structurally resembles LDL− isolated from plasma was evaluated after LDL treatment with snake venom phospholipase A2 (PLA2). PLA2 treatment of LDL increased its electrophoretic mobility in proportion to the amount of LDL− formed without evidence of lipid peroxidation. These changes dose-dependently correlated with the degree of phospholipid hydrolysis. Strong immunoreactivity of LDL− subfraction from plasma and PLA2-treated LDL (PLA2-LDL) to amyloid oligomer-specific antibody was observed. Higher β-strand structural content and unfolding proportionate to the loss of α-helical structure of apolipoprotein B-100 (apoB-100) of LDL− isolated from both native and PLA2-LDLs was demonstrated by circular dichroism (CD) spectropolarimetry. These structural changes resembled the characteristics of some oxidatively modified LDLs and soluble oligomeric aggregates of amyloidogenic proteins. PLA2-LDL was also more susceptible to nitration by peroxynitrite, likely because of exposure of otherwise inaccessible hydrophilic and hydrophobic domains arising from apoB-100 unfolding. This was also demonstrated for plasma LDL−. In contrast, PLA2-LDL was more resistant to copper-mediated oxidation that was reversed upon the addition of small amounts of unsaturated fatty acids.The observed similarities between PLA2-LDL−-derived LDL− and plasma LDL− implicate a role for secretory PLA2 in producing modified LDL− that is facilitated by unfolding of apoB-100.

The objective of the present study was to identify disturbances of nitric oxide radical (·NO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations of·NO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 ± 9.7 years; blood pressure, 148.3 ± 24.8/90.8 ± 10.2 mmHg) and in 11 healthy subjects (N: 48.4 ± 7.0 years; blood pressure, 119.4 ± 9.4/75.0 ± 8.0 mmHg).Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1% increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 ± 26.0, N: 54.2 ± 24.9 µM), urate (H: 108.5 ± 18.9, N: 156.4 ± 26.3 µM), ß-carotene (H: 1.1 ± 0.8, N: 2.5 ± 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 ± 0.2, N: 0.7 ± 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3ß,5,6ß-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 ± 0.2, N: 0.7 ± 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for ·NO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although ·NO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides.

Oxidized LDL (OxLDL) is composed of many potentially proatherogenic molecules, including oxysterols. Of the oxysterols, 7-ketocholesterol (7-KC) is found in relatively large abundance in OxLDL, as well as in atherosclerotic plaque and foam cells in vivo. Although there is evidence that 7-KC activates endothelial cells, its effect on monocytes is unknown. We tested the hypothesis that 7-KC may induce monocyte differentiation and promote foam cell formation. THP-1 cells were used as a monocyte model system and were treated with 7-KC over a range of concentrations from 0.5 to 10 μg/ml. Changes in cell adhesion properties, cell morphology, and expression of antigens characteristic of differentiated macrophages were monitored over a 7-day period. 7-KC promoted cells to firmly adhere and display morphologic features of differentiated macrophages; this effect was time and dose dependent and was markedly more potent than cholesterol treatment (45% of cells became adherent after 7 days of treatment with 7-KC at 10 μg/ml vs. less then 5% for control cells, P < 0.01). Similar effects were obtained when LDL enriched with 7-KC or OxLDL were added to THP-1 cells. 7-KC-differentiated cells expressed CD11b, CD36, and CD68, phagocytized latex beads, and formed lipid-laden foam cells after exposure to acetylated LDL or OxLDL. In contrast to 7-KC, oxysterols with known cell regulatory effects such as 25-hydroxycholesterol, 7β-hydroxycholesterol, and (22R)-hydroxycholesterol did not effectively promote THP-1 differentiation. In conclusion, these results demonstrate for the first time that 7-KC, a prominent oxysterol formed in OxLDL by peroxidation of cholesterol, may play an important role in promoting monocyte differentiation and foam cell formation. These studies also suggest that 7-KC induces monocyte differentiation through a sterol-mediated regulatory pathway that remains to be characterized. —Hayden, J. M., L. Brachova, K. Higgins, L. Obermiller, A. Sevanian, S. Khandrika, and P. D. Reaven. Induction of monocyte differentiation and foam cell formation in vitro by 7-ketocholesterol. J. Lipid Res. 2002. 43: 26–35.

