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PubMed2005

Influence of peroxisome proliferator-activated receptor gamma gene polymorphism on 24S-hydroxycholesterol levels in Alzheimer's patients.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear hormone receptor, that is involved in lipid and glucose metabolism, which both seem to influence the risk of Alzheimer's disease (AD). 24S-Hydroxycholesterol is the major cholesterol elimination product of the brain and plasma and CSF 24S-hydroxycholesterol levels are altered in patients with neurodegenerative diseases. We investigated the effect of the common Pro12Ala variant of the PPARgamma gene on plasma cholesterol levels and 24S-hydroxycholesterol/ cholesterol ratios in 124 AD patients and 77 healthy controls. Furthermore, the influence of PPARgamma polymorphism on the risk of AD in 247 AD patients and 324 healthy controls was investigated. We found that PPARgamma Pro12Ala polymorphism influenced plasma 24S-hydroxycholesterol/ cholesterol ratios in AD patients in that carriers of the Ala allele presented with higher ratios than homozygote carriers of the Pro-allele. PPARgamma polymorphism did not influence the risk of AD. These results might point to an influence of PPARgamma Pro12Ala polymorphism on the elimination of 24S-hydroxycholesterol.

Aged↗
PubMed2007

Mitochondrial perturbation, oxidative stress and lysosomal destabilization are involved in 7beta-hydroxysitosterol and 7beta-hydroxycholesterol triggered apoptosis in human colon cancer cells.

We reported previously that 7beta-hydroxysitosterol and 7beta-hydroxycholesterol induced apoptosis in Caco-2 cells. Apoptosis caused by 7beta-hydroxysitosterol but not by 7beta-hydroxycholesterol was related to a caspase-dependent process. In the present report, we compared the effects of both compounds on mitochondria integrity and on various modulators of apoptosis. When Caco-2 cells were exposed to both hydroxysterols, no changes in Bcl-2 and Bax expressions were detected indicating a Bcl-2/Bax-independent cell death pathway, whereas loss of mitochondrial membrane potential and cytochrome c release were observed. Endonuclease G expression and enhanced production of reactive oxygen species were detected in 7beta-hydroxycholesterol treated cells, but not with 7beta-hydroxysitosterol. Loss of mitochondrial membrane potential and cell death produced by both hydroxysterols were prevented by vitamin C. Lysosomal membrane integrity was altered with both hydroxysterols, but 7beta-hydroxysitosterol was significantly more active on than 7beta-hydroxycholesterol. Both hydroxysterols induced apoptosis by mitochondrial membrane permeabilization. However, 7beta-hydroxycholesterol exhibited a specific enhancement of oxidative stress and of endonuclease G expression despite its closely related chemical structure with 7beta-hydroxysitosterol. The two hydroxysterols exhibit different lipophilic properties which may explain their different biological effects.

Apoptosis↗
PubMed1977

Effects of 25-hydroxycholesterol and 7-ketocholesterol, inhibitors of sterol synthesis, administered orally to mice.

When 25-hydroxycholesterol or 7-ketocholesterol was fed to mice with the diet, growth was suppressed and mature mice lost weight. The effect of the 7-ketone upon body weight was effectively counteracted by cholesterol whereas cholestanol and beta-sitosterol were ineffective. Growth repression due to 25-hydroxycholesterol was only partially relieved by cholesterol. The effects of 25-hydroxycholesterol and 7-ketocholesterol upon body weight were related to an apparent effect upon appetite. However the sterols were not unpalatable since diets containing them were not rejected in favor of control diet. Intestinal sterol synthesis was inhibited soon after the administration of dietary 7-ketocholesterol or 25-hydroxycholesterol but inhibition decreased with prolonged feeding. When fed by gavage, the sterols suppressed intestinal sterol synthesis as soon as 2 h after administration. In contrast, cholesterol administered by gavage did not affect intestinal sterol synthesis during a 24 h test period. When fed with the diet 25-hydroxycholesterol and 7-ketocholesterol did not depress hepatic cholesterol synthesis beyond the low levels found in pair-fed controls. Inhibition of intestinal sterol synthesis was accompanied by a decrease in the concentration of cholesterol in the intestinal mucosa and, usually, by a drop in the molar ratio of cholesterol to phospholipids.

