Different patterns of IL-1β secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7α-, 7β-hydroxycholesterol, or 7-ketocholesterol

Among oxysterols oxidized at C7 (7α-, 7β-hydroxycholesterol, and 7-ketocholesterol), 7β-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-1β and/or tumor necrosis factor (TNF)-α secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7β-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1β secretion. TNF-α secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the α- or β-hydroxyl radical position plays a key role in apoptosis and IL-1β secretion.     

The role of oxysterols in control of endothelial stiffness

Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE^−/− mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.     

Relevance and mechanism of oxysterol stereospecifity in coronary artery disease

Cholesterol oxidation products (oxysterols) are markers for in vitro LDL oxidation. They are potent inducers of programmed cell death and are also found in high concentrations inside atherosclerotic lesions. Among physiologically occurring oxysterols, 7beta-OH-cholesterol suggests an increase of lipid peroxidation in vivo. In the underlying study, we quantified free plasma oxysterols by means of gas chromatography in patients with stable coronary artery disease (CAD). Total free plasma oxysterols were elevated more than 2-fold in patients with stable CAD (233 +/- 49 vs 108 +/- 19 ng/ml, n = 22, P < 0.05) compared to a control group (n = 20) with similar atherogenic risk profile and angiographically normal coronary arteries. We found that 7-ketocholesterol, as well as the beta-isomers of epoxide (25.7 +/- 10.0 vs 7.3 +/- 1.4 ng/ml, P = 0.07) and 7beta-OH-cholesterol (65.1 +/- 15.7 vs 19.4 +/- 8.9 ng/ml, P < 0.01), was mainly responsible for this increase. To elucidate a potential relevance of oxysterol stereospecificity in regard to endothelial damage, we further conducted in vitro experiments using human arterial endothelial cells (HAECs). Surprisingly, beta-isomers exerted an up to 10-fold higher amount of cell death in equivalent doses when compared to alpha-isomers. The greater cytotoxic potential of beta-isomers was due to increased apoptosis, preceded by mitochondrial release of cytochrome c with subsequent caspase-3 activation. Stereospecific release of cytochrome c depended on the presence of an intact cytoplasmic membrane, hinting at the existence of a putative oxysterol receptor or a direct stereospecific effect on membrane biology. Finally, both isoforms of oxysterols directly released cytochrome c only in conjunction with protein containing cytosol and endoplasmatic reticulum. Free plasma oxysterol levels, particularly 7-ketocholesterol, beta-epoxide and 7beta-OH-cholesterol, are elevated in patients with stable CAD, independent of their LDL cholesterol levels. Due to the highly increased cytotoxicity of oxysterol beta-isomers in vitro, they may represent important atherogenic risk factors.     

Comparison of the effects of O2*-/HO* free radical- and copper ions-oxidized LDL or lipoprotein(a) on the endothelial cell releases of tissue plasminogen activator and plasminogen activator inhibitor-1.

We investigated the effects of low density-lipoproteins (LDL) and lipoprotein(a) [Lp(a)] oxidized by O2*-/HO* free radicals generated by gamma radiolysis of water, on the release of tissue Plasminogen Activator (tPA) and of its main inhibitor Plaminogen Activator Inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC). These effects were compared to those of lipoproteins issued from the same preparations but oxidized by the classical copper ions procedure. The results showed that O2*-/HO* free radical oxidized LDL and Lp(a) led to a dramatic decrease of PAI-1 release but did not affect tPA release, whereas copper oxidation of lipoproteins resulted in an increase in PAI-1 release and a decrease in tPA release. Chemical analysis revealed that O2*-/HO* free radical oxidized lipoproteins exhibited very much lower levels of phosphatidylcholine hydroperoxides, lysophosphatidylcholine and oxysterols (7-ketocholesterol, 7beta-hydroxycholesterol, 5,6beta-epoxycholesterol) than copper oxidized LDL. Thus, the discordant effects of O2*-/HO* oxidized and copper oxidized LDL and Lp(a) on the endothelial releases of PAI-1 and tPA appeared to be due to qualitatively and/or quantitatively different formation of oxidized components by the two oxidation processes.     

In vivo interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol, potential surrogate markers for oxidative stress

The oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol are cholesterol autoxidation products. These two oxysterols are formed as a result of low density lipoprotein oxidation and in a study on biomarkers for oxidative stress in patients with atherosclerosis, 7beta-hydroxycholesterol was found to be the strongest predictor of progression of carotid atherosclerosis. Interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in vitro has been reported recently, using recombinant 11beta-hydroxysteroid dehydrogenase or rodent liver microsomes. In this study deuterium-labeled 7beta-hydroxycholesterol or 7-ketocholesterol was administered intravenously to two healthy volunteers and blood samples were collected at different time points. The mean half-life for elimination of 7beta-hydroxycholesterol from the circulation was estimated to be 1.9 h. The corresponding half-life for 7-ketocholesterol was estimated to be 1.5 h. Infusion of deuterium-labeled 7-ketocholesterol resulted in labeling of 7beta-hydroxycholesterol and vice versa. In addition, the biological within-day and between-day variations of the two oxysterols were determined. In summary, the present investigation clearly shows an interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in humans.     

