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PubMed1993

Induction of basic fibroblast growth factor mRNA and protein synthesis in smooth muscle cells by cholesteryl ester enrichment and 25-hydroxycholesterol.

Basic fibroblast growth factor (bFGF) is a potent smooth muscle cell mitogen. Smooth muscle cell and macrophage-derived foam cells, resulting from cholesteryl ester accretion, are hallmark characteristics of atherosclerosis. We wanted to determine if bFGF synthesis is altered during cholesteryl ester accumulation in smooth muscle cells. Cholesteryl ester enrichment causes a 3-fold increase in bFGF in cellular lysates and a 3-fold increase in steady state mRNA levels for bFGF, as compared with control cells. Conditioned media from cholesteryl ester-enriched smooth muscle cells contains 6 times more mitogenic activity than conditioned media from control cells; this activity is neutralized by an antibody directed against bFGF but not by an antibody directed against platelet-derived growth factor. These results suggest that cholesteryl ester enrichment also enhances bFGF release. Since oxysterols have been implicated in the pathogenesis of atherosclerosis, we determined if oxysterols could affect bFGF production and release. 25-Hydroxycholesterol also increases the release of bFGF-like mitogens from smooth muscle cells, as well as increasing mRNA transcript levels for bFGF. Cholesteryl ester enrichment and 25-hydroxycholesterol did not promote bFGF release secondary to cell injury. In conclusion, these data define a basic mechanism for smooth muscle cell hyperplasia during atherogenesis involving the generation of bFGF by smooth muscle cell-derived foam cells.

Animals↗
PubMed1996

LY295427, a novel hypocholesterolemic agent, enhances [3H]25-hydroxycholesterol binding to liver cytosolic proteins.

LY295427, (3 alpha,4 alpha,5 alpha)-4-(2-propenylcholestan-3-ol), acts through an unknown mechanism to derepress the transcription of the low density lipoprotein (LDL) receptor in the presence of 25-hydroxycholesterol (25-OH chol). Preincubation with LY295427 in Chinese hamster ovary (CHO) cells increased uptake of 25-OH chol in a time-dependent manner, suggesting that the drug interfered with the negative feedback mechanism of 25-OH chol on LDL receptor expression. To explore the mechanism by which LY295427 inhibited the suppressive actions of 25-OH chol, the radioactive ligand [3H]25-OH chol and specific antibodies to the oxysterol binding protein (OSBP) were used to identify possible drug:protein interactions. After separation by anion exchange chromatography, protein fractions from hamster liver cytosol were found to selectively bind [3H]25-OH chol with high affinity. In fractions in which 25-OH chol binding was evident, and in other distinct fractions that lacked specific binding, addition of LY295427 increased [3H]25-OH chol binding 2- to 5-fold. LY306039, the 3 beta-isomer of LY295427, failed to derepress the LDL receptor in CHO cells, and it had no effect on [3H]25-OH chol binding. Analysis of Western blots using polyclonal antibodies to OSBP showed that specific [3H]25-OH chol binding in the absence of LY295427 was present only in fractions containing OSBP. However, enhanced [3H]25-OH chol binding in the presence of LY295427 was evident in distinct fractions after immunodepletion of both the 90-100 kDa form of OSBP and a 170 kDa protein; and specific binding of a radioiodinated analog of LY295427 was detected in select fractions lacking [3H]25-OH chol binding in the absence of LY295427. Moreover, LY295427 did not displace or enhance [3H]25-OH chol binding to OSBP purified to near homogeneity. These data suggest that LY295427, while not dependent on the presence of oxysterol binding protein, binds to cytosolic protein(s) that interact with 25-hydroxycholesterol and other oxystcrols, thus preventing the repression of the LDL receptor.

Animals↗
PubMed1997

7 alpha-hydroxylation of 27-hydroxycholesterol: biologic role in the regulation of cholesterol synthesis.