Animals↗
PubMed1982

Inhibition of protein synthesis blocks the response to 25-hydroxycholesterol by inhibiting degradation of hydroxymethylglutaryl-CoA reductase.

The activity of the rate-limiting enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase in Chinese hamster ovary (CHO) cells decreased more rapidly in cells treated with 25-hydroxycholesterol alone (t 1/2 = 1.5 h) than in those incubated with cycloheximide alone (t 1/2 = 5 h). The inhibitory action of 25-hydroxycholesterol on reductase activity was reduced when the sterol and cycloheximide were added together, and was totally abolished when cells were preincubated with cycloheximide for 30 min before the addition of 25-hydroxycholesterol. The effect of puromycin was similar to that of cycloheximide. Treatment of cells with an inhibitor of RNA synthesis, i.e., actinomycin D or cordycepin, had little effect on hydroxymethylglutaryl-CoA reductase activity; however, preincubation of cells with these reagents also decreased the ability of 25-hydroxycholesterol to suppress the reductase activity. These data are consistent with a model which suggests (a) that 25-hydroxycholesterol inhibits the activity of hydroxymethylglutaryl-CoA reductase by repressing its synthesis, (b) that cycloheximide and puromycin affect hydroxymethylglutaryl-CoA reductase activity by blocking the de novo synthesis of the enzyme and by reducing the degradation of the preexisting enzyme, (c) that actinomycin D and cordycepin affect the supply of message for the continuous synthesis of at least one component of a system which degrades hydroxymethylglutaryl-CoA reductase, and (d) that one component of the degradative system has a half-life shorter than 0.5 h.

Animals↗
PubMed1995

Serum 7 alpha-hydroxycholesterol as a new parameter of liver function in patients with chronic liver diseases.

To examine bile acid synthesis in chronic liver diseases, serum total 7 alpha-hydroxycholesterol level was measured by gas-liquid chromatography-mass spectrometry in patients with cirrhosis (n = 23), patients with chronic hepatitis (n = 21), and control subjects (n = 18). The serum 7 alpha-hydroxycholesterol levels were significantly lower in patients with cirrhosis than the controls (78 +/- 59 pmol/mL vs. 237 +/- 97 pmol/mL; mean +/- SD). However, in patients with chronic hepatitis, the level was fully retained (262 +/- 102 pmol/mL). Serum 7 alpha-hydroxycholesterol levels of 17 patients with cirrhosis classified as Child B and C ranged from 33 to 69 pmol/mL, and all were less than the normal range (between 104 and 466 pmol/mL), however, those levels of some patients classified as Child A were within the normal range. Serum 7 alpha-hydroxycholesterol levels significantly correlated with serum albumin, cholinesterase, total bile acid, direct bilirubin, alkaline phosphatase, indocyanine green (ICG) retention rate, hepaplastin test, and lecithin-cholesterol acyltransferase activities. We conclude that bile acid synthesis is well preserved in patients with chronic hepatitis and that it is decreased in most patients with cirrhosis. Serum 7 alpha-hydroxycholesterol may be a new parameter of liver function testing to assess hepatic bile acid synthesis in patients with chronic liver diseases.

Adult↗
PubMed1993

Inhibition of hepatocyte gap junctional communication by 25-hydroxycholesterol may be mediated through free radicals.

25-Hydroxycholesterol, an autoxidation product of cholesterol, has been shown to inhibit gap junctional communication between rat hepatocytes. In this study, we investigated whether free radicals are responsible for the effect of 25-hydroxycholesterol on hepatocytes. Addition of superoxide dismutase, hydroxyl radical scavenger mannitol, or the antioxidants diphenylphenylenediamine and alpha-tocopherol markedly decreased the inhibitory effect of 25-hydroxycholesterol. However, addition of catalase or the catalase inhibitor aminotriazole did not influence the inhibition of gap junctional communication by 25-hydroxycholesterol. Data from this study suggest that free radicals other than hydrogen peroxide are involved in the mechanism of 25-hydroxycholesterol-induced inhibition of gap junctional communication.