ABCG1 mediated oxidized LDL-derived oxysterol efflux from macrophages

Objectives

The uptake of oxidized LDL (oxLDL) by macrophages is a key initial event in atherogenesis, and the removal of oxidized lipids from artery wall via reverse cholesterol transport is considered antiatherogenic. The aims of this study were to investigate the pathways mediating the removal of oxysterols from oxLDL-loaded macrophages, and the subsequent uptake of the oxysterols by hepatocytes.

Methods

LDL was labeled with [3H]cholesterol, and LDL-[3H]cholesterol was oxidized by copper using a standard method. [3H]oxysterol formation in oxLDL was analyzed by thin layer chromatography. oxLDL-[3H]sterol was incubated with macrophages, allowing the uptake of [3H]sterol by macrophages. [3H]sterol efflux from macrophages mediated by ATP binding cassette transporters (ABCA1, ABCG1), or scavenger receptor class B type I (SR-BI) was measured. The subsequent uptake of the [3H]sterol by hepatocytes was also determined.

Results

7-Ketocholesterol was the major oxysterol formed in oxLDL, and it was significantly higher in oxLDL compared with that in native LDL (naLDL). oxLDL-derived sterol efflux to HDL from macrophages was significantly increased compared with naLDL-derived sterol, and it was mainly mediated by ABCG1, but not by ABCA1 or SR-BI. Moreover, although HDL dose-dependently induced sterol efflux from macrophages, only the exported sterol by ABCG1 pathway was efficiently taken up by hepatocytes.

Conclusions

ABCG1 mediates oxysterol efflux from oxLDL-loaded macrophages, and the exported oxysterol by ABCG1 pathway can be selectively taken up by hepatocytes.     

Oxysterol mixtures, in atheroma-relevant proportions, display synergistic and proapoptotic effects

Apoptotic cells in atheroma lesions may contribute to plaque development and instability. Oxysterols constitute the major toxic component in oxLDL and are present in mixed forms in human atheroma lesions. However, the cellular effects of oxysterols have been mostly studied individually. In the present study, we investigated the cytotoxic effects of 7beta-hydroxycholesterol (7betaOH), 7-ketocholesterol (7keto), 25-hydroxycholesterol (25OH), and 27-hydroxycholesterol (27OH) on U937 monocytic cells, both individually and in atheroma-relevant mixtures mimicking the oxysterol composition reported in human atheroma lesions. Apoptosis and necrosis were studied by examining cell morphology, phosphatidylserine exposure, caspase activation, and the terminal dUTP nick end-labeling technique. Cellular reactive oxygen species and total amount of reduced thiols were measured by using fluorescence probes and 5,5'-dithiobis-(2-nitrobenzoic acid), respectively. We found that 7betaOH and 7keto induced caspase activation, ROS production, cellular thiol depletion, permeabilization of lysosomal and mitochondrial membranes, and cell death. 25OH and 27OH did not cause any of the above alterations, whereas 7betaOH and 7keto exerted synergistic toxic effects. Although single 25OH or 27OH exhibited quenching effects on both 7betaOH- and 7keto-induced cell death, the combination of all four oxysterols in atheroma-relevant proportions was proapoptotic. Our findings indicate that the major oxysterols accumulated in human atheroma are proapoptotic and may contribute to atherosclerotic lesion development.     

Ceramide generation occurring during 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis is caspase independent and is not required to trigger cell death

Biological activities of oxysterols seem tightly regulated. Therefore, the ability to induce cell death of structurally related oxysterols, such as those oxidized at C7(7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5-80 microg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7beta-hydroxycholesterol and 7-ketocholesterol, which trigger an apoptotic process characterized by the occurrence of cells with fragmented and/or condensed nuclei, and by various cellular dysfunctions: loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADP-ribose) polymerase, and increased accumulation of cellular C16 : 0 and C24 : 1 ceramide species. This ceramide generation is not attributed to caspase activation since inhibition of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 microM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7beta-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 microM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7alpha-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the alpha or beta hydroxyl radical position of oxysterols oxidized at C7 plays a key role in the induction of the apoptotic process. In addition, our findings demonstrate that 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis involve the mitochondrial signal transduction pathway and they suggest that C16 : 0 and C24 : 1 ceramide species generated through ceramide synthase play a minor role in the commitment of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death.     

Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking

Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured macrophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.     