The report of a novel cytochrome P450 enzyme in mouse hippocampus (cyp7b) with close homology to cholesterol 7 alpha-hydroxylase led us to determine the substrate specificity with respect to 27-hydroxycholesterol, known to be a potent inhibitor of cholesterol synthesis. Transfection of 293/T-cells with PcDNA3.1(+)-mcyp7b was followed by metabolism of 2.5 microM 27-hydroxycholesterol to the 7 alpha-hydroxy intermediate, cholest-5-ene,3 beta,7 alpha,27-triol, with complete loss of down-regulation of cholesterol synthesis. Addition of 5 microM and 10 microM concentrations of the triol to HepG2 and CHO cells, respectively, also did not reduce cholesterol synthesis. The contrast between the biologic effect on cholesterol synthesis by these two C27 hydroxysterols and the wide tissue distribution of both cholesterol 27-hydroxylase and hydroxysterol 7 alpha-hydroxylase implies local regulatory effects prior to their further catabolism in the liver to chenodeoxycholic and cholic acids.

Animals↗
PubMed1974

The stoichiometry of the conversion of cholesterol and hydroxycholesterols to pregnenolone (3beta-hydroxypregn-5-en-20-one) catalysed by adrenal cytochrome P-450.

The stoichiometry of the side-chain cleavage of cholesterol (cholest-5-en-3beta-ol), 20alpha-hydroxycholesterol (cholest-5-ene-3beta,20alpha-diol), and 20alpha,22-dihydroxycholesterol (cholest-5-ene-3beta,20alpha,22-triol) has been examined with respect to TPNH, oxygen and H(+). Side-chain cleavage of cholesterol can be described by the following equation:Cholesterol + 3TPNH + 3H(+) + 3O(2) --> Pregnenolone (3beta-hydroxypregn-5-ene-20-one) + isocapraldehyde + 3 TPN(+) 4H(2)OStoichiometry of 20 alpha-hydroxycholesterol is 2TPNH and 2O(2) per mole of cleavage and with 20alpha, 22-dihydroxycholesterol the values are 1:1:1. In addition 1 mole of H(+) is consumed per mole of TPNH oxidized in each of these reactions. These observations are in keeping with a mechanism previously proposed in the literature, namely:Cholesterol --> 20alpha-OH-Cholesterol --> 20alpha, 22-di-OH-Cholesterol --> pregnenoloneDiscordant observations are reviewed and it is concluded that insufficient data are available to sustain objections to this pathway.

Adrenal Glands↗
PubMed2003

Cholesterol is superior to 7-ketocholesterol or 7 alpha-hydroxycholesterol as an allosteric activator for acyl-coenzyme A:cholesterol acyltransferase 1.

We compared the abilities of cholesterol versus various oxysterols as substrate and/or as activator for the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), by monitoring the activity of purified human ACAT1 in response to sterols solubilized in mixed micelles or in reconstituted vesicles. The results showed that 5 alpha,6 alpha-epoxycholesterol and 7 alpha-hydroxycholesterol are comparable with cholesterol as the favored substrates, whereas 7-ketocholesterol, 7 beta-hydroxycholesterol, 5 beta,6 beta-epoxycholesterol, and 24(S),25-epoxycholesterol are very poor substrates for the enzyme. We then tested the ability of 7-ketocholesterol as an activator when cholesterol was measured as the substrate, and vice versa. When cholesterol was measured as the substrate, the addition of 7-ketocholesterol could not activate the enzyme. In contrast, when 7-ketocholesterol was measured as the substrate, the addition of cholesterol significantly activated the enzyme and changed the shape of the substrate saturation curve from sigmoidal to essentially hyperbolic. Additional results show that, as an activator, cholesterol is much better than all the oxysterols tested. These results suggest that ACAT1 contains two types of sterol binding sites; the structural requirement for the ACAT activator site is more stringent than it is for the ACAT substrate site. Upon activation by cholesterol, ACAT1 becomes promiscuous toward various sterols as its substrate.

Allosteric Regulation↗
PubMed2025

Role of oxysterol 4β-hydroxycholesterol and liver X receptor alleles in pre-eclampsia.