Animals↗
PubMed2005

Tocotrienols reduce 25-hydroxycholesterol-induced monocyte-endothelial cell interaction by inhibiting the surface expression of adhesion molecules.

The migration of circulating monocytes into the subendothelial space occurs through the expressing of some adhesion molecules on endothelial cells. In the present study, using human aortic endothelial cells (HAECs), we investigated whether a model compound for oxysterols, 25-hydroxycholesterol, can enhance the monocyte adherence to HAECs exposed to 25-hydroxycholesterol via increasing expression of vascular cell adhesion molecule-1 (VCAM-1). We also aimed to determine the in vitro effects of tocotrienols on the enhanced interaction between monocytes and endothelial cells. We found that 25-hydroxycholesterol enhances surface expression determined by ELISA, induces VCAM-1 mRNA expression by real time-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells in a dose-dependent fashion. The combination treatment with anti-VCAM-1 and anti-CD11b monoclonal antibodies significantly reduced the monocyte adherence to 25-hydroxycholesterol-stimulated HAECs. Compared to alpha-tocopherol, tocotrienols displayed a more profound inhibitory effect on adhesion molecule expression and monocytic cell adherence. We observed that delta-tocotrienol exerted a most profound inhibitory action on monocytic cell adherence when compared to alpha-tocopherol and alpha-, beta-, and gamma-tocotrienols. Tocotrienols accumulated in HAECs to levels approximately 25-95-fold greater than that of alpha-tocopherol. In conclusion, these results indicate that a model compound 25-hydroxycholesterol can enhance the interaction between monocytes and HAECs, and that tocotrienols had a profound inhibitory effect on monocytic cell adherence to HAECs relative to alpha-tocopherol via inhibiting the VCAM-1 expression. These superior inhibitory effects of tocotrienols may be dependent on their intracellular accumulation.

Antibodies, Monoclonal↗
PubMed2006

Induction of apoptosis by 25-hydroxycholesterol in adult rat Leydig cells: protective effect of 17beta-estradiol.

Testicular macrophages can convert cholesterol into 25-hydroxycholesterol which strongly stimulates Leydig cell testosterone production. We demonstrated that 25-hydroxycholesterol reduced cholesterol biosynthesis in adult rat Leydig cells. This oxysterol can also be cytotoxic. As hydroxylated cholesterol can induce apoptosis in various cells, we investigated cell death produced by 25-hydroxycholesterol. Apoptosis was characterized by TUNEL assay and by DAPI test. Addition of 25-hydroxycholesterol, during 24h, induced a dose dependent increase of apoptosis. This effect was reduced by a treatment with a caspase-3 inhibitor (Ac-DEVD-CHO). 25-Hydroxycholesterol is known to stimulate testosterone production, but an increase of intracellular or culture medium testosterone level does not modify significantly the percentage of apoptotic cells. In contrast, addition of 17beta-estradiol (2 nM) induced a decrease of apoptotic cells. These data suggested that this oxysterol can be used by rat Leydig cells in culture for sterol metabolism, but also induces apoptosis which could be inhibited by 17beta-estradiol.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Effect of naturally reduced ovarian function on plasma lipoprotein and 27-hydroxycholesterol levels in baboons (Papio sp.).

