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L Kritharides

Publications and source records attributed to L Kritharides.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of cholesterol efflux by 7-ketocholesterol: comparison between cells, plasma membrane vesicles, and liposomes as cholesterol donors.

Cholesterol removal from lipid-loaded macrophages is an important, potentially antiatherogenic process, and we have previously shown that an oxysterol, 7-ketocholesterol (7K), can impair efflux to lipid-free apoprotein A-1 (apoA-1). This publication investigates whether incorporation of 7K into membranes could account for this impairment of cholesterol efflux. Cholesterol efflux was studied from lipoprotein-loaded THP-1 cells, from plasma membrane vesicles obtained from these cells, and from artificial, protein-free liposomes. Impairment of cholesterol efflux by 7K was observed for all cholesterol donor systems whether measured as decline in cholesterol removal rates or as the percentage mass of total cellular cholesterol exported. 7-Ketocholesterol itself was not removed by apoA-1 from any of the cholesterol donor systems. Increasing membrane cholesterol content increased the rate of cholesterol removal by apoA-1 (as seen with plasma membrane vesicles), the quantity of cholesterol removed at equilibrium (liposomes), or both (whole cells). Although the minimum inhibitory 7K concentrations varied between the cholesterol donor systems, 7K inhibited cholesterol efflux in all systems. It was concluded that 7K induces alteration in membranes which decreased the efficiency of cholesterol efflux and the quantity of removed cholesterol induced by apoA-1. As cell membrane proteins are not essential for cholesterol efflux in these systems, the impairment of such by 7K suggests that its effect on membrane lipid composition and its structure are key regulatory elements in this efflux process.

Biological Transport↗
PubMed2001

A kinetic model to evaluate cholesterol efflux from THP-1 macrophages to apolipoprotein A-1.

The kinetics (0 to 3 h) of cholesterol efflux to delipidated apolipoprotein A-1 were investigated, and the experimental data were best fitted to a mathematical model that involves two independent pathways of cholesterol efflux. The first pathway with a rate constant of 4.6 h(-1) is fast but removes only 3-5% of total cholesterol. After preconditioning apoA-1, it was found that this pathway remains, and hence it is a property of the cholesterol-loaded cells rather than due to modification on the apolipoprotein. This fast initial efflux does not seem to contribute to cholesterol efflux at later stages (>1 h) where a second pathway predominates. However, the fast initial efflux pool can be restored if apoA-1 is withdrawn. The second slower pathway (k(membrane--media) = 0.79 h(-1)) is associated with cholesterol ester hydrolysis whose rate constant could be experimentally verified (k(cal) = 0.43, k(exp) = 0.38 +/- 0.05). The model suggests that two different plasma membrane domains are involved in the two pathways. Loading of the cells with an oxysterol, 7-ketocholesterol (7K), inhibits efflux from both pathways. The model predicts that 7K decreases the initial efflux by decreasing the available cholesterol (by possibly affecting lipid packing), while all rate constants in the second pathway are decreased. In conclusion, the kinetic model suggests that cholesterol efflux to apoA-1 is a two-step process. In the first step, some of the plasma membrane cholesterol contributes to a fast initial efflux (possibly from lipid rafts) and leads to a second pathway that mobilizes intracellular cholesterol mobilization.

Acetylation↗
PubMed2000

Oxidation of methionine residues to methionine sulfoxides does not decrease potential antiatherogenic properties of apolipoprotein A-I.

The initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I(+32)) may affect the antiatherogenic properties of HDL, because it has been suggested that Met(86) and Met(112) are important for cholesterol efflux and Met(148) is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I(+32) and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I(+32) to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I(+32) from reconstituted HDL. Met(86) and Met(112) were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I(+32). Selective oxidation did not alter the alpha-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I(+32). Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I(+32) than unoxidized apoA-I. Also, compared with native apoA-I, apoA-I(+32) had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [(3)H]cholesterol, [(3)H]phospholipid, and [(14)C]alpha-tocopherol from lipid-laden human monocyte-derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and alpha-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic.

Apolipoprotein A-I↗
PubMed2000

Metabolism of oxidized LDL by macrophages.