BACKGROUND: Liver X receptors (LXRs) are expressed in placenta and may be associated with pre-eclampsia (PE). Oxysterols act as agonists for LXRs. We recently proposed a new blood pressure-regulating circuit with oxysterol 4β-hydroxycholesterol (4βHC) acting as a hypotensive factor via LXRs. MATERIALS AND METHODS: This study investigated the association between maternal plasma 4βHC, blood pressure (BP) indices, placental expression of LXR target genes, and patient characteristics using data from the Finnish Genetics of Pre-Eclampsia Consortium (FINNPEC) cohort. Plasma samples of 144 women with PE and 38 healthy pregnant controls as well as 44 PE and 40 control placental samples were available. In addition, genetic data from the FinnGen project was utilized to explore the associations of LXR alleles with PE and pregnancy hypertension. RESULTS: There were no significant associations between 4βHC and BP or maternal and perinatal characteristics in FINNPEC cohort. However, plasma 4βHC was inversely correlated with the maternal body mass index. There were no associations with the genetic variants of LXRs with PE in FinnGen. LXR target genes APOD, SCARB1, TGM2, and LPCAT3 were expressed differently between PE and normal pregnancies in placental samples of FINNPEC. CONCLUSIONS: Our results demonstrate that plasma 4βHC and genetic LXR variants do not play a major role in PE and BP regulation during pregnancy. However, key LXR target genes involved in lipid metabolism were expressed differently in normal and PE pregnancies. Further research is needed to understand the complexities of oxysterols, LXRs, and their potential contributions to placental function and pregnancy outcomes.

Humans↗
PubMed2006

Bio-markers of lipid peroxidation in vivo: hydroxyoctadecadienoic acid and hydroxycholesterol.

The biological role of lipid peroxidation products has continued to receive a great deal of attention not only for the elucidation of pathological mechanisms but also for their practical application to clinical use as bio-markers. In the last fifty years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. In this decade, F2-isoprostanes from arachidonates and neuroprostanes from docosahexanoates have been proposed as bio-markers. Although these markers are formed by a free radical-mediated oxidation, the yields from the parent lipids are minimal. Compared to these markers, hydroperoxy octadecadienoates (HPODE) from linoleates and oxysterols from cholesterols are yielded by much simpler mechanisms from more abundant parent lipids in vivo. Recently, the method in which both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as total hydroxyoctadecadienoic acid (tHODE) and 7-hydroxycholesterol (t7-OHCh), respectively, was proposed. The concentrations of tHODE and t7-OHCh determined by GC-MS analysis from physiological samples were much higher than that of 8-iso-prostagrandin F(2alpha). In addition to this advantage, hydrogen-donor activity of antioxidants in vivo could be determined by the isomeric-ratio of HODE (9- and 13-(Z,E)-HODE/9- and 13-(E,E)-HODE).

Animals↗
PubMed2004

Generation of an oxidative stress precedes caspase activation during 7beta-hydroxycholesterol-induced apoptosis in U937 cells.

The oxysterol 7beta-hydroxycholesterol (7beta-OH) has been shown to induce apoptosis in a number of cell lines. Though not fully elucidated, the mechanism through which this oxysterol induces cell death is thought to involve the generation of an oxidative stress leading to perturbation of the mitochondrion and release of cytochrome c into the cytosol. Cytochrome c together with Apaf-1 causes activation of the initiator caspase, caspase-9, which in turn activates caspase-3 ultimately leading to the degradation of poly(ADP-ribose) polymerase (PARP). The objective of the present study was to investigate the signalling pathway in 7beta-OH-induced apoptosis in U937 cells, a human monocytic blood cell line known to undergo apoptosis upon treatment with 7beta-OH, over a time course of 48 h. Apoptosis was evident after 24 h incubation. Glutathione levels were decreased after 6 h and this was coupled with an increase in SOD activity. Through western blot analysis we examined expression of caspase-3, -8, and -9 and cleavage of the caspase-3 substrate PARP. The sequence proceeded with activation of caspase-9 after 9 h, caspase-3 at the 12 h timepoint, and cleavage of PARP after 24 h treatment with 7beta-OH. Caspase-8 did not appear to play a major role in this particular apoptotic pathway.

Apoptosis↗
PubMed2005

Activation of caspase-3-dependent and -independent pathways during 7-ketocholesterol- and 7beta-hydroxycholesterol-induced cell death: a morphological and biochemical study.

On treatment with 7-ketocholesterol (7-keto) or 7beta-hydroxycholesterol (7beta-OH), which are major oxysterols in atherosclerotic plaques, the simultaneous identification of oncotic and apoptotic cells suggests that these compounds activate different metabolic pathways leading to various modes of cell death. With U937, MCF-7 (caspase-3 deficient), MCF-7/c3 cells (stably transfected with caspase-3), we demonstrate that caspase-3 is essential for caspase-9, -7, -8 activation, for Bid degradation mediating mitochondrial cytochrome c release, for cleavage of poly(ADP-ribose) polymerase and inhibitor of the caspase-activated deoxyribonuclease, and, at least in part, for internucleosomal DNA fragmentation. The crucial role of caspase-3 was supported by the use of z-VAD-fmk and z-DEVD-fmk, which abolished apoptosis and the associated events. However, inactivation or lack of caspase-3 did not inhibit 7-keto- and 7beta-OH-induced cell death characterized by staining with propidium iodide, loss of mitochondrial potential. The mitochondrial release of apoptosis-inducing factor and endonuclease G was independent of the caspase-3 status, which conversely played major roles in the morphological aspects of dead cells. We conclude that caspase-3 is essential to trigger 7-keto- and 7beta-OH-induced apoptosis, that these oxysterols simultaneously activate caspase-3-dependent and/or -independent modes of cell death.

Apoptosis↗
PubMed1991

Collisional spectroscopy approaches in the characterization of 7-alpha- and 7-beta-hydroxycholesterol.

High- and low-energy collision experiments, by means of double-focusing and ion-trap mass spectrometers, respectively, performed on the M+ and [M-H2O]+. ions of 7-alpha- and 7-beta-hydroxycholesterol lead to a clear structural characterization of the two stereoisomers. In particular, the beta isomer shows peculiar fragmentation pathways due to interaction between the hydroxyl group in position 7-beta and the hydrogen atom on C(15).

Hydroxycholesterols↗
PubMed1998

Determination of 7alpha-hydroxycholesterol in dog plasma by high-performance liquid chromatography with fluorescence detection.

A new method was developed for the determination of 7alpha-hydroxycholesterol (7-HC) in dog plasma by means of high-performance liquid chromatography (HPLC) with fluorescence detection. 7-HC extracted with organic solvent from plasma was purified with Bond Elut 2OH and converted to a sensitive fluorescent derivative containing double coumarin groups at the C-3 and C-7 positions of the steroid nucleus with 7-methoxycoumarin-3-carbonyl azide. After removal of the excess reagent with Bond Elut NH2, the 7-HC derivative was separated by reverse-phase HPLC method. The detection limit of the authentic 7-HC-3,7-coumarin derivative was 4 pg (S/N = 5), approximately four times less than that of the 7-HC-3-anthroyl derivative yielded by reaction of 7-HC with 1-anthroylcyanide. The newly developed method was used to investigate the effects of consecutive oral administrations of cholestyramine (CA) on 7-HC levels in dog plasma. The plasma 7-HC levels of the CA-treated group were two times greater than those of the control group.

Animals↗
PubMed2000

25-Hydroxycholesterol activates a cytochrome c release-mediated caspase cascade.

We have previously shown that 25-hydroxycholesterol (25-OHC) treated CHO-K1 cells could be used as a model to investigate the signaling pathway of apoptosis induced by oxidized LDL in vascular cells. In the present study, we examine the execution phase of the apoptotic pathway in CHO-K1 cell death induced by 25-OHC. Oxysterol-induced apoptosis in CHO-K1 was accompanied by caspase activation and was preceded by mitochondrial cytochrome c release. The addition of a competitive caspase-3 inhibitor, Ac-DEVD-CHO, prevented 25-OHC-induced apoptotic cell death. Furthermore, immunoblot analysis showed that 25-OHC treatment induced the degradation of poly(ADP-ribose) polymerase (PARP)-a substrate for caspase 3 and a key enzyme involved in genome surveillance and DNA repair. Thus, we could demonstrate in CHO-K1 cells that 25-OHC activates the apoptotic machinery through induction of the release of cytochrome c from mitochodria into the cytosol and activation of a typical caspase cascade.

Animals↗
PubMed1997

Characteristics of 25-hydroxycholesterol-induced apoptosis in the human leukemic cell line CEM.

Cholesterol and related compounds can give rise to oxygenated sterol molecules (oxysterols) which are potent regulators of lymphoid cell growth. Oxysterols added exogenously cause cell death of several lines of cultured cells, and on the basis of limited criteria, it has been suggested that this death is apoptosis. In the present study, we show definitive evidence that 25-hydroxycholesterol (25OHC) kills cells of the clone CEM-C7 by apoptosis and establish the temporal sequence of related cellular and biochemical events. Cell shrinkage was evident as early as 12 h, while cell death was not evident until after 24 h. It mounted rapidly after that, and by 72 h, virtually all cells were dead. Electron microscopic analysis shows that by 24 h after treatment and before the onset of cell death, early ultrastructural features typical of apoptosis were present. DNA breaks were detected by TUNEL assay prior to the onset of cell death. Two types of specific DNA pieces often associated with apoptosis were found as increasing numbers of cells died. DNA fragments of 300 and 50 kbp were not appreciable until 42 h, and internucleosomal cleavage was observed by 48 h after oxysterol addition. None of these effects were seen in an oxysterol-resistant CEM subclone, establishing the specificity for apoptosis of the biochemical and morphological events. z-VAD.FMK, a peptide inhibitor of ICE-related proteases delayed but did not prevent the apoptosis of CEM-C7 cells induced by 25OHC. The addition of mevalonate partially protected CEM-C7 cells from apoptosis but did not restore cell growth.

Amino Acid Chloromethyl Ketones↗
PubMed1991

Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 7 beta-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity, and on cell surface protein expression.

The cytotoxicity of 7 beta-hydroxycholesterol (7 beta-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 microM 7 beta-OHC over a period of 72 h whereas 30 microM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7 beta-OHC added to the cultures at concentrations of 20 microM or 30 microM. Cellular fractionation after incubation with 20 microM or 30 microM 7 beta-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7 beta-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 microM 7 beta-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 microM 7 beta-OHC treatment. Fluorescence anisotropy measurements indicated that 20 microM 7 beta-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 microM 7 beta-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 microM 7 beta-OHC. These findings demonstrate that 7 beta-OHC is lethal to highly proliferating cultured glial cells. The high accumulation of 7 beta-OHC in the plasma membrane and its decrease in fluidity, by themselves, do not seem to be involved in the processes leading to cellular death. However, increase of plasma membrane fragility associated with the decrease of CH/PL, which occurs exclusively in plasma membranes isolated from 7 beta-OHC treated transformed cells together with high 7 beta-OHC uptake, are probably implicated in 7 beta-OHC cytotoxicity. The possibility of an additional action mechanism is discussed.

Animals↗
PubMed1987

Acetoacetyl-CoA ligase activity in the isolated rat hepatocyte: effects of 25-hydroxycholesterol and high density lipoprotein.

The activity of acetoacetyl-CoA (AcAc-CoA) ligase (E.C.6.2.1.16) in hepatocytes from rats was shown to be the same as the activity in homogenates of their livers. In hepatocytes treated with 25-hydroxycholesterol, AcAc-CoA ligase, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and rates of sterol synthesis were substantially decreased. Hepatocytes treated with high density lipoprotein (HDL) exhibited a 2 to 4 fold induction of HMG-CoA reductase activity; however an accompanying increase in AcAc-CoA ligase activity and the rate of cholesterol synthesis was not observed. We conclude (a) that increases in the activity of HMG-CoA reductase when mediated by HDL in hepatocytes do not result in a corresponding change in the capacity for sterol synthesis and (b) that changes in the activity state of HMG-CoA reductase can be dissociated from that of AcAc-CoA ligase.

Animals↗
PubMed1989

Toxic effects of 7 beta-hydroxycholesterol on rat liver primary cultures, epithelial lines and co-cultures.

The toxic effects of 7 beta-hydroxycholesterol (7 beta-OHC) on cultures and co-cultures of rat hepatocytes, rat liver epithelial cell lines, and rat liver fibroblast lines were investigated. Hepatocytes in primary culture or co-cultured with proliferative epithelial cells, were not affected by the presence of 7 beta-OHC at a concentration of 400 microM over a period of 72 hours. In contrast, proliferative cultures of liver epithelial cell lines and liver fibroblast lines were killed by 50 microM 7 beta-OHC within the first 24 hours. Established liver epithelial cells (hyperploid) were more sensitive to 7 beta-OHC than the same line at early passages (diploid). When hepatocytes and liver epithelial cells were co-cultured and treated with 100 microM 7 beta-OHC, only epithelial cells were lysed. A concentration of 50 microM 7 beta-OHC was toxic to co-cultures of liver epithelial cell and fibroblasts together. In a serum-free medium, the cytotoxic concentration of 7 beta-OHC was lower than that in the serum-supplemented medium. Thus, liver epithelial cells cultured alone or co-cultured with hepatocytes were killed at 12.5 microM and 50 microM 7 beta-OHC, respectively. Finally, cholesterol concentrations four-fold that of 7 beta-OHC antagonized the lethal effects of 7 beta-OHC in the serum-free medium.

Albumins↗
PubMed1985

Lysis of platelets and erythrocytes by the incorporation of a unique oxygenated sterol: 22R-hydroxycholesterol.

We have found that an oxygenated sterol, 22R-hydroxycholesterol [(22R)-5-cholestene-3 beta, 22-diol], lyses not only platelets but also erythrocytes in a dose-dependent manner. The lysis of platelets and erythrocytes were evidenced by the release of intracellular proteins, lactate dehydrogenase and hemoglobin, respectively. Their morphological change was shown by scanning electron microscopy. Elevated temperature was required for the lysis, probably to redistribute the sterol in the lipid bilayers in the plasma membranes. When the sterol was incorporated at low temperature, the temperature had to be raised to readily lyse cells. This lytic effect was, surprisingly enough, restricted to the R-isomer; the S-isomer was only marginally effective. Furthermore, sitosterol and other oxygenated sterols, with a hydroxyl group at different positions in the side chain of cholesterol, were much less lytic, regardless of the configuration of the hydroxyl group introduced. A possible mechanism for this interesting phenomenon will be discussed in relation with a structural alteration in lipid bilayers in plasma membranes brought about by the incorporation of this unique oxygenated sterol.

Animals↗
PubMed1992

Effect of 25-hydroxycholesterol on cholesteryl ester formation in Caco-2 cells.

Incubation of Caco-2 cells, a human intestinal cell line, with 25-hydroxycholesterol (25-HOC) marked enhanced cellular cholesteryl ester formation determined by incorporation of [14C]oleic acid into intracellular cholesteryl [14C]oleate. The stimulation by 25-HOC of cholesteryl ester formation was suppressed by staurosporine, a kinase inhibitor, but not by cycloheximide or actinomycin D. The specific activity of microsomal acyl-coenzyme A:cholesterol acyltransferase (ACAT) increased two-fold in cells treated with 10 microM 25-HOC for 5 h. ACAT activity decreased when microsomes were incubated without sodium fluoride, a phosphatase inhibitor, but the decrease in ACAT activity in cells stimulated with 25-HOC was more pronounced. The results suggest that protein phosphorylation may be involved in the stimulation of cholesteryl ester formation by 25-HOC in Caco-2 cells.

Alkaloids↗