Female baboons over 15 years of age develop irregular menstrual cycles, an indication of declining ovarian function similar to that occurring in perimenopausal women. To determine the effect of declining ovarian function on plasma lipoprotein metabolism and plasma oxysterols, we measured plasma lipoprotein and 27-hydroxycholesterol levels in 86 female baboons from 15-28 years of age with regular (n = 51) and irregular (n = 35) menstrual cycles. We sampled blood and liver while they were consuming a basal diet and after consuming a high cholesterol and high fat diet for 7 weeks. On the basal diet, baboons with irregular cycles had higher VLDL + LDL/HDL cholesterol ratios (P = 0.034). After consuming the HCHF diet for 7 weeks, total plasma (P < 0.001) and VLDL + LDL (P < 0.001) cholesterol concentrations and VLDL + LDL/HDL sterol ratios (P < 0.001) increased in both cycle groups; whereas HDL cholesterol concentrations increased only in baboons with regular cycles (P = 0.009). As a result, HDL cholesterol concentrations (P = 0.006) were lower and VLDL + LDL/HDL cholesterol ratios (P = 0.002) were higher in baboons with irregular cycles on the HCHF diet. Plasma 27-hydroxycholesterol concentrations were higher in baboons with regular cycles than in those with irregular cycles on both basal (P = 0.018) and HCHF (P = 0.037) diets and were positively correlated (P < 0.001) with hepatic sterol 27-hydroxylase activities on both diets. Hepatic sterol 27-hydroxylase activities were negatively correlated with the VLDL + LDL/HDL cholesterol ratios on the HCHF diet (r = -0.342, P = 0.033). These results suggest that declining ovarian function changes the plasma lipoprotein pattern to one that is more atherogenic. Ovarian failure is also associated with decreased concentrations of plasma 27-hydroxycholesterol (the major oxysterol of plasma), and the decrease in plasma 27-hydroxycholesterol concentration was due to the decrease in hepatic sterol 27-hydroxylase activity. The effects of ovarian failure on plasma lipoprotein metabolism and plasma 27-hydroxycholesterol may be mediated by the decreased production of estrogen in perimenopausal baboons. Thus, the perimenopausal baboon is an excellent model for menopause and can be used for studies that cannot be conducted in women.

Animals↗
PubMed1998

Biosynthesis of digitalis-like compounds in rat adrenal cells: hydroxycholesterol as possible precursor.

The biosynthesis of digitalis-like compounds (DLC) was determined in bovine and rat adrenal homogenates, as well as in primary rat adrenal cells, by following changes in the concentration of DLC using three independent sensitive bioassays: inhibition of [3H]-ouabain binding to red blood cells and competitive ouabain and bufalin ELISA. The amounts of DLC in bovine and rat adrenal homogenates, as measured by the two first bioassays, increased with time when the mixtures were incubated under tissue culture conditions. Rat primary adrenal cells were incubated in the presence of [1,2-(3)H]-25-hydroxycholesterol, [26,27-(3)H]-25-hydroxycholesterol or [7-(3)H]-pregnenolone. The radioactive products, as well as the digitalis-like activity, were fractionated by three sequential chromatography systems. When [1,2-(3)H]-25-hydroxycholesterol or [7-(3)H]-pregnenolone was added to the culture medium, the radioactivity was co-eluted with digitalis-like activity, suggesting that at least one of the DLC might originate in hydroxycholesterol. In contrast, when the culture medium was supplemented with [26,27-(3)H]-25-hydroxycholesterol, the radioactivity was not co-eluted with the digitalis-like activity, indicating that side chain cleavage is the first step in the synthesis of digitalis-like compounds by rat adrenal.

Adrenal Cortex↗
PubMed2002

Cerebrospinal fluid 24S-hydroxycholesterol is increased in patients with Alzheimer's disease compared to healthy controls.

Experiments in cell cultures indicate that accumulation of cholesterol in hippocampal neurons results in an accelerated cleavage of amyloid precursor protein into amyloidogenic components. To be eliminated from the brain, cholesterol is converted to 24S-hydroxycholesterol which may reflect cerebral cholesterol turnover. We investigated cerebrospinal fluid (CSF) concentrations of 24S-hydroxycholesterol in a group of 14 Alzheimer's disease (AD) patients and ten healthy controls without any cognitive deficits or psychiatric or neurological disorders. To exclude potential effects of circulating plasma cholesterol on CSF 24S-hydroxycholesterol levels, only patients and controls with cholesterol levels in the normal range of 150-230 mg/dl were included. We found significantly elevated 24S-hydroxycholesterol CSF but not plasma levels in AD patients compared with healthy controls. Our results demonstrate that CSF 24S-hydroxycholesterol is increased in AD. This effect does not seem to be triggered by plasma cholesterol levels since the latter did not significantly differ between groups.

Age Factors↗
PubMed1995

3'-untranslated sequences mediate post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA by 25-hydroxycholesterol.

In an earlier study [Choi, Lundquist and Peffley (1993) Biochem. J. 296, 859-866], we determined that 25-hydroxycholesterol regulates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA through a post-transcriptional mechanism that requires protein synthesis. To investigate whether 3'-untranslated sequences play a role in 25-hydroxycholesterol-mediated post-transcriptional control, we ligated approx. 1400 bp of the 3'-untranslated region of HMG-CoA reductase cDNA to the coding region of human beta-globin DNA. beta-Globin-3'-untranslated reductase fusion constructs were then transiently expressed in Chinese hamster ovary fibroblasts under conditions known to regulate reductase mRNA. There were no differences in beta-globin RNA levels in transfected cells incubated with or without lovastatin, a competitive inhibitor of reductase. However, in the presence of lovastatin and an oxysterol, 25-hydroxycholesterol, beta-globin RNA levels were decreased approx. 2-fold. Inhibition of protein synthesis with cycloheximide blocked the effects of 25-hydroxycholesterol on beta-globin RNA. Moreover, replacing the 3'-untranslated sequences with 1367 bp of the simian virus 40 enhancer region eliminated the regulatory effect of 25-hydroxycholesterol. Because the fusion construct has no sterol regulatory elements necessary for transcription, our results indicate that the change in beta-globin RNA occurred at a post-transcriptional level. In addition, we have shown that the 3'-untranslated region of HMG-CoA reductase cDNA imparted oxysterol-mediated post-transcriptional regulation to beta-globin RNA, an effect that required protein synthesis.

Animals↗
PubMed1998

Cholesterol regulates oxysterol binding protein (OSBP) phosphorylation and Golgi localization in Chinese hamster ovary cells: correlation with stimulation of sphingomyelin synthesis by 25-hydroxycholesterol.

Sphingomyelin (SM) and cholesterol content is positively correlated in cellular membranes, and in several pathological and experimental conditions there is evidence for coregulation. The potential role of oxysterols and oxysterol binding protein (OSBP) in mediating the coregulation of cholesterol and SM was examined using Chinese hamster ovary (CHO) and cholesterol auxotrophic, sterol regulatory defective (SRD) 6 cells. SRD 6 cells grown in the presence or absence of cholesterol for 24 h displayed a 30-50% reduction in SM synthesis compared with control CHO 7 cells. SM synthesis in CHO 7 and cholesterol-supplemented SRD 6 cells was stimulated 2-fold by 25-hydroxycholesterol, but cholesterol-starved SRD 6 cells were unresponsive. Basal and 25-hydroxycholesterol-stimulated SM synthesis was also inhibited in lovastatin-treated wild-type CHO-K1 cells. Lack of 25-hydroxycholesterol activation of SM synthesis in cholesterol-starved SRD 6 and lovastatin-treated CHO-K1 cells was correlated with dephosphorylation of OSBP. In SRD 6 cells, this was evident after 12 h of cholesterol depletion, it occurred equally at all phosphorylation sites and was exacerbated by 25-hydroxycholesterol. Unlike CHO 7 cells, where OSBP was observed in small vesicles and the cytoplasm, OSBP in cholesterol-starved SRD 6 cells was constitutively localized in the Golgi apparatus. Supplementation with non-lipoprotein cholesterol promoted redistribution to vesicles and the cytoplasm. Similarly, OSBP in CHO-K1 cells grown in delipidated serum was predominantly in the Golgi apparatus. Low-density lipoprotein (LDL) supplementation of CHO-K1 cells caused the redistribution of OSBP to the cytoplasm and small vesicles, and this effect was blocked by pharmacological agents ¿3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one and progesterone¿, which inhibited LDL cholesterol efflux from lysosomes. The results showed that localization of OSBP between the Golgi apparatus and a cytoplasmic/vesicular compartment was responsive to changes in cholesterol content and trafficking. In cholesterol depleted SRD 6 cells, this was accompanied by dephosphorylation of OSBP and attenuation of 25-hydroxycholesterol activation of SM synthesis.

Animals↗
PubMed2006

24(S)-hydroxycholesterol participates in a liver X receptor-controlled pathway in astrocytes that regulates apolipoprotein E-mediated cholesterol efflux.

Both apolipoprotein E (apoE) and 24(S)-hydroxycholesterol are involved in the pathogenesis of Alzheimer disease (AD). It has been hypothesized that apoE affects AD development via isoform-specific effects on lipid trafficking between astrocytes and neurons. However, the regulation of the cholesterol supply of neurons via apoE-containing high density lipoproteins remains to be clarified. We show for the first time that the brain-specific metabolite of cholesterol produced by neurons, i.e. 24(S)-hydroxycholesterol, induces apoE transcription, protein synthesis, and secretion in a dose- and time-dependent manner in cells of astrocytic but not of neuronal origin. Moreover, 24(S)-hydroxycholesterol primes astrocytoma, but not neuroblastoma cells, to mediate cholesterol efflux to apoE. Similar results were obtained using the synthetic liver X receptor (LXR) agonist GW683965A, suggesting involvement of an LXR-controlled signaling pathway. A 10-20-fold higher basal LXRalpha and -beta expression level in astrocytoma compared with neuroblastoma cells may underlie these differential effects. Furthermore, apoE-mediated cholesterol efflux from astrocytoma cells may be controlled by the ATP binding cassette transporters ABCA1 and ABCG1, since their expression was also up-regulated by both compounds. In contrast, ABCG4 seems not to be involved, because its expression was induced only in neuronal cells. The expression of sterol regulatory element-binding protein (SREBP-2), low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and SREBP-1c was transiently up-regulated by GW683965A in astrocytes but down-regulated by 24(S)-hydroxycholesterol, suggesting that cholesterol efflux and synthesis are regulated independently. In conclusion, evidence is provided that 24(S)-hydroxycholesterol induces apoE-mediated efflux of cholesterol in astrocytes via an LXR-controlled pathway, which may be relevant for chronic and acute neurological diseases.

ATP Binding Cassette Transporter, Subfamily G↗
PubMed1981

Up-regulation in vascular endothelial cells of binding sites of high density lipoprotein induced by 25-hydroxycholesterol.

Exposure of bovine vascular endothelial cell cultures to 25-hydroxycholesterol (50--100 microgram/ml) result in a 5--10-fold increase in cell surface binding sites of high density lipoprotein (HDL). This increase in HDL-binding sites was dependent on time and temperature. After a 48-h exposure to the oxygenated sterol, a maximal increase in HDL binding could be observed, and newly binding sites disappeared rapidly once 25-hydroxycholesterol was removed from the medium. No increase in HDL-binding sites was observed when cells were maintained at 4 degrees C. In contrast, cultures maintained at 37 degrees C did show an increase in HDL-binding sites when exposed to 25-hydroxycholesterol. Since simultaneous exposure of the cells to 25-hydroxycholesterol and cycloheximide resulted in an inhibition of HDL binding to the cells, it is suggested that de novo synthesis of HDL-binding sites is induced by 25-hydroxycholesterol. When the abilities of HDL2 and HDL3 to bind to newly synthesized HDL-binding sites were compared, HDL3 was found to bind more efficiently than HDL2. It is therefore unlikely that apoprotein E plays a major role in the binding of HDL to newly synthesized HDL-binding sites. When the properties of newly synthesized HDL-binding sites were analyzed, they were found to have a high affinity for HDL, since half-maximal binding was reached at a concentration as low as 5 microgram HDL protein/ml, and were saturable. Such HDL-binding sites had a relaxed specificity, since they were capable of binding low density liprotein (LDL). However, when LDL bound to newly synthesized HDL binding sites, it was no longer internalized, as reflected by a 90% reduction of LDL degradation, and instead of being cytotoxic it became mitogenic.

Animals↗
PubMed1999

Characterization and comparison of the mode of cell death, apoptosis versus necrosis, induced by 7beta-hydroxycholesterol and 7-ketocholesterol in the cells of the vascular wall.