Oxidation products of lipids and proteins are found in atherosclerotic plaque and in macrophage foam cells. Macrophages avidly endocytose in-vitro oxidized LDL and accumulate sterols. What is the evidence that such a process is involved in in-vivo foam cell formation? The present review surveys current knowledge on the metabolism of oxidized LDL by macrophages, and the types, amounts and location of oxidation products that accumulate in these cells. Comparable studies of lesion lipoproteins and foam cells indicate that limited extracellular lipoprotein oxidation, perhaps followed by more extensive intracellular oxidation subsequent to uptake by macrophages, is a more likely scenario in vivo.

Animals↗
PubMed2000

Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes.

Cholesterol- and cholesteryl ester-rich macrophage foam cells, characteristic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL). However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationship to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxLDL-loaded macrophages, and to compare this with macrophages loaded with acetylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized lipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), the major oxysterol present in OxLDL). Both cholesterol and 7KC and their esters were measured in macrophages after uptake of these modified lipoproteins. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, with no evidence of retention of either free sterol within lysosomes. The cells also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholesteryl esters. The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by fatty acid limitation. OxLDL-loaded macrophages also contained large (approximately 40-50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensitive to ACAT inhibition, very stable and located in lysosomes, indicating resistance to lysosomal esterases. Macrophages loaded with OxLDL do not accumulate free sterols in their lysosomal compartment, but do accumulate lysosomal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam cells would represent a pool of sterols that is particularly resistant to removal.

Animals↗
PubMed1999

Apolipoprotein A-I stimulates secretion of apolipoprotein E by foam cell macrophages.

Apolipoprotein A-I (apoA-I) overexpression inhibits atherogenesis in mice, and apolipoprotein E (apoE) secreted by foam cell macrophages may exert antiatherogenic effects within the arterial wall. We hypothesized that interaction between apoA-I and apoE contributed to the antiatherogenic properties of apoA-I, and therefore investigated whether apoA-I stimulated secretion of apoE by foam cell macrophages. Cholesterol enrichment of primary murine and human macrophages increased spontaneous apoE secretion 2-fold, as quantified by Western blot and chemiluminescence detection. Human apoA-I caused a further marked increase of apoE secretion from both murine (3.8-fold, p < 0.01) and human (3.2-fold, p = 0.01) foam cells in a time- and concentration- dependent manner, and this increase was confirmed by immunoprecipitation of [(35)S]methionine-labeled macrophage apoE. The protein synthesis inhibitor cycloheximide, but not the transcription inhibitor actinomycin D, markedly inhibited apoE secretion to apoA-I (73.1 +/- 9.8% inhibition at 4 h) and completely suppressed apoE secretion beyond 4 h. Pretreatment of macrophages with Pronase inhibited initial apoA-I-mediated apoE secretion by 70.5 +/- 6.5% at 2 h, but by 8 h apoA-I-induced apoE secretion was the same in Pronase-pretreated and non-pretreated cells. Non-apolipoprotein-mediated cholesterol efflux induced by trimethyl-beta cyclodextrin did not enhance apoE secretion, whereas phospholipid vesicles inducing the same degree of cholesterol efflux substantially enhanced apoE secretion, and apoA-I and phospholipid vesicles in combination demonstrated additive induction of apoE secretion. We conclude that apoA-I concurrently stimulates apoE secretion and cholesterol efflux from foam cell macrophages and that lipoprotein-derived apoA-I may enhance local secretion and accumulation of apoE in atherosclerotic lesions.

Animals↗
PubMed1998

Cholesterol metabolism and efflux in human THP-1 macrophages.

