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L Kritharides

Publications and source records attributed to L Kritharides.

31 records · Page 2Linked to original sources
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↗
PubMed1995

Apolipoprotein A-I-mediated efflux of sterols from oxidized LDL-loaded macrophages.

Although oxidized low-density lipoprotein (Ox-LDL) can accumulate in macrophages in vitro, generating cholesterol-loaded cells, little attention has been paid to the capacity of such macrophages loaded with OxLDL to export cholesterol and oxidized sterol moieties. In vitro lipid-loaded cells were generated by incubating primary cultures of mouse peritoneal macrophages with acetylated LDL (AcLDL) or OxLDL for 24 hours. The cellular content of native cholesterol, individual cholesteryl esters, and 7-ketocholesterol was determined by high-performance liquid chromatography. These cells were then incubated with medium containing apolipoprotein (apo) A-I and albumin or albumin alone for up to 24 hours; cholesterol and oxidized sterol efflux were measured both in terms of intracellular depletion and extracellular accumulation. Macrophages loaded with AcLDL accumulated cholesterol and large quantities of cholesteryl esters, whereas OxLDL-loaded cells accumulated cholesterol, a number of oxidized compounds (predominantly 7-ketocholesterol), and a relatively small quantity of cholesteryl esters. AcLDL-derived cells released approximately 50% of their total cholesterol (unesterified and esterified) to apo A-I-containing medium over 24 hours in the form of unesterified cholesterol, whereas OxLDL-derived cells released approximately 30% of their total cholesterol and 7% of their total content of 7-ketocholesterol over the same period. There was minimal efflux of any sterol in the absence of apo A-I. The proportions of cholesterol and 7-ketocholesterol released by either AcLDL- or OxLDL-loaded cells were not reduced by inhibiting cellular acyl-CoA:cholesterol acyl transferase using Sandoz 58-035, despite substantial alterations in the proportions of both free cholesterol and (in OxLDL-loaded cells) free 7-ketocholesterol in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

EDTA differentially and incompletely inhibits components of prolonged cell-mediated oxidation of low-density lipoprotein.

The extent to which cells can oxidize LDL may be underestimated because of the use of standard and arbitrary 24 hour in vitro incubations of cells with LDL. Such incubations have resulted in inconsistent results regarding the ability of cell-mediated LDL oxidation to generate relatively advanced oxidation products such as 7-ketocholesterol (7-KC). We studied prolonged oxidation of low density lipoprotein (LDL) by mouse peritoneal macrophages using HPLC measurement of cholesterol, cholesteryl esters and their oxidation products 7-KC and cholesteryl linoleate hydroperoxide (CL-OOH). Cell-mediated oxidation in Ham's F10 consistently followed the successive stages previously described during 24 hour-10 microM copper-mediated LDL oxidation, always generating 7-KC if allowed to proceed for sufficient time. The degree of inhibition of LDL oxidation achieved by metal chelators EDTA and DTPA at more advanced stages of cell-mediated LDL oxidation was not predictable from the published effects of such chelators upon early stages of metal-mediated and cell-mediated LDL oxidation. EDTA and DTPA only incompletely prevented the consumption of cholesteryl esters and the loss of performed CL-OOH when added after cell-mediated LDL oxidation was established, while effectively concurrently inhibiting the generation of 7-KC. These data indicate that progressive cell-mediated peroxidation of LDL cholesteryl esters and decomposition of CL-OOH may be less dependent upon a continuing supply of redox active metals than is the generation of 7-KC. In addition, they confirm the plausibility of prolonged cell-mediated oxidation of LDL as a source of oxysterols found in human atherosclerotic plaque, and imply that active redox cycling of metals is particularly important for their generation in vivo.

Animals↗
PubMed1993

A method for defining the stages of low-density lipoprotein oxidation by the separation of cholesterol- and cholesteryl ester-oxidation products using HPLC.

A new high-performance liquid chromatographic system for the identification of some of the lipid oxidation products of low-density lipoprotein (LDL) oxidized by copper is described. Using a reversed-phase C-18 column and an isocratic solvent system of acetonitrile/isopropanol/water (44/54/2, v/v/v), a number of oxidized lipid moieties were resolved and detected simply by their 234-nm absorbance. The nature of several of these compounds was determined by chromatographic criteria, chemiluminescence, and mass spectrometry. The production of compounds within 4 h oxidation corresponded to the production of lipid hydroperoxides, the quantitatively most important of which is cholesteryl linoleate hydroperoxide, and to the rapid decrease in the cholesteryl ester content of LDL detected at 210 nm. More prolonged copper oxidation (up to 48 h) of LDL resulted in decreased quantities of lipid hydroperoxide moieties and increased amounts of a number of other, nonhydroperoxide, compounds. 7-Ketocholesterol and cholesterol linoleate hydroxide are two of the major products of prolonged oxidation. The detection of oxidation products correlates with the modification of LDL protein, permits a four-stage definition of metal-mediated LDL oxidation, and enables the calculation of a quantitative index of oxidation (lipoprotein oxidation index). This method will be generally applicable to cell- and copper-mediated oxidation, and will enable standardization of, and direct comparison between, different preparations of oxidized LDL.

Apolipoproteins B↗
PubMed1992

Late development of conduction block over the Mahaim fibers after electrical atrioventricular junction ablation for Mahaim fiber tachycardia.

A patient suffering from so called "Mahaim fiber" tachycardia is presented who developed complete heart block over Mahaim fibers after 18 months of AV junction ablation. Antegrade conduction along the Mahaim fiber was intact immediately after the procedure. This finding has not been previously described and suggests that permanent pacemaker implantation is mandatory in such patients.

Adult↗
PubMed1988

Paracetamol-associated coma, metabolic acidosis, renal and hepatic failure.

A case of metabolic acidosis, acute renal failure and hepatic failure following paracetamol ingestion is presented. The diagnostic difficulty at presentation is highlighted. Continuous arteriovenous haemofiltration proved a valuable means of maintaining fluid and electrolyte balance. The patient recovered.

Acetaminophen↗