Báo cáo khoa học: Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophages

Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophages Bronwyn E. Brown1, Imran Rashid1, David M. van Reyk2 and Michael J. Davies1,3

1 Free Radical Group, The Heart Research Institute, Camperdown, Sydney, NSW, Australia 2 Department of Health Sciences, University of Technology Sydney, NSW, Australia 3 Faculty of Medicine, University of Sydney, NSW, Australia

Keywords aldehydes; atherosclerosis; foam cells; human monocyte-derived macrophages; low-density lipoproteins

Correspondence M. J. Davies, 114 Pyrmont Bridge Road, Camperdown, Sydney, NSW 2050, Australia Fax: +61 2 95655584 Tel: +61 2 82088900 E-mail: daviesm@hri.org.au

(Received 12 December 2006, accepted 15 January 2007)

doi:10.1111/j.1742-4658.2007.05699.x

Nonenzymatic covalent binding (glycation) of reactive aldehydes (from glu- cose or metabolic processes) to low-density lipoproteins has been previ- ously shown to result in lipid accumulation in a murine macrophage cell line. The formation of such lipid-laden cells is a hallmark of atheroscler- osis. In this study, we characterize lipid accumulation in primary human monocyte-derived macrophages, which are cells of immediate relevance to human atherosclerosis, on exposure to low-density lipoprotein glycated using methylglyoxal or glycolaldehyde. The time course of cellular uptake of low-density lipoprotein-derived lipids and protein has been character- ized, together with the subsequent turnover of the modified apolipoprotein B-100 (apoB) protein. Cholesterol and cholesteryl ester accumulation occurs within 24 h of exposure to glycated low-density lipoprotein, and increases in a time-dependent manner. Higher cellular cholesteryl ester lev- els were detected with glycolaldehyde-modified low-density lipoprotein than with methylglyoxal-modified low-density lipoprotein. Uptake was signifi- cantly decreased by fucoidin (an inhibitor of scavenger receptor SR-A) and a mAb to CD36. Human monocyte-derived macrophages endocytosed and degraded significantly more 125I-labeled apoB from glycolaldehyde-modified than from methylglyoxal-modified, or control, low-density lipoprotein. Dif- ferences in the endocytic and degradation rates resulted in net intracellular accumulation of modified apoB from glycolaldehyde-modified low-density lipoprotein. Accumulation of lipid therefore parallels increased endocytosis and, to a lesser extent, degradation of apoB in human macrophages exposed to glycolaldehyde-modified low-density lipoprotein. This accumula- tion of cholesteryl esters and modified protein from glycated low-density lipoprotein may contribute to cellular dysfunction and the increased atherosclerosis observed in people with diabetes, and other pathologies linked to exposure to reactive carbonyls.

Complications associated with diabetes are the major cause of mortality and morbidity in people with this disease. These include microvascular complications that

induce damage to the retina, nephrons and peripheral nerves, and macrovascular disease that is associated with accelerated atherosclerosis (deposition of lipids in

Abbreviations AGE, advanced glycation end-products; apoB, apolipoprotein B-100; HBSS, Hank’s balanced salt solution; HMDM, human monocyte-derived macrophage; HSA, human serum albumin; LDL, low-density lipoprotein.

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hyde and methylglyoxal, respectively) [22], providing strong evidence for the formation and subsequent reac- tions of these aldehydes in atherosclerotic lesions. The plasma concentrations of these aldehydes are elevated in people with diabetes [23,24], although the concentra- tions of these materials present in the artery wall, and in atherosclerotic lesions, are unknown.

the cellular handling of

The role of glycation and the two facets of glycoxida- tion in generating modified LDLs and lipid-laden (foam) cells, in vitro or in vivo, is incompletely under- stood. Most studies have employed conditions under which both processes have occurred, or where the nat- ure and extent of modifications have not been quanti- fied adequately [15,25]. It is therefore unclear as to whether glycation of LDL, in the absence of oxidation, results in foam cell formation in cell types of direct rele- vance to human atherosclerosis. It is also not known whether the protein and lipid components of modified LDL accumulate in synchrony, or to similar levels, due to differences in the rates of cellular proteolysis and lipolysis. Furthermore, the resulting glycated apoB has not been well characterized. Modified proteins have been shown to have different susceptibilities to proteolysis than native proteins, with both enhanced and decreased rates having been charac- terized [26,27]. The latter may result in the accumula- tion of modified proteins within cells, and subsequent perturbation of cellular metabolism [16,19–21].

the artery wall) in the coronary, peripheral and carotid arteries [1]. Factors that may contribute to this acceler- ated atherosclerosis include chronic elevated glucose levels (hyperglycemia) and insulin resistance, dyslipide- mias, and abnormalities of homeostasis [2]. Macrovas- cular disease has been reported to appear in people with type 2 diabetes at, or near the time of, first diag- nosis of diabetes, consistent with a shared underlying pathogenesis [2]. An early and persistent feature of the atherosclerotic lesion is the presence of lipid-laden (foam) cells in the intima of the artery wall, arising from cholesterol and cholesteryl ester accumulation by macrophage cells present in the artery wall [3]. Low- density lipoproteins (LDLs) are the likely source of this lipid, with unregulated LDL uptake occurring via receptors other than the native LDL receptor, including CD36 and class A scavenger receptors [4,5]. These receptors recognize abnormal LDL species, including those modified by oxidation, aggregation, chemical modification and formation of immune complexes [4,6]. Elevated glucose levels are strongly linked to the incidence and severity of atherosclerosis [7,8]. Of par- ticular relevance is the potential role of glucose (or species derived from glucose) in LDL modification [9,10]. Previous studies have identified multiple poten- tial mechanisms of LDL modification, including gly- cation and glycoxidation [9]. Glycation involves the covalent adduction of an aldehyde (from glucose or related species) to a reactive amine (e.g. Lys and Arg side chains, N-terminus [11–13]) or thiol (Cys) groups on proteins [14], such as those of the single protein molecule of LDL, apolipoprotein B-100 (apoB). The initial Schiff base undergoes subsequent rearrangement to yield Amadori products (e.g. fructose-lysine). Glyc- oxidation consists of two related processes ) oxidation of protein-bound sugars (from glycation), and oxida- tion of free glucose and its products. Both processes can generate radicals that modify LDL, and hence potentially contribute to the enhanced uptake of such particles by macrophages [12,15–17].

Previously, we have characterized conditions that yield glycated, but nonoxidized, LDL [28], and have shown that such particles give rise to lipid accumula- tion in cultured mouse macrophage-like cells [29]. In the current study, we have determined whether lipid accumulation also occurs in a more relevant cell type ) human monocyte-derived macrophages (HMDMs) ) on exposure to LDL glycated using methylglyoxal or glycolaldehyde. The time course of cellular uptake of LDL-derived lipid and protein has been characterized, as well as the subsequent turnover of the apoB protein. It is shown that both lipid and protein are taken up, in a time-dependent manner, via scavenger receptor SR-A- and CD36-mediated processes, and that the uptake of lipid and protein occurs in synchrony. Fur- thermore, it is shown that both lipid and modified pro- tein accumulate in cells, despite significant proteolytic degradation of the modified protein.

Results

LDL characterization

Glycated LDL particles were prepared using meth- ylglyoxal, glycolaldehyde and glucose, as described

The species formed by glycation and glycoxidation undergo subsequent reactions to give a heterogeneous and complex mixture of materials often called advanced glycation end-products (AGEs) [9,12]. Eleva- ted levels of AGEs have been reported in people with diabetes compared to controls [18], with some of these materials (e.g. Ne-carboxymethyl-lysines and Ne-carboxy- ethyl-lysines and pentosidine) being known to accumu- late with age on tissue proteins, and at an increased rate in LDL and atherosclerotic lesions in people with diabetes [16,19–21]. Ne-carboxymethyl-lysine and Ne-carboxyethyl-lysine can arise from reaction of Lys residues with reactive aldehydes (glyoxal ⁄ glycolalde-

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Table 1. Lipid composition (nmol lipidÆmg)1 apo B) of native, control and glycated LDL. LDL (1 mg proteinÆmL)1) was incubated with 50 lM EDTA (control LDL) or 100 mM modifying agent ± 1 lM Cu2+, in NaCl ⁄ Pi (pH 7.4), for 7 days at 37 (cid:2)C. Values are means ± SEM from three experiments, each with triplicate samples. None of the treatments resulted in significantly different values compared to the native LDL (P > 0.05).

Total cholesterol

Free cholesterol

Cholesteryl ester

Triglyceride

Phospholipid

Native LDL LDL plus EDTA Methylglyoxal-LDL Glycolaldehyde-LDL Glucose-LDL Glucose-LDL + Cu2+

3006 ± 214 3028 ± 11 2968 ± 150 2940 ± 134 3289 ± 85 2982 ± 111

935 ± 209 950 ± 99 870 ± 167 948 ± 81 982 ± 138 935 ± 116

2005 ± 144 2080 ± 82 1960 ± 51 1903 ± 92 2243 ± 42 2146 ± 29

287 ± 58 300 ± 70 286 ± 67 285 ± 66 319 ± 78 279 ± 57

875 ± 73 911 ± 123 790 ± 67 761 ± 51 833 ± 53 764 ± 23

previously [28,29]. This method results in minimal oxidation of apoB, cholesterol, cholesteryl esters, or a-tocopherol [28,29], and does not affect the relative cholesterol, cholesteryl ester, phospholipid or triglycer- ide composition of the particles (Table 1). In contrast, significant time- and concentration-dependent glyca- tion of apoB occurs with methylglyoxal or glycolal- dehyde when compared to control or glucose-modified particles, as indicated by particle charge, aggregation, and amino acid modification [28,29]. The relative elec- trophoretic mobility of the particles used in the current study was not significantly different to that reported irrespective of LDL iodination (data previously [29], not shown).

In contrast to the above, significant time-dependent accumulation of cholesteryl esters in HMDMs was observed on incubation with glycolaldehyde- or methyl- glyoxal-modified LDL (Fig. 1B). Glycolaldehyde-modi- fied LDL induced the greatest accumulation, with this being significantly higher than for methylglyoxal-modi- fied LDL, or control LDL, at all time points. Methyl- glyoxal-modified LDL induced significantly greater cholesterol ester accumulation than control LDL at the 48 h and 96 h time points, with the majority of this accumulation occurring over the first 24 h. There was no significant difference in cellular cholesterol ester content between cells incubated with unmodified LDL and cells not incubated with LDL, at all time points.

Lipid accumulation in HMDMs

cholesterol

incubated with LDL,

Glycolaldehyde-modified LDL induced a steady increase in the percentage of total cholesterol present as esters over the 96 h period, reaching a value of 53 ± 7% (Fig. 1C). Similar levels were detected with acetylated LDL (data not shown). Methylglyoxal- modified LDL also induced a significant increase in the percentage of cholesterol esters when compared to control LDL at all time points, with this reaching 23 ± 6% at 96 h. There was no significant difference in the percentage of esters between HMDMs incubated with unmodified LDL and those not indicating an absolute requirement for LDL modification for significant lipid accumulation in these cells.

Accumulation and turnover of apoB in HMDMs

HMDMs were exposed to glycated 125I-labeled LDL, with the levels of cell surface, endocytosed, degraded and intracellular accumulated apoB being determined by radioactive counting. 125I-Labeled acetylated LDL was used as a positive control (data not shown), and gave similar results to those observed for glycol- aldehyde-modified LDL. The extent of endocytosis, degradation and intracellular accumulation of apoB increased over time in HMDMs exposed to control

Lipid accumulation was quantified after exposure of HMDMs (1 · 106 cells per well) at 37 (cid:2)C, for up to 96 h, to LDL (0 or 100 lgÆmL)1) previously modified by methylglyoxal (100 mm), glycolaldehyde (100 mm) or glucose (100 mm ± 1 lm Cu2+), or control LDL incubated with EDTA (50 lm). No change in cell viab- ility or protein was detected in comparison to control cells not exposed to LDL. LDL chemically modified by acetylation was employed as a positive control. Cells exposed to glucose (± Cu2+)-modified LDL did not contain significantly elevated cellular cholesterol or cholesteryl ester levels in comparison to control cells incubated with LDL exposed to EDTA (data not shown). No increase in cellular free cholesterol levels was observed on exposure of HMDMs to methylgly- oxal- or glycolaldehyde-modified LDL for 0–96 h in comparison to incubation controls (LDL incubated with EDTA; Fig. 1A), although these values were sig- nificantly higher than in cells exposed to no LDL. No significant difference was observed in free cholesterol levels of HMDMs incubated with unmodified LDL, compared to no LDL, except at the 96 h time point (Fig. 1A).

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LDL (Fig. 2A), methylglyoxal-modified LDL (Fig. 2B), glycolaldehyde-modified LDL (Fig. 2C) and LDL modified by glucose ± Cu2+ (similar to control LDL; data not shown). However, the absolute amount of apoB endocytosed, degraded and accumulated was dependent upon the nature of the LDL modification; these were quantified at the 96 h time point (Fig. 2D). The extent of protein endocytosis, degradation and intracellular accumulation of apoB was increased in HMDMs exposed to glycolaldehyde-modified LDL in comparison to those exposed to control LDL. HMDMs exposed to methylglyoxal-modified LDL showed signifi- cantly increased endocytosis and degradation when compared to those exposed to control LDL, although this was less marked than with glycolaldehyde-modified LDL. These parameters were not elevated for HMDMs exposed to glucose (± Cu2+)-modified LDL when compared to those exposed to control LDL. In each case, amounts of cell surface (bound) apoB were min- imal, remained constant over time, and did not vary between conditions (Fig. 2A–D).

The turnover of intracellular (accumulated) apoB was examined over a 24 h chase period using LDL-free medium following exposure of HMDMs to labeled gly- colaldehyde- and methylglyoxal-modified LDL, and control LDL, for 96 h. The use of LDL-free medium during the chase period allows the turnover of preaccu- mulated protein to be studied in the absence of further cellular uptake. In these studies, cell death was < 12% as measured by the appearance of nondegraded apoB in the medium. In each case, a time-dependent decrease in (previously nondegraded) intracellular apoB concen- trations was detected, and was matched by an increase in the concentration of degraded apoB (i.e. peptides) in the medium (Fig. 3A–C). In all cases, only 20–30% of the apoB present at the start of the chase period was degraded. The absolute concentration of apoB turned over decreased in the order glycolaldehyde-modi- (P < 0.05). fied > methylglyoxal-modified > control With a 24 h loading period with 125I-labeled LDL prior to a 24 h chase period, a greater turnover of intracellu- lar apoB was observed, with 35–55% of the nondegrad- ed intracellular apoB being turned over (data not shown). The absolute concentration of apoB turned over was lower under these conditions, due to the lower initial accumulation of nondegraded intracellular apoB (data not shown).

Investigation of the nature of the receptors responsible for uptake of glycated LDL

Fig. 1. Cellular free cholesterol (A), total cholesteryl esters (B) and percentage cholesteryl esters of total cholesterol (sum of free cho- lesterol plus total cholesteryl esters) (C) present in HMDMs after exposure to no LDL (circles), incubation control LDL (LDL + EDTA; triangles), methylglyoxal-modified LDL (squares), or glycolaldehyde- modified LDL (diamonds). HMDMs (1.0 · 106 cells per well) were exposed to 100 lgÆmL)1 modified LDL (1 mg proteinÆmL)1, incuba- ted with 100 mM modifying agent or 50 lM EDTA, in NaCl ⁄ Pi, pH 7.4, for 7 days at 37 (cid:2)C) for up to 96 h in medium containing 10% lipoprotein-deficient serum (with fresh medium and LDL added at 48 h) before extraction and analysis by HPLC with UV detection. Values are means ± SEM from three or more experi- ments, each with triplicate samples. *, # and + indicate statistically elevated values (P < 0.05) compared to the control cells (no LDL), LDL plus EDTA-treated cells, and methylglyoxal-modified LDL- treated cells, respectively, at each time point.

HMDMs were exposed to methylglyoxal- or glycol- aldehyde-modified LDL in the absence or presence of

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(B) or glycolaldehyde (C)

Fig. 3. Turnover of accumulated apoB in HMDMs after exposure to 50 lgÆmL)1 incubation control [125I]LDL (A) or 50 lgÆmL)1 [125I]LDL modified by methylglyoxal (B) or glycolaldehyde (C) for 96 h. The preparation and cellular exposure to [125I]LDL were performed as described in Fig. 1, after iodination of the LDL, and were followed by cell washing and exposure to LDL-free chase medium (DMEM containing 1 mgÆmL)1 BSA in place of serum). At the appropriate chase times, cells were lysed and processed to determine nonde- graded intracellular apoB (triangles), degraded intracellular apoB (open circles), and degraded extracellular apoB (squares). Values are means ± SEM from three experiments, each with triplicate samples. Note different axis scales. * and # indicate statisti- cally elevated values (P < 0.05) compared to 0 h chase time for nondegraded intracellular apoB and extracellular degraded apoB, respectively.

Fig. 2. Time course of endocytosis (open circles), surface binding (triangles), degradation (diamonds) and intracellular accumulation [125I]LDL (A) and (squares) of apoB from incubation control [125I]LDL modified by methylglyoxal in HMDMs. (D) compares the data obtained at the 96 h time point on the cellular handling of apoB in HMDMs exposed to 50 lgÆmL)1 incubation control [125I]LDL (white) or 50 lgÆmL)1 [125I]LDL modi- fied by methylglyoxal (black), glycolaldehyde (horizontal stripes) or glucose in the absence (dots) or presence (vertical stripes) of Cu2+. HMDMs (1.0 · 106 cells per well) were exposed to 50 lgÆmL)1 modified [125I]LDL for up to 96 h (with fresh medium and [125I]LDL added at 48 h) before analyses. The preparation and cellular expo- sure to [125I]LDL were performed as described in Fig. 1, after iodi- is the sum of degraded nation of the LDL. Endocytosed material and intracellular measurements. Values are means ± SEM from three experiments, each with triplicate samples. Note different axis scales. *, # and + (A–C) indicate statistically elevated values (P < 0.05) compared to the 0 h time point for apoB endocytosis, degradation and intracellular accumulation, respectively. Columns (D) with different letters above them are significantly different by one-way ANOVA (P < 0.05) for that apoB measurement.

mAb to CD36, fucoidin or AGE–human serum albu- min (HSA) for 48 h, and changes in total cellular cho- lesteryl esters were determined using HPLC. Exposure of cells to methylglyoxal-modified LDL (Fig. 4A) or glycolaldehyde-modified LDL (Fig. 4B) and the mAb to CD36 or fucoidin resulted in significantly decreased cellular cholesteryl ester accumulation in comparison to cells exposed only to modified LDL. Cells exposed to modified LDL in the presence of AGE–HSA had lower cholesteryl ester levels, but this decrease was not significantly different in comparison to cells exposed to modified LDL alone.

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confounding

a potential

incubation of HMDMs with LDL methylglyoxal, modified by glucose, or glucose plus Cu2+ (with a con- centration of Cu2+ similar to that detected in advan- ced atherosclerotic lesions [30]), did not result in significant cellular sterol accumulation. This is in con- trast to the results of a previous study, in which a two- fold increase in cholesteryl ester synthesis was observed in HMDMs exposed to glucose-modified LDL [25]. No characterization data were presented for the LDL used in this previous study, so this discrepancy may arise from the nature of the modified LDL used, with oxidation being factor. Uptake of oxidized LDL has been previously shown to result in foam cell formation [17]. The lack of choleste- ryl ester accumulation resulting from LDL being incu- bated with glucose, in the presence or absence of Cu2+, is consistent with our previous studies using murine macrophage-like cells [29].

by macrophage

scavenger

(approximately 50% of

sterol

total

Exposure of LDL to 100 mm glycolaldehyde has been shown previously to result in extensive modifica- tion of the Lys residues present on the apoB protein [29]. Such modification has been reported to result in recognition receptors [15,29,31]. The cellular accumulation of cholesteryl esters levels) observed in the present study is consistent with that reported with cultured murine macrophage-like cells [29]. The proportion of total cholesterol present as cholesterol esters in these HMDMs is of a similar mag- nitude to that detected in human atherosclerotic lesions [32].

in HMDMs exposed to changes Fig. 4. Cholesteryl ester 100 lgÆmL)1 LDL modified by methylglyoxal (A) or glycolaldehyde (B) for 48 h in the absence (control) or presence of mAb to CD36 (2 lgÆmL)1), fucoidin (200 lgÆmL)1), or AGE–HSA (200 lgÆmL)1). Modified LDL was prepared and incubated with cells as described in Fig. 1. Values are means ± SEM from three experiments, each with triplicate samples. *Significantly decreased (P < 0.05) cellular cholesteryl esters levels compared to cells incubated with the modified LDL in the absence of any receptor inhibitors.