This study has investigated in detail factors regulating accumulation, esterification, and mobilization of cholesterol in human THP-1 macrophages. Human THP-1 monocytes were differentiated into macrophages and then cholesterol enriched by exposure to acetylated LDL (AcLDL), together with [3H]free cholesterol (FC). Although THP-1 macrophages accumulated FC and esterified cholesterol (EC), assessed by both mass and radioactivity, cellular EC always demonstrated a much lower specific activity (cpm/ microg) than did cellular FC, and several potential causes of this finding were investigated. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) during loading decreased cell [3H]EC by 95+/-1.4% but decreased cell EC mass by only 66.0+/-4.0%, indicating that some intracellular undegraded AcLDL-derived EC was present in these cells. Esterification of [3H]oleate to EC in THP-1 cells loaded with AcLDL was 2.0 nmol x mg-1 x h-1, consistent with previous literature. However, EC, triglyceride, and phospholipid fractions respectively contained 1.0+/-0.07%, 80.0+/-0.5%, and 18.9+/-0.3% of cell [3H]oleate, indicating triglycerides were much more metabolically active than EC. In addition, the mass of triglyceride in THP-1 macrophages exceeded that of EC both before and after exposure to AcLDL. Esterification of nonlipoprotein-derived cholesterol was compared in THP-1 cells and nonhuman Fu5AH, CHO, and RAW macrophage cells. Whereas the nonhuman cell lines all esterified over 30% of 2-hydroxypropyl-beta-cyclodextrin (hp-ss-CD)-delivered cholesterol within 6 hours, THP-1 cells esterified <8.0% of incorporated cholesterol. Kinetics of cholesterol efflux from AcLDL-loaded THP-1 cells were first investigated after loading with only FC, and interactions between efflux and EC hydrolysis were further assessed after loading cells with both EC and FC. Over 24 hours, human apolipoprotein (apo) A-I, apoHDL reconstituted with phosphatidylcholine, and HDL3 respectively removed 46.6+/-3.7%, 61. 3+/-3.4%, and 76.4+/-10.1% of [3H]FC from FC-enriched THP-1 cells. Cholesterol efflux to apoA-I was saturated by 24 hours and was enhanced by using apoA-I-phospholipid instead of pure apoA-I. Kinetic modeling identified that 97% of effluxed FC derived from a slow pool, with a T1/2 ranging from 27.7 hours for HDL to 69.3 hours for apoA-I. Although efflux enhanced net clearance of EC, hydrolysis of EC during concurrent inhibition of ACAT was unaffected by cholesterol efflux. Supplementation of THP-1 cultures with cAMP to stimulate hormone-sensitive lipase did not significantly enhance net hydrolysis of EC or cholesterol efflux. In conclusion, human THP-1 macrophages contain a large and metabolically active pool of triglyceride and a relatively inactive pool of EC. The low specific activity of EC relative to FC is contributed to by reduced esterification of FC, slow hydrolysis of EC, and accumulated lipoprotein EC. The relative inactivity of the EC pool may further contribute to already impaired cholesterol efflux from these cells. Net cholesterol efflux from human macrophages is achieved by pure apoA-I and is substantially further enhanced by the presence of phospholipid in acceptor particles.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1998

Accumulation and metabolism of low density lipoprotein-derived cholesteryl linoleate hydroperoxide and hydroxide by macrophages.

Cholesteryl linoleate hydroperoxide (CLOOH) and hydroxide (CLOH) are present in human atheroma. The intracellular metabolism of low density lipoprotein (LDL)-derived CLOOH and CLOH remain undefined because extensive free radical-mediated LDL oxidation, which modifies LDL apolipoprotein B sufficiently to allow endocytosis by the scavenger receptor (ScR), also degrades CLOOH and CLOH. This problem was approached by first acetylating LDL lysine residues (AcLDL) to achieve protein modification, then exposing AcLDL to the aqueous radical donor 2,2'-azobis(2-amidinopropane) HCl (AAPH), to generate mildly oxidized AcLDL (OxAcLDL). Murine peritoneal macrophages incubated with OxAcLDL accumulated large quantities of CE and small, non-toxic quantities of CLOOH and CLOH in a time- and concentration-dependent manner, and accumulation was inhibited by fucoidin. Inhibition of acyl CoA: cholesterol acyltransferase during loading did not inhibit the accumulation of either CLOOH or CLOH, whereas NH4Cl decreased intracellular clearance of accumulated CLOOH from 68.3 +/- 1.7% to 35.3 +/- 1.0% over 12 h, suggesting lysosomal or pre-lysosomal accumulation. Intracellular clearance of unoxidized lipoprotein-derived CE decreased from 84.0 +/- 5.9% to 43.1 +/- 2.3% over 12 h when cells were loaded with AcLDL or OxAcLDL, respectively. Aggregation of mildly oxidized LDL, even without acetylation, also promoted cellular accumulation of CLOOH and CLOH. We conclude that intracellular accumulation of cholesteryl linoleate hydroperoxide and cholesteryl linoleate hydroxide can follow charge modification or aggregation of mildly oxidized LDL, and that LDL-derived oxidation products may inhibit hydrolysis of LDL-derived CE in foam cell macrophages.

Acetylation↗
PubMed1997

Effects of CSF-1 on cholesterol accumulation and efflux by macrophages.

To assess whether human monocyte-specific colony-stimulating factor (CSF-1) might influence atherogenesis, CSF-1-induced macrophage responses that might contribute to enhanced clearance of low-density lipoprotein (LDL) or modified LDL were investigated. Careful account was made of cell preservation and increases in cell volume and protein (representing increased cell surface area, and thus endocytically active membrane) during culture with CSF-1. This permitted distinction between selective and nonspecific effects of CSF-1, the latter paralleling increases in cellular mass and volume. CSF-1 enhanced mouse peritoneal macrophage survival in vitro during exposure to lipoprotein-deficient serum with or without native LDL or acetylated LDL (Ac-LDL), as judged by maintenance of cellular DNA and cell numbers. In the presence of copper-oxidized LDL (Ox-LDL), such effects were very slight. In all conditions, CSF-1 increased cellular protein content. CSF-1 increased the uptake of both Ac-LDL and Ox-LDL calculated per culture, but this was entirely explicable by the increased cell protein, indicating that there was no selective enhancement of scavenger receptor or other routes for uptake of the modified LDLs. Similarly, CSF-1 also increased the accumulation of cholesterol and its esters nonspecifically. CSF-1 did have a marked and specific effect on the composition of cholesterol esters, decreasing the proportion of polyunsaturated esters relative to monounsaturated and saturated esters. Finally, cholesterol efflux induced by apolipoprotein A1 from Ac-LDL-loaded macrophages was not influenced by CSF-1. Thus, the enhanced macrophage catabolism of modified LDLs by CSF-1 is part of a nonspecific action on the cells but could contribute to a reduction in circulating cholesterol, observed in some situations of CSF-1 presentation in humans.

Animals↗
PubMed1996

Hydroxypropyl-beta-cyclodextrin-mediated efflux of 7-ketocholesterol from macrophage foam cells.

Atherosclerosis involves the arterial accumulation of lipid-laden "foam cells" containing oxidized and unoxidized sterols and their esters (Mattsson-Hulten, L., Lindmark, H., Diczfalusy, U., Bjorkhem, I., Ottosson, M., Liu, Y., Bondjers, G., and Wiklund, O. (1996) J. Clin. Invest. 97, 461-8). Oxidized sterols are probably critical to atherogenesis because they inhibit cholesterol removal from cells and are cytotoxic. We recently reported that there is deficient induction of cellular cholesterol efflux by apolipoprotein A-I, the main initial acceptor of cellular cholesterol from macrophages loaded in vitro with oxidized low density lipoprotein (Kritharides, L., Jessup, W., Mander, E., and Dean, R. T. (1995) Arterioscler. Thromb. 15, 276-289). There was an even more marked impairment of the release of 7-ketocholesterol which is a major oxysterol in these cells and in human atherosclerotic lesions. Here we show that hydroxypropyl-beta-cyclodextrin can induce selective efflux of 7-ketocholesterol. Efflux of 7-ketocholesterol was time and concentration dependent, and the rate of its removal was 50-fold greater for hydroxypropyl-beta-cyclodextrin than for apolipoprotein A-I. Over a defined range of concentrations (0-5 mg/ml), efflux of 7-ketocholesterol was preferred over that of cholesterol and occurred without cell toxicity. Efflux of free 7-ketocholesterol was associated with decreased intracellular free and esterified 7-ketocholesterol. Hydroxypropyl-beta-cyclodextrin also enhanced efflux of other oxysterols. The physical solubilization of 7-ketocholesterol by the cyclodextrin was much greater than that of cholesterol, in accordance with its differential effects on efflux. These data highlight the importance of extracellular sterol solubilization in the efflux of cellular oxysterols and the mobilization of intracellular free and esterified oxysterol pools in macrophages loaded with oxidized low density lipoprotein. Synthetic sterol-solubilizing agents such as hydroxypropyl-beta-cyclodextrin are thus potential prototypes for the further development of oxysterol-removing agents.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1996

Sterol efflux is impaired from macrophage foam cells selectively enriched with 7-ketocholesterol.