Cytotoxic oxysterols induce caspase-independent myelin figure formation and caspase-dependent polar lipid accumulation

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7beta-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7beta-hydroxycholesterol, cholesterol-5beta, 6beta-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7beta-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.     

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.     

Trojan horse-like behavior of a biologically representative mixture of oxysterols

Oxysterols, 27-carbon atoms cholesterol oxidation products, are consistently detectable in minimally oxidized low density lipoproteins (oxLDLs) and accumulate in the core of fibrotic plaques. Several oxysterols of pathophysiological interest have been shown to possess many and diverse biochemical activities. In particular, 7-ketocholesterol (7K), a major cholesterol oxide both in oxLDLs and in atherosclerotic lesions, is able to lead vascular cells to apoptosis. Indeed, when 7K is added to cells of the macrophage lineage, in a concentration range actually detectable in hypercholesterolemic patients, a marked apoptotic effect was observed. However, when identical concentrations of 7K are given to the same cells in a mixture with other oxysterols, also detectable in human low density lipoprotein (LDL), cell apoptosis was dramatically reduced. Of note, identical amounts of unoxidized cholesterol did not show any significant pro-apoptotic effect. With the aim to investigate the mechanisms underlying the quenching of 7K-dependent apoptosis by the oxysterol mixture, we found that the combined oxysterol mixture counteracted the ability of 7K given alone to strongly increase the steady-state level of reactive oxygen species (ROS) in macrophages as well as the up-regulation of the pro-apoptotic factor p21 and the triggering of the mitochondria-dependent pathway of apoptosis. Competition among oxysterols, apparently at NADPH oxidase level, diminishes the macrophage ROS production and direct toxicity that is evoked by defined oxysterols, as for instance, 7-ketocholesterol.     

Measurement of oxysterols and alpha-tocopherol in plasma and tissue samples as indices of oxidant stress status

Oxidant stress seems to play a role in several setting of human pathology, such as atherosclerosis, cancer, and aging. The study of oxidant stress in human disease should be based on the evaluation of either sensitive and specific markers of enhanced oxidant stress, such as oxysterols, or antioxidant defense, by measuring alpha-tocopherol. We have developed a rapid method to measure the oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol in plasma (50 healthy subjects) and tissue as an index of oxidant stress in vivo, and from the same sample alpha-tocopherol content. The mean plasma concentration of 7beta-hydroxycholesterol and 7-ketocholesterol was 4.6+/-1.1 and 13.4+/-7.6 ng/mL, respectively. Plasma alpha-tocopherol concentration was 5.8+/-1.0 micromol/mol cholesterol. Samples from atherosclerotic plaques contained 20 times more cholesterol, about 45 times higher oxysterols levels, and 600 times more alpha-tocopherol compared to normal arteries. No significant difference in cholesterol and oxysterol content was observed between cirrhotic and normal liver. However, cirrhotic liver contained significantly smaller concentration of alpha-tocopherol compared to normal liver. In conclusion, we have developed a rapid and reliable method for the assay of cholesterol oxidation products and alpha-tocopherol in plasma and tissue useful for estimation of oxidant stress/antioxidant balance.     

Effects of oxidatively modified LDL on cholesterol esterification in cultured macrophages

Oxidative modification of low density lipoproteins (LDL) has been shown to cause accelerated degradation of LDL via the scavenger receptor pathway in cultured macrophages, and it has been proposed that this process might lead to cholesterol accumulation in macrophages in the arterial wall in vivo. However, oxidation of LDL is accompanied by a substantial reduction in LDL total cholesterol content and hence the amount of cholesterol delivered by oxidatively modified LDL may be less than that delivered by scavenger receptor ligands such as acetyl LDL which results in massive cholesterol accumulation in cultured macrophages. The present studies were done to determine whether the decrease in total cholesterol content during LDL oxidation was due to oxidation of cholesterol and cholesteryl ester, and to determine whether the resulting oxidized sterols could affect cholesterol esterification in cultured macrophages. It was found that when LDL prelabeled with [3H]cholesteryl linoleate was oxidized, there was a decrease in cholesterol mass but no change in radioactivity. The radioactive substances derived from cholesteryl linoleate appeared more polar than the parent compound when analyzed by reverse-phase liquid chromatography, but were not identical with free cholesterol. Thin-layer chromatography of oxidized LDL lipids confirmed the loss of esterified cholesterol, and revealed multiple new bands, some of which matched reference oxysterols including 7-ketocholesterol, 5,6-epoxycholesterol, and 7-hydroxycholesterol. In addition to oxysterols, oxidized cholesteryl esters were also present. Quantitation by gas chromatography indicated that 7-ketocholesterol was the major oxysterol present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of monocyte differentiation and foam cell formation in vitro by 7-ketocholesterol.

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 microg/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 microg/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, 7beta-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.     

Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase

The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS+nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS+nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions (()O(2)(-)) and peroxynitrite (ONOO(-)) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS+nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers. Our results suggest that reduction of functional NO in response to a combination of sbPBS+nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation.     

Rapid hepatic metabolism of 7-ketocholesterol in vivo: implications for dietary oxysterols.

7-Ketocholesterol is a major dietary oxysterol and the predominant non-enzymically formed oxysterol in human atherosclerotic plaque. We tested the hypothesis that 7-ketocholesterol is preferentially retained by tissues relative to cholesterol in vivo. To ensure rapid tissue uptake, acetylated low density lipoprotein, labeled with esters of [(14)C]-7-ketocholesterol and [(3)H]cholesterol, was injected into rats via a jugular catheter. At timed intervals (2 min to 24 h) rats (n = 48 total) were exsanguinated and tissues were dissected and assayed for radioactivity. In two experiments the majority of both radiolabels appeared in the liver after 2 min. In all tissues, (14)C appeared transiently and did not accumulate. Rather, it was metabolized in the liver and excreted into the intestine mainly as aqueous-soluble metabolites (presumably bile acids). By 9 h, (14)C in the liver had decreased to 10% of the injected dose while 36% was present in the intestine. In contrast, at 9 h 38% of (3)H was evident in the liver while only 5% was found in the intestine. Unlike [(3)H]cholesterol, little (14)C was found to re-enter the circulation, indicating that enterohepatic recycling of 7-ketocholesterol was negligible.This is the first report of the distribution of an oxysterol relative to cholesterol, administered simultaneously, in a whole animal model. The finding that [(14)C]-7-ketocholesterol is rapidly metabolized and excreted by the liver suggests that diet may not be a major source of oxysterols in atherosclerotic plaque, and that perhaps dietary oxysterols make little or no contribution to atherogenesis.     

Oxidized low density lipoprotein inhibits prostacyclin generation by rat aorta in vitro: a key role of lysolecithin.

The objective of this study was to examine the effect of oxLDL on prostacyclin (PGI2) generation by rat aortic segments and to see whether the lipid fraction of oxLDL or its components are responsible for that effect. We also tested if antioxidants have any protective role. LDL oxidized by copper was characterized by higher TBARS, conjugated diene, lysophosphatidylcholine (lyso PC), oxysterols and less polyunsaturated fatty acids (PUFA) than nLDL. Preincubation of aortas with oxLDL caused a significant inhibition of PGI2 generation compared to aortas preincubated with nLDL or buffer only. The percent inhibition was dependent on the concentration of oxLDL. Most of the inhibitory effect of oxLDL resided in its lipid moiety while the lipid fraction of nLDL, as well as native LDL had no effect. Preincubation of aortas with 10 microg/ml of 7-ketocholesterol the major oxysterol in oxLDL reduced the amount of PGI2 generated by aorta at all times tested; however that decrease did not reach a significant level. Aortas preincubated with 10 microg/ml of lyso PC showed a 21-36% inhibition of PGI2 generation which was comparable to the inhibition produced by preincubating the aortas with 50 microg protein/ml of oxLDL (containing about 7.5 microg lyso PC). This indicated that most of the inhibitory effect of oxLDL was due to its lyso PC. The small molecular weight fraction (< 10 kDa) with a high level of TBARS (TBARS solution) also significantly decreased the PGI2 generation by aorta. Addition of superoxide dismutase (SOD) + catalase or vitamin E simultaneously with oxLDL or TBARS solution in the preincubation medium did not reverse their inhibitory effects. This indicated that oxygen free radicals are not a contributing factor to the inhibitory effect of oxLDL but lyso PC and the lipid peroxides and probably other components already present within oxLDL are the important inhibitors.     

Serum-induced arachidonic acid release and prostaglandin biosynthesis are potentiated by oxygenated sterols in NRK 49F cells

Fetal calf serum is able to activate arachidonic acid release from phospholipids in NRK 49F cells. We showed that this phenomenon can be potentiated by adding oxysterols to the culture medium. The oxysterol effect was dose-dependent and was not observed in the absence of fetal calf serum. Greater amounts of prostaglandin E2 and prostaglandin F2 alpha were released into the medium in the presence of oxysterols without apparent modification of the cyclooxygenase activity. The most effective oxysterols, in descending order, were the following: calcitriol greater than 7 alpha-hydroxycholesterol greater than 7 beta-hydroxycholesterol greater than 25-hydroxycholesterol. Cholesterol and 7-ketocholesterol were unable to activate phospholipase activity. The mechanism of this activation by oxysterols is still unknown.     

Oxysterols are substrates for cholesterol sulfotransferase

Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7alpha/7beta-hydroxycholesterol and 5alpha,6alpha/5beta,6beta-epoxycholesterol as well as the 7alpha-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds.