| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Experimental studies on dissolution of cholesterol gallstones using MTBE Yamashita, N. (1990), Nippon Shokakibyo Gakkai Zasshi 87(5): 1183-90. Abstract: Recently, methyl tertiary butyl ether (MTBE) has been evaluated as a new cholesterol gallstone dissolving agent. The dissolution rate of cholesterol monohydrate (ChM) by MTBE was measured and effective types of MTBE administration in vivo were studied. When the dissolution rate of ChM by MTBE was compared with that of d-limonene and monooctanoin used clinically at present, MTBE was found to be the most excellent dissolving agent. However, the dissolving effect of MTBE can be suppressed by water (a component of wet gallstones and 97% of bile) because MTBE is insoluble in water. Therefore, MTBE mixed with a small amount of surfactants (MTBEP) was prepared. The dissolving effect of MTBE alone and MTBEP on dry and wet gallstones were measured. The dissolving effect of MTBE alone on the wet stones was markedly decreased, while MTBEP suppressed the decrease of dissolving effect on wet stones significantly. Furthermore, the dissolving effect of MTBE alone and MTBEP on gallstones in water were measured. MTBEP significantly improved the decrease of dissolving effect on stones by MTBE alone, because the addition of a small amount of a surfactant suppressed the influence of water for dissolving effect of MTBE. MTBE emulsion, newly type of solvent, was prepared. Although the dissolution rate of ChM in MTBE emulsion was low, the dissolving effect of MTBE emulsion was also better than that of MTBE in animal experiments. In this study, emulsion type can be expected to become excellent dissolving agents, because emulsion type suppressed the influence of water for dissolving effect of solvent. |
| Exploration of molecular interactions in cholesterol superlattices: effect of multibody interactions Huang, J. (2002), Biophys J 83(2): 1014-25. Abstract: Experimental evidences have indicated that cholesterol may adapt highly regular lateral distributions (i.e., superlattices) in a phospholipid bilayer. We investigated the formations of superlattices at cholesterol mole fraction of 0.154, 0.25, 0.40, and 0.5 using Monte Carlo simulation. We found that in general, conventional pairwise-additive interactions cannot produce superlattices. Instead, a multibody (nonpairwise) interaction is required. Cholesterol superlattice formation reveals that although the overall interaction between cholesterol and phospholipids is favorable, it contains two large opposing components: an interaction favoring cholesterol-phospholipid mixing and an unfavorable acyl chain multibody interaction that increases nonlinearly with the number of cholesterol contacts. The magnitudes of interactions are in the order of kT. The physical origins of these interactions can be explained by our umbrella model. They most likely come from the requirement for polar phospholipid headgroups to cover the nonpolar cholesterol to avoid the exposure of cholesterol to water and from the sharp decreasing of acyl chain conformation entropy due to cholesterol contact. This study together with our previous work demonstrate that the driving force of cholesterol-phospholipid mixing is a hydrophobic interaction, and multibody interactions dominate others over a wide range of cholesterol concentration. |
| Exploring detergent insolubility in bovine hippocampal membranes: a critical assessment of the requirement for cholesterol Pucadyil, T. J. and A. Chattopadhyay (2004), Biochim Biophys Acta 1661(1): 9-17. Abstract: The phenomenon of detergent insolubility of bovine hippocampal membranes in Triton X-100 was monitored by estimating the presence of phospholipids in the insoluble pellet. This represents a convenient and unambiguous assay and reports the dependence of the extent of phospholipid solubilization on detergent concentration. The advantage of this approach is its ability to accurately determine the extent of detergent insolubility in natural membranes. Importantly, our results show that when suboptimal concentrations of Triton X-100 are used for solubilization, interpretations of the mechanism and extent of detergent insolubility should be made with adequate caution. At concentrations of Triton X-100 that leads to no further solubilization, approximately 44% of phospholipids are left insoluble at 4 degrees C in bovine hippocampal membranes. Cholesterol depletion using methyl-beta-cyclodextrin enhanced phospholipid solubilization at low detergent concentrations but produced no significant change in the amount of insoluble phospholipids at saturating detergent concentration. Progressive solubilization by the detergent resulted in insoluble membranes that contained lipids with higher fatty acyl chain order as reported by fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH). These results suggest that it is the presence of such lipids rather than their association with cholesterol that determines detergent insolubility in membranes. |
| Exponential relationship between plasma cholesterol levels and atherosclerotic lesion size in hyperlipidemic swine Schmee, J., D. N. Kim, et al. (1993), Exp Mol Pathol 59(3): 177-85. Abstract: The effect of fish oil supplements on atherogenesis is controversial, especially when fish oil does not lower plasma cholesterol. Some studies in swine have shown that a fish oil supplement to a butter-cholesterol diet reduces atherogenesis. The fish oil supplement also frequently reduces average plasma cholesterol levels. The reduction in lesion size has been shown to be greater than can be expected from average plasma cholesterol reductions, if a linear relationship between lesion size and plasma cholesterol was assumed. However, in an experiment in which we equalized time-weighted average plasma cholesterol levels, there was no significant reduction in lesion size in the fish oil supplemented group. This led us to question the validity of the linear relationship between lesion size and plasma cholesterol level. In this study we have combined the results of eight study blocks with a total of 76 swine fed a similar hyperlipidemic, butter-cholesterol diet. Of these, 24 received a fish oil supplement (BT+fish oil) and 52 swine received no fish oil supplement (BT). The average lesion size as measured by nuclear profiles per cross section of a fixed site in the abdominal aorta (ABNpCx) was 7704 +/- 778 (mean +/- SEM) for the BT group and 2360 +/- 1145 for the BT+fish oil group. Total plasma cholesterol levels were measured at the outset and at monthly intervals until sacrifice. For each animal we obtained a time-weighted average based on the trapezoidal rule.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Exposure of phosphatidylinositol transfer proteins to sphingomyelin-cholesterol membranes suggests transient but productive interactions with raft-like, liquid-ordered domains Miller, E. C. and G. M. Helmkamp, Jr. (2003), Biochemistry 42(45): 13250-9. Abstract: Both isoforms of rat phosphatidylinositol transfer protein (PITP) mediate the intermembrane transfer of sphingomyelin (CerPCho). In the plasma membrane, CerPCho often segregates with cholesterol into microdomains such as lipid rafts and caveolae. To test the hypothesis that PITP exhibits a preference for CerPCho- and cholesterol-rich membranes, we prepared unilamellar vesicles containing variable amounts of these two lipids. We also used CerPCho species with different acyl composition and treated vesicles with agents known to sequester and remove cholesterol. We observed that the beta isoform of rat PITP was more sensitive to membrane cholesterol than was the alpha isoform, as shown by increases in specific activities of lipid transfer of 2-6-fold. A relatively high membrane content of cholesterol (mole fraction > 0.4) was required to elicit such enhancements. Treatment of cholesterol-rich membranes with a series of beta cyclodextrins demonstrated that, upon depletion of cholesterol from participating membranes, the PITPbeta activity changes were fully reversible. We finally noted that the mechanism by which cholesterol enhances the activity of PITPbeta appeared to involve a decreased affinity of the protein for the membrane surface, in a manner that was independent of vesicle size and membrane microviscosity. We conclude that PITPbeta interacts transiently but productively with the liquid-ordered phase formed by CerPCho and cholesterol and discuss the possibility of PITP interactions in vivo with sphingolipid- and cholesterol-rich membrane microdomains. |
| Exposure of rat peritoneal macrophages to acetylated low density lipoprotein results in release of plasma membrane cholesterol. An efficient substrate for esterification by acyl-CoA:cholesterol acyltransferase Shinohara, M., A. Miyazaki, et al. (1992), J Biol Chem 267(3): 1603-8. Abstract: In J774 macrophages and murine macrophages stimulated with acetylated low density lipoprotein (acetyl-LDL), the plasma membrane free cholesterol (FC) became accessible to acyl-CoA:cholesterol acyltransferase (ACAT) as substrate, the result being an accumulation of cholesteryl esters (CE) (Tabas, I., Rosoff, W. J., and Boykow, G. C (1988) J. Biol. Chem. 263, 1266-1272). As the route of delivery of FC to ACAT was not well characterized, we examined this route in the present study. In foam cells derived from rat peritoneal macrophages by preincubation with acetyl-LDL, esterification of the exogenously labeled 3HFC was low (1.3% of total labeled cholesterol). In contrast, when cells were first labeled with exogenous 3HFC and then chased with acetyl-LDL, the esterification was more extensive (9.2% of the total labeled cholesterol). During this experiment a significant portion of cellular 3HFC was released into the medium (up to 33.4% of the total labeled cholesterol). In experiments using a two-compartment chamber in which cells in the lower and upper chambers were separated by filter paper yet the cells in both compartments could communicate without direct contact, 3HFC released into the medium was biologically active and could serve as an efficient substrate for ACAT. Thus, when acetyl-LDL is not included in culture medium, FC delivery from the macrophage plasma membrane to ACAT is not enhanced, whereas in the presence of acetyl-LDL, plasma membrane FC released and bound to acetyl-LDL may re-enter the cells, possibly through the scavenger receptor. This would provide a significant route for CE synthesis in macrophages. |
| Expression and characterization of recombinant caveolin. Purification by polyhistidine tagging and cholesterol-dependent incorporation into defined lipid membranes Li, S., K. S. Song, et al. (1996), J Biol Chem 271(1): 568-73. Abstract: Caveolin, a 22-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin has been proposed to function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Because of its unusual membrane topology, both the N- and C-terminal domains of caveolin remain entirely cytoplasmic and are not subject to luminal modifications that are accessible to other integral membrane proteins. Under certain conditions, caveolin also exists in a soluble form as a cytosolic protein in vivo. These properties make caveolin an attractive candidate for recombinant expression in Escherichia coli. Here, we successfully expressed recombinant full-length caveolin in E.coli. A polyhistidine tag was placed at its extreme C terminus for purification by Ni(2+)-nitrilotriacetic acid affinity chromatography. Specific antibody probes demonstrated that recombinant caveolin contained a complete N and C terminus. Recombinant caveolin remained soluble in solutions containing the detergent octyl glucoside and formed high molecular mass oligomers like endogenous caveolin. By electron microscopy, recombinant caveolin homo-oligomers appeared as individual spherical particles that were indistinguishable from endogenous caveolin homo-oligomers visualized by the same technique. As recombinant caveolin behaved as expected for endogenous caveolin, this provides an indication that recombinant caveolin can be used to dissect the structural and functional interaction of caveolin with other protein and lipid molecules in vitro. Recombinant caveolin was efficiently incorporated into lipid membranes as assessed by floatation in sucrose density gradients. This allowed us to use defined lipid components to assess the possible requirements for insertion of caveolin into membranes. Using a purified synthetic form of phosphatidylcholine (1,2-dioleoylphosphorylcholine), we observed that incorporation of caveolin into membranes was cholesterol-dependent; the addition of cholesterol dramatically increased the incorporation of caveolin into these phosphatidylcholine-based membranes by approximately 25-30-fold. This fits well with in vivo studies demonstrating that cholesterol plays an essential role in maintaining the structure and function of caveolae. Further functional analysis of these reconstituted caveolin-containing membranes showed that they were capable of recruiting a soluble recombinant form of G(i)2 alpha. This is in accordance with previous studies demonstrating that caveolin specifically interacts directly with multiple G protein alpha-subunits. Thus, recombinant caveolin incorporated into defined lipid membranes provides an experimental system in which the structure, function, and biogenesis of caveolin-rich membrane domains can be dissected in vitro. |
| Expression and localization of matrix metalloproteinase-12 in the aorta of cholesterol-fed rabbits: relationship to lesion development Matsumoto, S., T. Kobayashi, et al. (1998), Am J Pathol 153(1): 109-19. Abstract: Degradation of extracellular matrix (ECM) proteins in the aorta is a critical step for the development of atherosclerosis. Expression of matrix metalloproteinase (MMP)-12 (macrophage elastase), an elastin-degrading proteinase in the MMP family, was investigated in the thoracic aorta of rabbits fed a 1% cholesterol-containing diet for 16 weeks. In the atherosclerotic lesions, MMP-12 was produced abundantly at both the mRNA and protein levels, whereas no expression was observed in the normal rabbit aortas. The principal source of MMP-12 was macrophage foam cells (MFCs) that had infiltrated the atherosclerotic intima; this was demonstrated in both in vitro culture studies of MFCs purified from atherosclerotic lesions and immunohistochemical studies of aortic lesions. Additional biochemical studies using recombinant rabbit MMP-12 revealed that MMP-12 digested elastin, type IV collagen, and fibronectin and also activated MMP-2 and MMP-3. Expression of MMP-12 by human macrophage cell lines was increased by stimulation with acetylated low-density lipoprotein, implying augmentation of MMP-12 production during foam cell formation. Increased expression of MMP-12 in atherosclerotic lesions, concomitant with foam cell generation, which triggers the acceleration of ECM breakdown, is likely to be a critical step in the initiation and progression of the atherosclerotic cascade. |
| Expression and localization of the proteoglycan decorin during the progression of cholesterol induced atherosclerosis in Japanese quail: implications for interaction with collagen type I and lipoproteins Jarrold, B. B., W. L. Bacon, et al. (1999), Atherosclerosis 146(2): 299-308. Abstract: The temporal and spatial distribution, and relative levels of the proteoglycan decorin and collagen type I were examined during the progression of atherosclerosis in the dorsal aortas of Japanese quail selected for cholesterol induced atherosclerosis. The quail were placed on either a control or 0.5% added cholesterol diet at approximately 16 weeks of age. Dorsal aortas were collected at 1- or 2-week intervals over a 15-week period after initiating cholesterol feeding. Biochemical analysis for decorin and collagen type I showed that both increased in the cholesterol-fed birds compared to control-fed birds beginning at 9 weeks and continued through the duration of the study. Through immunohistochemical staining for decorin and collagen type I, the spatial localization of decorin and collagen type I in control and less severe plaques in cholesterol-fed birds was most prominent in the arterial adventitia. However, in severe atherosclerotic plaques, decorin was localized in foam cell regions and collagen type I was found surrounding the foam cell regions where decorin accumulated. These results demonstrated a localization of decorin in the core of the atherosclerotic plaque foam cell region with collagen type I being located on the plaque surface. |
| Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms that stimulate cellular cholesterol efflux to high density lipoprotein Nakamura, K., M. A. Kennedy, et al. (2004), J Biol Chem 279(44): 45980-9. Abstract: The murine Abcg1 gene is reported to consist of 15 exons that encode a single mRNA (herein referred to as Abcg1-a) and protein. We now demonstrate that (i) the murine gene contains two additional coding exons downstream of exon 1, (ii) transcription involves the use of multiple promoters, and (iii) the RNA undergoes alternative splicing reactions. As a result, three mRNAs are expressed that encode three putative protein isoforms that differ at their amino terminus. ABCG1 transcripts are induced in vivo in multiple tissues in response to the liver X receptor ligand T0901317. Identification and characterization of four liver X receptor response elements in the intron downstream of exon 2 provides a mechanism by which this induction occurs. Importantly, cholesterol efflux to high density lipoprotein was stimulated following transfection of Hek293 cells with plasmids encoding individual ABCG1 isoforms. In situ hybridization studies in embryonic day 11.5-15.5 mouse embryos revealed strong expression of ABCG1 transcripts in the olfactory epithelium, hind brain, eye, and dorsal root ganglia. The relatively high levels of expression in neuronal tissues and the eye suggest that ABCG1-dependent cholesterol efflux may be critical for normal neuronal function in addition to its role in macrophages. |
| Expression of 7 alpha-hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression Dueland, S., J. D. Trawick, et al. (1992), J Biol Chem 267(32): 22695-8. Abstract: The goal of this study was to understand why the expression of low density lipoprotein (LDL) receptors by the liver is poorly down-regulated by cholesterol. We examined the hypothesis that 7 alpha-hydroxylase may indirectly induce the expression of the LDL receptor by metabolizing, i.e. inactivating oxysterol repressors. Non-hepatic Chinese hamster ovary cells, transfected with a plasmid encoding 7 alpha-hydroxylase, expressed both the mRNA and functional activity of this liver-specific enzyme. In the presence of 5% serum, expression of the LDL receptor by transfected cells was > 20 times that of non-transfected cells despite a 50% increased content of cholesterol ester. Both cell types displayed an almost complete repression of the LDL receptor by the oxysterol 25-hydroxycholesterol, suggesting that transcriptional control of the LDL receptor gene remained intact in the transfected cells. However, only cells expressing 7 alpha-hydroxylase showed a derepression of the LDL receptor with time. This transient sensitivity to 25-hydroxycholesterol repression was attributed to a 3-fold greater rate of metabolism of 3H25-hydroxycholesterol. The paradoxical induction of LDL receptor mRNA in transfected cells having greater amounts of cholesterol esters suggests that 7 alpha-hydroxylase may preferentially use oxysterols rather than cholesterol as substrates. The combined data are consistent with the proposal that 7 alpha-hydroxylase indirectly induces the LDL receptor gene by metabolizing (inactivating) oxysterol repressors. Liver-specific expression of 7 alpha-hydroxylase can account for the relative resistance of hepatic LDL receptors to down-regulation. |
| Expression of ABCG5 and ABCG8 is required for regulation of biliary cholesterol secretion Yu, L., S. Gupta, et al. (2005), J Biol Chem 280(10): 8742-7. Abstract: The major pathway for elimination of cholesterol in mammals is via secretion into bile. Biliary cholesterol secretion is mediated by the ATP-binding cassette (ABC) transporters ABCG5 (G5) and ABCG8 (G8) and is stimulated by cholesterol and by the non-cholesterol steroids cholate and diosgenin. To define the relationship between G5G8 expression and biliary cholesterol secretion, we measured G5 and G8 mRNA levels and biliary cholesterol concentrations in genetically manipulated mice expressing 0, 1, 2, 5, 10, or 16 copies of the two genes. Biliary cholesterol levels varied directly with G5G8 copy number and hepatic mRNA levels over a >16-fold range. Thus neither delivery of cholesterol to the transporter nor levels of cholesterol acceptors in bile were limiting under these conditions. In wild-type mice, cholate and diosgenin both increased biliary cholesterol concentrations 2-3-fold. The increase in biliary cholesterol content was dependent on expression of G5 and G8; neither steroid increased biliary cholesterol levels in G5G8-/- mice. Cholate treatment was associated with a farnesoid X receptor (FXR)-dependent increase in hepatic mRNA and protein levels of G5 and G8. In contrast to cholate, diosgenin treatment did not affect G5G8 expression. Diosgenin increased the expression of several pregnane X receptor (PXR) target genes and the choleretic effect of diosgenin was reduced by approximately 70% in PXR knock-out mice. Thus G5 and G8 are required to modulate biliary cholesterol secretion in response to cholate and diosgenin, but the choleretic effects of these two steroids are mediated by different mechanisms requiring FXR and PXR, respectively. |
| Expression of apolipoprotein E in cholesterol-loaded macrophages of extrahepatic tissues during experimental hypercholesterolemia Polanco, J. I., M. T. Berciano, et al. (1995), Life Sci 56(22): 1865-75. Abstract: To study the expression of extrahepatic apolipoprotein E (apoE) under hypercholesterolemic conditions, apoE mRNA levels were evaluated in 14 tissues of rabbits fed on a cholesterol rich diet and compared to age-matched control animals. In hypercholesterolemic rabbits apoE expression was significantly induced in adipose tissue, adrenals, aorta, lung and spleen. The increase in apoE mRNA levels in lung and spleen was associated with the presence of cholesterol-loaded macrophages. These cells were found to express high levels of apoE mRNA as demonstrated by in situ mRNA hybridization. Our results suggest that extrahepatic tissues would be partially responsible for the rise in serum apoE levels detected under hypercholesterolemic conditions. |
| Expression of caveolin-1 enhances cholesterol efflux in hepatic cells Fu, Y., A. Hoang, et al. (2004), J Biol Chem 279(14): 14140-6. Abstract: HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane. |
| Expression of cholesterol 7alpha-hydroxylase restores bile acid synthesis in McArdle RH7777 cells Labonte, E. D., Q. Li, et al. (2000), Arch Biochem Biophys 381(2): 273-7. Abstract: Bile acid synthesis involves several enzymes and occurs only in liver cells. The first and rate-determining step is catalyzed by cholesterol 7alpha-hydroxylase (cyp7a). McArdle RH7777 hepatoma cells do not synthesize bile acids and do not express the cyp7a gene. A synthetic cyp7a gene was stably expressed in this cell line to determine if restoration of cyp7a activity is sufficient to reconstitute the bile acid synthetic pathway. The transfected cells contained the recombinant cyp7a mRNA and the corresponding protein. Microsomes from recombinant cells converted cholesterol into 7alpha-hydroxycholesterol, indicating that the recombinant enzyme was active. Radiolabeled bile acids, originated from exogenously supplied radiolabeled cholesterol, were detected in the culture medium of recombinant cells. Thus, expression of cyp7a is sufficient in restoring bile acid synthesis in McArdle RH7777 cells. The results also show that the additional complement of enzymatic activities required to convert cholesterol into bile acids has remained active in this cell line. |
| Expression of cholesterol sulfotransferase (SULT2B1b) in human platelets Yanai, H., N. B. Javitt, et al. (2004), Circulation 109(1): 92-6. Abstract: BACKGROUND: Cholesterol sulfate, the most important sterol sulfate in the human circulation, has emerged as a multifaceted molecule. Among its many demonstrated regulatory actions is its ability to influence blood clotting and fibrinolysis. Additionally, cholesterol sulfate is a constituent of human platelets, where it has been shown to support platelet aggregation. METHODS AND RESULTS: We have documented the presence of the enzyme (SULT2B1b) that sulfonates cholesterol in human platelets and examined the influence of plasma lipoproteins on the expression and activity of this enzyme. SULT2B1b mRNA was detected by reverse transcription-polymerase chain reaction and found to be the only steroid/sterol sulfotransferase expressed in these discoid anucleate particles. Using real-time polymerase chain reaction for quantification, we found that the level of SULT2B1b mRNA in platelets was maintained at 4 degrees C but substantially diminished over a period of 4 hours at 37 degrees C. The loss of SULT2B1b mRNA, however, was markedly reduced in the presence of HDL but not LDL. The stabilizing influence of HDL was attributable specifically to its apolipoprotein (apo) A-I component, whereas apoA-II and apoE were without effect. Importantly, there was a direct correlation between platelet SULT2B1b mRNA and protein levels in the presence or absence of lipoprotein that was reflected in enzymatic activity and cholesterol sulfate production. CONCLUSIONS: Human platelets selectively express SULT2B1b, the physiological cholesterol sulfotransferase. Furthermore, the stability of SULT2B1b mRNA and protein in platelets maintained at 37 degrees C is subject to regulation by the apoA-I component of HDL. |
| Expression of cholesterol sulfotransferase (SULT2B1b) in human skin and primary cultures of human epidermal keratinocytes Higashi, Y., H. Fuda, et al. (2004), J Invest Dermatol 122(5): 1207-13. Abstract: Cholesterol sulfate is a highly amphipathic molecule that is present in a relatively high concentration in the epidermis of human skin, particularly in the granular layer. The physiologic significance of this finding, however, is not well-understood. Therefore, we examined expression of the gene encoding for the enzyme that sulfonates cholesterol (SULT2B1b). Of the three enzymes known to sulfonate steroids/sterols, only the SULT2B1b isozyme was detected in cultures of normal human epidermal keratinocytes (NHEK) in response to Ca(2+)-induced terminal differentiation as well as by normal human epidermal tissue. Immunocytochemical analysis of normal skin as well as specific skin disorders was carried out. In normal skin, the expression of SULT2B1b was localized to the granular layer of the epidermis similar to that of filaggrin, an acknowledged late marker of differentiation and in contrast to that of involucrin, an early marker of terminal differentiation, which was expressed throughout the suprabasal region. The confinement of SULT2B1b to the granular layer coincides with this being the area with the highest cholesterol sulfate content suggesting that the physiologic action of cholesterol sulfate is likely carried out in this region of the living epidermis. Additionally, 88% of cholesterol sulfate in NHEK was membrane-associated further suggesting a cellular location for cholesterol sulfate action. |
| Expression of cholesterol-7alpha-hydroxylase in murine macrophages prevents cholesterol loading by acetyl-LDL Moore, G. L. and R. A. Davis (2002), J Lipid Res 43(4): 629-35. Abstract: Unlike macrophages, the hepatic parenchymal cells express cholesterol-7 alpha-hydroxylase (CYP7A1) which regulates the conversion of cholesterol into bile acids, the major quantitative pathway maintaining cholesterol homeostasis. We examined if CYP7A1 expression in RAW 264.7 macrophages could prevent the accumulation of cholesterol when they were incubated with acetyl-LDL. A single cell clone (designated as 7 alphaRAW cells) that stably expresses rat CYP7A1 displayed similar rates of growth as non-transfected RAW cells. The CYP7A1 enzymatic activity expressed by microsomes obtained from 7 alphaRAW cells was similar to that expressed by microsomes obtained from mouse liver. Incubating non-transfected RAW with increasing amounts of acetyl-LDL caused a parallel accumulation of cholesterol, whereas 7 alphaRAW cells displayed a complete resistance to cholesterol accumulation. 7 alphaRAW cells displayed increased expression of both ABCA1 mRNA (3.1-fold, P < 0.001) and ABCG1 mRNA (2.2-fold, P < 0.01), whereas the expression of scavenger receptor class A mRNA was unchanged. 7 alphaRAW cells also displayed small but significant increases in the rate of efflux of (3)Hcholesterol to both delipidated apolipoprotein A1 and to HDL.Thus, CYP7A1 expression in RAW cultured macrophages prevented the accumulation of cholesterol from acetyl-LDL via both increased metabolism and efflux of cholesterol. |
| Expression of cholesteryl ester transfer protein in human atherosclerotic lesions and its implication in reverse cholesterol transport Zhang, Z., S. Yamashita, et al. (2001), Atherosclerosis 159(1): 67-75. Abstract: Reverse cholesterol transport (RCT) is the major protective system against atherosclerosis. In this system, cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of neutral lipids between lipoproteins in plasma. We reported the pathophysiological significance of CETP by clinical studies with genetic CETP deficiency, showing that this protein plays a crucial role in the RCT system. However, information about the expression of this protein in the initial step of RCT, macrophages (Mphi) in the blood vessels, is still very limited. In the present study, we have performed immunohistochemical analyses on the expression of CETP in human atherosclerotic lesions. The immunoreactive mass of CETP was abundantly detected in foam cells in human aortic and coronary atherosclerotic lesions, but not in the normal arterial wall. A double immunostaining showed that the majority of CETP-positive foam cells were derived from Mphi and a minor population appeared to derive from smooth muscle cells. Transient transfection of CETP cDNA into COS-7 cells showed that high density lipoprotein (HDL)-mediated efflux of free cholesterol from the cells expressing CETP was much higher than that from mock-transfected cells, while uptake of HDL-lipids was not affected in cells transfected with CETP cDNA. Efflux of free cholesterol from the Mphi obtained from CETP deficiency was significantly decreased compared with that from normal subjects. These data indicate that CETP is expressed in Mphi in the atherosclerotic lesions and may possess an anti-atherogenic function to remove cholesterol from the cells, suggesting another role of CETP at the initial step of RCT. |
| Expression of elastase activity by human monocyte-macrophages is modulated by cellular cholesterol content, inflammatory mediators, and phorbol myristate acetate Rouis, M., F. Nigon, et al. (1990), Arteriosclerosis 10(2): 246-55. Abstract: We investigated the effects of a number of stimulatory agents on the production of both cell-associated and extracellular elastase-type enzymes on human monocyte-macrophages in vitro and of the modulation of such effects by modification of cellular cholesterol content. The stimulatory agents included phorbol myristate acetate (PMA) and the inflammatory mediators, lipopolysaccharide (LPS), opsonized zymosan (OZ), and platelet activating factor (PAF). Using the synthetic substrate, N-succinyl-trialanyl-paranitroanilide (SANA), we detected cell-associated elastase-like activity in monocyte-derived macrophages. Such activity increased markedly with cell maturation over the period from 5 to 15 days of adherence culture. While PAF (10 micrograms/ml) and LPS (10 micrograms/ml) were without effect on cell-associated elastase-like activity in macrophages, PMA (100 ng/ml) and OZ (1 mg/ml) markedly stimulated such activity in cells cultured for 15 days. Furthermore, a fivefold increase in the cell-associated elastase-like activity of macrophages occurred upon cholesterol loading of the cells with acetylated low density lipoprotein (AcLDL). By contrast, this activity was markedly diminished upon depletion of cellular cholesterol content after incubation with high density lipoprotein (HDL3). Latent elastinolytic activity in the culture medium was detected by use of a radioactive substrate, insoluble 3H-elastin, after initial tryptic treatment of the medium. Such latent elastase activity was secreted only by activated macrophages; the relative potency of stimulation was: PMA greater than LPS = PAF greater than OZ. Increase in cellular cholesterol content alone markedly enhanced the secretion of elastase (from undetectable levels to 28 ng of 3H-elastin degraded/hr/micrograms DNA). In all cases, both the cell-associated and secreted latent elastinolytic activities were due to metalloproteases, in view of their 90% inhibition by 2 mM EDTA. Cholesterol-loaded macrophages, which displayed an approximately 40-fold increase in total cholesterol content as compared to control cells, remained sensitive to the action of activators of OZ and PMA, while LPS and PAF exerted only weak effects. Our data indicate that cellular cholesterol content and inflammatory mediators are effective stimulants of the production and secretion of elastase-type enzymes by human monocyte-macrophages. Among these factors, cellular cholesterol content, OZ, PAF, and LPS may represent factors of relevance to the inflammatory role of the macrophage in atherogenesis and more specifically to the alteration of elastin structure in the extracellular matrix of the vessel wall. |