Oxidized LDL (oxLDL) produced a rapid depletion of intracellular glutathione (GSH) followed by an adaptive increase in J774 A.1 macrophages. OxLDL also induced a transient increase in the levels of γ-glutamylcysteine synthetase heavy subunit (γ-GCS-HS), representing the catalytic subunit of the rate-limiting enzyme for de novo GSH synthesis. The induction took place within 3 h, with maximum levels observed by 10 h of treatment. Pretreatment of oxLDL with ebselen inhibited GSH depletion and attenuated the γ-GCS-HS induction. OxLDL-associated lipid hydroperoxides and their decomposition product aldehydes are two major components thought to account for GSH depletion in macrophages. Ebselen pretreatment had only a minor effect on malondialdehyde levels, whereas peroxide content was essentially abolished, suggesting that oxLDL-associated hydroperoxides may mediate both GSH depletion and γ-GCS-HS induction. Acetylated LDL (AcLDL) also caused a moderate induction of γ-GCS-HS protein along with a 30% transient increase in GSH by 3–6 h, suggesting a minor involvement of scavenger receptor-mediated signaling of GSH synthesis. However, the level of γ-GCS induction by AcLDL was insufficient to cause a sustained increase in GSH. Macrophages with higher glutathione peroxidase (GPx) activity experienced a more rapid and extensive depletion of GSH when treated with oxLDL under similar conditions, along with greater resistance to oxLDL- or peroxide-induced cytotoxicity.We conclude that oxLDL-associated peroxides are primarily responsible for GSH depletion, creating an oxidizing environment required for γ-GCS induction and compensatory GSH synthesis. This is facilitated in cells expressing high GPx activity through a rapid depletion of GSH in the face of a peroxide challenge.

Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC. —Navab, M., S. Y. Hama, G. M. Anantharamaiah, K. Hassan, G. P. Hough, A. D. Watson, S. T. Reddy, A. Sevanian, G. C. Fonarow, and A. M. Fogelman. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3. J. Lipid Res. 2000. 41: 1495–1508.

A subclass of LDL described on the basis of its greater electronegativity and oxidative status is further characterized using a new, highly sensitive single photon counting technique to measure lipid hydroperoxides. We describe in this report that these particles, which we refer to as LDL-, are enriched in lipid peroxides and other peroxidation products as compared to the bulk of the unmodified, normal LDL (nLDL) recovered from human plasma. This chemiluminescence-based, single photon counting technique has unique advantages in that analyses are performed on whole LDL, thus avoiding artifactual lipid peroxidation during lipid extraction. Evidence for increased amounts of lipid hydroperoxides in LDL- versus nLDL are in agreement with other analytical methods such as measurement of conjugated dienes as well as cholesterol oxidation products. LDL- also has lower proportions of polyunsaturated fatty acids than nLDL. Analysis of the amino acid composition of apoB-100 and fatty acid composition of total LDL lipids also revealed major differences between nLDL and LDL- consistent with an oxidative modification of the latter. Thus, LDL- has significantly lower proportions of the oxidizable amino acids histidine and lysine, and marked differences in other neutral and acidic amino acids. The deficit in specific amino acids is in agreement with a reduced TNBS reactivity and increased relative electrophoretic mobility of LDL-. We postulate that LDL- is a major carrier of lipid hydroperoxides associated with plasma LDL and may arise from oxidative events in the vasculature and/ or by ingestion of peroxide-enriched meals.

Planar oriented membranes of 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) containing cholesterol, 19-hydroxycholesterol, 22S-hydroxycholesterol, or 25-hydroxycholesterol in concentrations up to 5 mol % were investigated with angle-resolved fluorescence depolarization and electron spin resonance measurements. Analyses of the data with the Brownian diffusion model show that the oxysterols have structural effects similar to those of cholesterol: an increase in molecular order and no change in the rotational diffusion coefficients of the probe molecules. Time-resolved fluorescence anisotropy measurements on diphenylhexatriene (DPH) in small unilamellar vesicles of POPC and DOPC were performed using oxysterols commonly found in oxidized low density lipoproteins (LDL) in comparison to membranes containing cholesterol or no sterols. Analyses using the Brownian rotational diffusion model show that most LDL-oxysterols affect the vesicle physical structure in a manner similar to cholesterol, viz. an increase in molecular order and a decrease in the dynamics. Cholesterol-alpha-epoxide has a much smaller ordering effect than cholesterol in POPC-vesicles. A similar effect was found for 7 beta-hydroxycholesterol in DOPC-vesicles. The tendency of the oxysterols to influence the molecular order as compared to pure cholesterol may contribute to cell membrane permeability changes affecting crucial cell functions and events leading to vascular cell injury. Increased LDL oxysterol levels may account for some of the structural changes noted for oxidatively modified LDL as well as its toxicity to vascular cells.