The aim of the present study was to investigate whether impairment of cholesterol efflux previously found from mouse peritoneal macrophages loaded with oxidized low density lipoprotein (OxLDL) could be ascribed to the presence of oxysterols in these cells. 7-Ketocholesterol (7KC), the major oxysterol present in OxLDL-loaded cells, was selectively incorporated into unoxidized LDL, which was subsequently acetylated to produce a high uptake form. Mouse macrophages incubated with 7KC-enriched acetylated LDL (7kAcLDL) did not reveal cytotoxicity judged by cell protein and trypan blue exclusion. A large proportion of cellular 7KC was esterified, indicating that it is a substrate for acyl CoA:cholesterol acyltransferase. Cholesterol efflux from mouse macrophages loaded with 7kAcLDL, using apoA-I as a sterol acceptor, was impaired in cells containing >50 nmol of 7KC/mg of cell protein compared with cells loaded with oxysterol-free acetylated LDL. Thus impairment of cholesterol efflux could be reproduced in cells loaded with 7kAcLDL containing similar proportions of 7KC as OxLDL. 7KC itself was exported very poorly, even when the levels of 7KC in the cells were low. These results suggest that oxysterols present in foam cells in vitro can affect reverse sterol transport and may be potentially important in foam cell formation in vivo.

Animals↗
PubMed1996

Assessment of left ventricular function after radiofrequency and direct current atrioventricular node ablation.

BACKGROUND: There is limited information available regarding the effect of catheter ablation of the antioventricular (AV) junction on left ventricular (LV) function. Both deterioration and improvement in LV function have been reported following direct current (DC) ablation of the AV junction. The deterioration of LV function following DC ablation of the AV junction may be due to the accompanying barotrauma, DC arcing and direct coagulation, or even the effects of chronic ventricular pacing. If this deterioration of LV function was a result of the accompanying effects of DC shock, the use of radiofrequency ablation (RF) should not result in deterioration of LV function. AIM: To study LV function before and after different methods of AV junction ablation and in patients with chronic ventricular pacing without AV junction ablation. MATERIAL: This study assessed LV function in patients following RF ablation, low energy DC ablation of the AV junction and compared the results with our previously reported finding in patients who had AV junction ablation using high energy DC shock. A group of patients undergoing permanent single chamber ventricular pacemaker implantation without AV junction ablation were selected as controls. METHODS: All patients were paced in the ventricle at 110 beats/minute during LV function assessment by radionuclide angiography. Global LV function and segmental wall motion abnormalities were assessed before, immediately following and three months after ablation. RESULTS: In the high energy DC ablation group, a fall in global LV function (50 +/- 3.0% to 43 +/- 3.0%, p = 0.02) and impairment of segmental wall motion were detected. Low energy DC ablation resulted in segmental wall motion impairment similar to high energy DC but without affecting global ejection fraction (47.0% +/- 6.7 to 45.5% +/- 3.1, p > 0.05). Neither RF ablation (44.0% +/- 3.3 to 45.3% +/- 3.5, p > 0.05), nor chronic pacing (46.7% +/- 4.9 to 47.0% +/- 2.9 p > 0.05) had any effect on global or segmental LV function. CONCLUSIONS: Low energy DC or RF ablation of the AV junction does not affect global LV ejection fraction. The deterioration of global LV function after high energy DC shock ablation appears to be related to the accompanying effects of DC energy and not to the effects of chronic ventricular pacing.

Adult↗
PubMed1995

Macrophages require both iron and copper to oxidize low-density lipoprotein in Hanks' balanced salt solution.

The oxidation of low-density lipoprotein (LDL) may be important in the pathogenesis of atherosclerosis. However, the interactions between cells and metals in promoting LDL oxidation are inadequately understood. A sensitive high-performance liquid chromatography analysis of cholesterol, cholesteryl esters, and their oxidation products was used to identify and accurately measure LDL oxidation achieved in thiol-free Hanks' balanced salt solution (HBSS) at pH 7.4. Mouse peritoneal macrophages inhibited LDL oxidation when incubated in HBSS containing either 10 microM iron or 1 microM copper, but were markedly prooxidant in the presence of both metals. The prooxidant effect of macrophages in the presence of both iron and copper did not require the provision of added disulfides or thiols. Both Fe2+ and macrophages were demonstrated to independently reduce Cu2+ to Cu1+ in HBSS, indicating that the direct reduction of copper by cells or iron may underlie the observed promotion of LDL oxidation by macrophages in this system. We conclude that macrophages can either promote or inhibit metal-mediated LDL oxidation and that externally supplied thiols are not essential to the promotion of LDL oxidation by cells. The presence of both iron and copper may be particularly important for macrophages to promote LDL oxidation in vivo.

Animals↗