Oxidized low density lipoproteins (LDLs) play a central role in atherosclerosis, and their toxicity is due, at least in part, to the formation of oxysterols that have been shown to induce apoptosis in various cell types. As 7beta-hydroxycholesterol and 7-ketocholesterol are the major oxysterols found in oxidized LDLs, we have investigated and compared the mode of cell death, apoptosis versus necrosis, that they induce in the cells of the vascular wall, ie, endothelial cells, smooth muscle cells, and fibroblasts. To this end, human vascular endothelial cells from umbilical cord veins (HUVECs), human artery smooth muscle cells, A7R5 rat smooth muscle cells, MRC5 human fibroblasts, and human fibroblasts isolated from umbilical cord veins were taken at confluence and incubated for 48 hours with 7beta-hydroxycholesterol or 7-ketocholesterol (concentration range, 5 to 80 microg/mL). In all cells, both 7beta-hydroxycholesterol and 7-ketocholesterol exhibited toxic effects characterized by a loss of cell adhesion and an increased permeability to propidium iodide. In oxysterol-treated endothelial and smooth muscle cells, typical features of apoptosis were revealed: condensed and/or fragmented nuclei were detected by fluorescence microscopy after staining with Hoechst 33342, oligonucleosomal DNA fragments were visualized in situ in the cell nuclei by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method, and internucleosomal DNA fragmentation was found on agarose gel. In contrast, in oxysterol-treated fibroblasts, fragmented and/or condensed nuclei were never revealed, and no DNA fragmentation was observed either by the TUNEL method or by DNA analysis on agarose gel, indicating that these oxysterols induced necrosis in these cells but not apoptosis. In addition, acetylated Asp-Glu-Val-L-aspartic acid aldehyde (an inhibitor of Asp-Glu-Val-L-aspartic acid-sensitive caspases) prevented 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death in HUVECs and smooth muscle cells but not in fibroblasts. Thus, 7beta-hydroxycholesterol and 7-ketocholesterol have dual cytotoxic effects on the cells of the vascular wall by their ability to induce apoptosis in endothelial and smooth muscle cells and necrosis in fibroblasts.

Animals↗
PubMed1994

The influence of 25-hydroxycholesterol on phosphatidylinositol content in cultured smooth muscle cells.

The effect of 25-hydroxycholesterol on phosphatidylinositol content was studied in subconfluent human arterial smooth muscle cells exposed to myo-[2-3H]inositol for 90 min and then to 25-hydroxycholesterol at different concentrations or for different incubating periods. Ethanol at a concentration of 0.05% in culture medium was used as control. The results demonstrate that an increased incubation time or an increase in the concentration of 25-hydroxycholesterol produced an increase in phosphatidylinositol and its degraded products. The ratio of precursor to product at each stage of phosphatidylinositol turnover in 25-hydroxycholesterol-treated cells, however, was not different from that in control cells. The data indicate that the presence of 25-hydroxycholesterol in the media induced general activation of phosphatidylinositol synthesis and turnover.

Cells, Cultured↗
PubMed1990

26-Hydroxycholesterol: synthesis, metabolism, and biologic activities.

Cholest-5-ene-3 beta,26-diol (26-hydroxycholesterol) is synthesized by a mitochondrial P-450 enzyme that appears to be widely distributed in tissues. Together with other C-27 steroid intermediates it is transported to the liver and metabolized to bile acids. Although 26-hydroxycholesterol is transported in plasma lipoproteins mostly as the fatty acid ester, neither its assembly and orientation within lipoproteins nor its mechanism of transport across the sinusoidal liver membrane is known. Cell culture studies indicate that 26-hydroxycholesterol can inhibit both cholesterol synthesis and low density lipoprotein (LDL) receptor activity. Inhibition of DNA synthesis also occurs and may not be related to the reduction in HMG-CoA reductase activity. The relationship of these in vitro activities to the physiologic role(s) of 26-hydroxycholesterol remains to be clarified. A clue to its biologic role is the knowledge that markedly decreased 26-hydroxylase activity appears to be the molecular basis of cerebrotendinous xanthomatosis, an inborn error of metabolism characterized by a significant decrease in 26-hydroxycholesterol and bile acid synthesis and an increase in cholesterol synthesis.

Animals↗