The present study describes the toxicity of oxidized LDL towards rabbit aortic endothelial cells in terms of its lipid components with specific attention to the cholesterol oxidation products (ChOx) found in oxidized LDL isolated from human plasma. Measurements of the major ChOx associated with freshly isolated unmodified LDL, those found in oxidized LDL isolated from human plasma and LDL subjected to oxidation in vitro are described. We have confirmed previous findings that most of the cytotoxicity of freshly isolated human LDL may be attributable to a minor fraction that appears to be oxidatively modified by several criteria. Moreover, this plasma-derived oxidized LDL (referred to as LDL) is highly enriched in ChOx, whereas the content of lipid peroxides or derived products (measured as conjugated dienes and thiobarbituric acid reacting products) are much lower, particularly when compared to copper-induced LDL oxidation. Much of the ChOx found in plasma are associated with LDL, however, the levels and proportions of the various ChOx found in LDL differ from those produced after extensive copper-induced oxidation but resemble those produced after moderate oxidation with copper. The species and concentrations of ChOx found in LDL when applied as a mixture exhibit considerably more toxicity than any individual ChOx alone. At non-toxic levels this ChOx mixture causes an increased influx of several ions, including calcium, an effect not seen with individual ChOx at comparable doses. Perturbations in ionic homeostasis, and particularly the sustained increase in intracellular calcium concentrations, are associated with much of the cytotoxicity, an effect attributable to the membrane disruptive action of ChOx leading to altered ion transporter activity. The effect of the ChOx mixture (but not any individual ChOx) on sodium and potassium flux appears to be due to enhanced Na+/K(+)-ATPase activity based on the complete inhibition produced by ouabain under all treatment conditions. These findings also show that the levels of cholesterol oxidation products found in normal LDL are not cytotoxic whereas those present in oxidized LDL exceed the toxic threshold for endothelial cells and account for most of the cytotoxicity produced by this modified lipoprotein.

Using ion exchange high pressure liquid chromatography, total plasma low density lipoprotein (LDL) from 30 hypercholesterolemic and 10 normocholesterolemic cynomolgus monkeys was subfractionated into unmodified LDL (n-LDL) and more negatively charged LDL (LDL-). In hypercholesterolemic monkeys, the absolute LDL-cholesterol level was 16.54 +/- 2.82 mg/dl (mean +/- SE) whereas in normocholesterolemic monkeys it was 2.39 +/- 0.12 mg/dl (P < 0.0001); the percentage of LDL- was 5.2 +/- 0.71% and 4.9 +/- 0.19% of the total LDL for hypercholesterolemic versus normocholesterolemic monkeys, respectively. LDL- averaged 5% and n-LDL 95% of the total plasma LDL cholesterol. To confirm and further elucidate the oxidative nature of LDL-, cholesterol and cholesterol oxide contents of LDL- and n-LDL were determined by capillary gas chromatography; 53.98 +/- 2.24% (mean +/- SE) of the LDL- cholesterol was oxidized whereas in n-LDL only 10.70 +/- 1.06% of the cholesterol was oxidized (P < 0.00001). The spectrum of oxysterols identified, which was similar for LDL- and n-LDL, suggested a free radical-mediated process for cholesterol oxidation. The principal oxysterols identified were: cholest-5-ene-3 beta, 7 alpha-diol, cholesta-3,5-diene-7-one, cholest-5-ene-3 beta, 7 beta-diol, 5,6 beta-epoxy-5 beta-cholestan-3 beta-ol, 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol, 5 alpha-cholestan-3 beta,5,6 beta-triol, 3 beta-hydroxycholest-5-ene-7-one, and cholest-5-ene-3 beta,25-diol. To model one of the steps in the possible mechanism of atherogenesis, the cytotoxicity of LDL- was demonstrated to be greater against subconfluent than confluent aortic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidized L-[alpha]-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoryl-choline (OxPAPC), a component of minimally modified LDL, induces production of proinflammatory cytokines and development of atherosclerotic lesions. We tested the hypothesis that OxPAPC alters expression, phosphorylation, and localization of tight junction (TJ) proteins, particularly occludin, a transmembrane TJ protein. OxPAPC reduced total occludin protein and increased occludin phosphorylation dose dependently (10-50 [micro]g/ml) and time dependently in bovine aortic endothelial cells. OxPAPC decreased occludin mRNA and reduced the immunoreactivity of zonula occludens-1 at the cell-cell contacts. Furthermore, OxPAPC increased the diffusive flux of 10-kDa dextran in a dose-dependent manner. [O.sup.-.sub.2]. production by bovine aortic endothelial cells increased nearly twofold after exposure to OxPAPC. Also, enzymatic generation of Of. by xanthine oxidase-lumazine and [H.sub.2][O.sub.2] by glucose oxidase-glucose increased occludin phosphorylation, implicating reactive oxygen species as modulators of the OxPAPC effects on occludin phosphorylation. Superoxide dismutase and/or catalase blocked the effects of OxPAPC on occludin protein content and phosphorylation, occludin mRNA, zonula occludens-1 immunoreactivity, and diffusive flux of 10-kDa dextran. These findings suggest that changes in TJ proteins are potential mechanisms by which OxPAPC compromises the barrier properties of the vascular endothelium. OxPAPC-induced disruption of TJs, which likely facilitates transmigration of LDL and inflammatory cells into the subendothelial layers, may be mediated by reactive oxygen species. oxidized L-[alpha]-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoryl-choline; reactive oxygen species

Nutritional, or dietary oxidative stress denotes a disturbance of the redox state resulting from excess oxidative load or from inadequate nutrient supply favoring prooxidant reactions. Low intake or impaired availability of dietary antioxidants including vitamins E and C, carotenoids, polyphenols, and other micronutrients (e.g., selenium) weakens the antioxidant network. Postprandial oxidative stress, as a subform of nutritional oxidative stress, ensues from sustained postprandial hyperlipidemia and/or hyperglycemia and is associated with a higher risk for atherosclerosis, diabetes, and obesity. In Western societies, a significant part of the day is spent in the postprandial state. Unsaturated fatty acids incorporated into LDL and oxidized LDL are an atherogenic factor. Lipid hydroperoxides present in the diet are absorbed, contributing to the prooxidant load. In hyperlipidemic and hyperglycemic subjects, endothelium-dependent vasodilation is impaired in the postprandial state, making postprandial oxidative stress an important factor modulating cardiovascular risk. Postprandial oxidative stress is attenuated when dietary antioxidants are supplied together with a meal rich in oxidized or oxidizable lipids. Ingestion of dietary polyphenols, e.g., from wine, cocoa, or tea, improves endothelial dysfunction and lowers the susceptibility of LDL lipids to oxidation. Polyphenols affect endothelial function not solely as antioxidants but also as modulatory signaling molecules. KEY WORDS: * antioxidants * diet * flavonoids * nutritional oxidative stress

The isomeric cholesterol-5,6-epoxides represent two common cholesterol autoxidation products and along with their principal metabolic product, 3 beta,5 alpha,6 beta-cholestane triol, are purportedly angiotoxic. The uptake and cytotoxic action of these compounds was examined in cultured rabbit aortic endothelial cells emphasizing mechanisms of uptake and metabolic fate. The isomeric cholesterol epoxides are incorporated with equal facility and in a dose-dependent manner. The pattern of uptake, which is markedly influenced by media serum concentration and by temperature, suggests that these compounds are partly incorporated through association with serum lipoproteins. After incorporation, both epoxide isomers are rapidly converted to cholestane triol which readily exits the cells. Cholestane triol is further metabolized to an ester-type product representing up to 10% of the added cholesterol epoxides by 24 h of incubation. The order of cytotoxic potency of these cholesterol oxides is: cholestane triol greater than cholesterol-beta-epoxide greater than cholesterol-alpha-epoxide, with LD50 concentrations ranging from 23 to greater than 150 microM in confluent cells. Cholestane triol and cholesterol-beta-epoxide are twice as cytotoxic to subconfluent cells as compared to confluent cells, whereas cholesterol-alpha-epoxide is essentially equitoxic to confluent and subconfluent cells. Cholesterol epoxide cytotoxicity is significantly reduced by treatments in the absence of serum in accord with substantial reduction in uptake when incubations are performed in serum-free media. Our findings show that these cytotoxic cholesterol oxides are incorporated by endothelial cells through a combination of receptor-mediated and nonspecific or passive mechanisms; however, the efficacy of uptake and resulting toxicity is substantially influenced by serum lipoproteins.

Hormone replacement therapy does not slow the progression of existing coronary artery disease, according to a study of 226 postmenopausal women. This is true of both estrogen-only therapy and estrogen-progestin therapy. This study actually compared the coronary angiograms of women who took hormone replacement therapy and those who did not. There was no significant difference between the two groups during an average follow-up of 3.3 years.

A study of the role played by polyphenols in human nutrition finds that their redox chemistry accounts for their observed antioxidant effect and thus for their prevention of atherosclerosis. Optimal nutrition should include a 'recommended optimal intake' of polyphenols and plasma antioxidant capacity is suggested as a suitable index for such intake.

Multifrequency phase fluorometry was used to determine the lifetime distributions of 1,6 diphenyl-1,3,5-hexatriene in 1-palmitoyl-2 linoleoyl phosphatidylcholine small unilamellar vesicles containing 2% incorporation of phospholipid hydroperoxides. A biexponential decay was observed in both vesicle preparations over a temperature range of 5 to 35 degrees C. Vesicles containing phospholipid hydroperoxides showed an overall longer lifetime as well as a greater distribution in width. These findings suggest that phospholipid hydroperoxides create structural heterogeneity in membrane structure.

Supplementary doses of vitamin E may slow the progression of coronary artery disease. A total of 156 men between the ages of 40 to 49 who previously had undergone coronary bypass surgery and had participated in the Cholesterol Lowering Atherosclerosis Study were enrolled. The men each received three kinds of treatment: a high dose or a low dose of vitamin E; a high dose or a low dose of vitamin C; and, either drug therapy or a placebo as a control. Coronary angiography was used to measure narrowing of vessel diameter which indicates the progression of the disease.The men who took high doses vitamin E a day showed reduced progress toward coronary artery disease compared to patients who took the lower dose. No benefit was found for patients in the study who took supplementary vitamin C., Objective. - To explore the association of supplementary and dietary vitamin E and C intake with the progression of coronary artery disease. Design. - A subgroup analysis of the on-trial antioxidant vitamin intake database acquired in the Cholesterol Lowering Atherosclerosis Study, a randomized, placebo-controlled, serial angiographic clinical trial evaluating the risk and benefit of colestipol-niacin on coronary artery disease progression. Setting. - Community-and university-based cardiac catheterization laboratories. Subjects. - A total of 156 men aged 40 to 59 years with previous coronary artery bypass graft surgery. Intervention. - Supplementary and dietary vitamin E and C intake (nonrandomized) in association with cholesterol-lowering diet and either colestipol-niacin or placebo (randomized). Outcome. - Change per subject in the percentage of vessel diameter obstructed because of stenosis (%S) determined by quantitative coronary angiography after 2 years of randomized therapy on all lesions, mild/moderate lesions (

The effect of phospholipid hydroperoxides and alcohols on membrane order and dynamics was studied by using membrane spin label 3-doxyl-5-alpha-cholestane for the angle-resolved electron spin resonance measurements and fluorescent probe 1,6-diphenyl-1,3,5-hexatriene for the angle-resolved fluorescence depolarization measurements. The effect on membrane reorientation dynamics is negligible. However, small amounts of oxidatively modified phospholipids within the phosphatidylcholine or dilinoleoylphosphatidylcholine membranes have significant structural effects as the molecular orientation order of the probes is strongly decreased.