Discussion

The present study has shown that incubation of pri- mary HMDMs with glycated, but nonoxidized, LDL can give rise to time-dependent lipid loading, with this lipid accumulation occurring in parallel with the endo- cytosis and degradation of the protein (apoB) compo- nent of the LDL. This uptake of glycated LDL occurs primarily via scavenger receptor SR-A and CD36 endocytosis, as demonstrated by receptor-blocking experiments. The rates of uptake of both the lipid and protein components are not matched by the rate of cel- lular metabolism of these species, resulting in the accu- mulation of both unmodified cholesteryl esters and glycated apoB in the cells. The rate of removal of the latter species is slow, with only 20–30% of the glycated protein being degraded over a 24 h chase period.

In contrast to the rapid and extensive lipid accumu- lation induced by LDL modified by glycolaldehyde or

It has been reported that LDL modified by 10 mm methylglyoxal for 3 days is recognized by macrophage scavenger receptors, but results in decreased intracellu- lar cholesteryl ester synthesis in comparison to controls [33]. This is in contrast to the situation with cultured murine cells, where exposure to LDL modified with methylglyoxal for 14 days (with approximately 80% of Lys residues modified) resulted in significant choleste- ryl ester accumulation, with approximately 25% of the total cellular sterol being present as esters [29]. In the present study, HMDMs exposed to LDL modified by methylglyoxal for 7 days accumulated significant levels of cholesteryl ester within 24 h, with approximately 25% of total sterols being present as cholesteryl esters by 96 h. Thus, modification of LDL by methylglyoxal appears to result in macrophage scavenger receptor recognition, and significant cholesteryl ester accumula- tion, in human macrophages. It has been reported that LDL isolated from people with diabetes can stimulate cholesteryl ester synthesis in HMDMs, although the level of modification reported (approximately 5% of Lys residues [34]) is lower than that used in the current

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the cessation of

study. However, direct comparison between these two sets of data is not possible, as the extent of other mod- ifications present on these in vivo-modified particles is not known. We have suggested that it may be the nat- ure of the products arising from glycation, rather than purely the loss of the parent amino acid, which is the key factor in terms of receptor recognition [29]. There are no data available on the extent of Lys (and other in amino acid) modification, arising from glycation, LDL isolated from human atherosclerotic lesions, so it is not possible to judge the extent, or type, of amino acid modification on LDL to which macrophage cells might be exposed in vivo. Further studies are required to fully elucidate this point.

dehydrogenase,

lactate

in HMDMs,

been shown to accumulate in secondary lysosomes in macrophages because of inefficient degradation [39], although the extent of (labeled) apoB turnover in the chase period (i.e. after loading) observed in the current study with glycated LDL is much lower than that observed previously for some forms of oxidized LDL (e.g. that generated on expo- sure to 10 lm Cu2+ for 4 h [36]), consistent with poor cellular handling of the glycated apoB protein. This may be partly explained by the resistance of the modi- fied apoB to degradation by lysosomal cathepsins [40]. In addition we have also shown that glycated ⁄ glycoxi- dized proteins can inhibit thiol-dependent lysosomal cathpesins [41], as well as other intracellular enzymes, including glyceraldehyde- 3-phosphate dehydrogenase, and glutathione reductase [42]. The inhibition of the thiol-dependent lysosomal cathepsins by glycated proteins may be of particular importance in apoB turnover.

The accumulation of glycated apoB within HMDMs may be related to that of the cholesteryl esters observed under identical conditions, as a result of an interdependence of proteolysis and lipolysis. Jessup et al. have postulated, on the basis of studies with oxidized LDL, that failure of macrophages to degrade oxidized apoB may protect LDL cholesteryl esters in the core of the particle from lysosomal esterases, or that impaired lipolysis of LDL lipids may block pro- teolysis of apoB [36]. This may arise as a result of the failure of hydrophobic regions of apoB, which have been reported to be recognition signals for proteolysis, to become exposed [43,44]. The accumulation of such AGE-modified proteins may have significant cellular and atherogenic effects, and requires further study.

The increased rates of endocytosis and intracellu- lar degradation of methylglyoxal- and glycolaldehyde- is modified apoB protein from LDL, consistent with particle recognition by macrophage scavenger [15,29,31,33], or other receptors [35]. These data are in agreement with previous, more limited, stud- ies with glycolaldehyde-modified LDL [15,31]. The cel- lular uptake and turnover of apoB in macrophages exposed to methylglyoxal-modified LDL has not been examined previously, although increased endocytosis and degradation of apoB modified by other aldehydes (e.g. 4-hydroxynonenal, malondialdehyde) has been reported [36]. The pattern of uptake and degradation of apoB from the various types of modified LDL examined here mirrors cholesterol ester accumulation, with glycol- aldehyde inducing the largest changes, glucose (with or without Cu2+) the least, and methylglyoxal showing intermediate behavior. Previous studies have reported both decreased [15] and increased [25] degradation of apoB from glucose-modified LDL when compared to native LDL; however, the nature and extent of modifi- cation (or oxidation) of these particles are not known.

responsible for

Interestingly, apoB from glycolaldehyde-modified LDL accumulated in HMDMs over time. Accumula- tion of modified proteins has been previously implica- ted in diseases such as atherosclerosis and diabetes [16,19–21], and reported to have a variety of cellular effects. It has been shown that moderately oxidized proteins are more sensitive to proteolysis [37], and are endocytosed more quickly than native proteins, which in turn are more rapidly removed than heavily oxid- ized proteins [27,37]. Previous studies have shown that some proteins that contain AGEs (e.g. pyrraline-modi- fied albumin) accumulate in macrophages because of decreased cellular degradation rates and a reduced sus- ceptibility of this glycated protein to lysosomal proteo- lytic enzymes [38]. Thus, glycation alone appears to be sufficient to inhibit lysosomal degradation of modified proteins. Interestingly, apoB from oxidized LDL has

SR-A and CD36 have previously been reported to account for 75–90% of the uptake and degradation of acetylated or oxidized LDL [45]. Glycated ⁄ glycoxi- dized LDL has also been previously reported to be recognized by macrophage scavenger receptors, although data on which specific scavenger receptors were involved have not been reported [15,31,33]; the current data are consistent with SR-A and CD36 being key species. Greater than 60% modification of parent apoB Lys residues has been reported to result in macro- phage scavenger receptor recognition for both glycated and acetylated LDL [31,46]. Lys data previously repor- ted by our group [28,29] show that that greater than 60% Lys modification is observed for methylglyoxal- and glycolaldehyde-modified LDL under the condi- tions used in these consistent with this studies, previous conclusion. To investigate the types of recep- tor the uptake of glycated LDL observed in the current study, cells were incubated

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accumulation of glycated proteins observed in people with diabetes.

Experimental procedures

Materials

with LDL glycated using glycolaldehyde or methylgly- oxal, and either a mAb to CD36 [47], the SR-A inhib- itor fucoidin, or AGE–HSA, which is known to bind to RAGE [48]. The inhibition of uptake observed with the mAb to CD36 or fucoidin indicates that SR-A and CD36 are responsible for most of the observed uptake. Inhibition of RAGE by AGE–HSA did not decrease uptake significantly. Although RAGE is not an endo- cytotic receptor, binding of AGE ligands to RAGE has been shown to activate signaling pathways [49] that potentially could have affected LDL uptake.

Reagents were obtained from the following sources. Sigma- Aldrich (Castle Hill, NSW, Australia): methylglyoxal, glycolaldehyde, fatty acid-free BSA, HSA, fucoidin, tryp- sin [type I, Na-benzoyl-l-arginine ethyl esters, (cid:2) 10 000 unitsÆ(mg protein))1], EDTA, Hank’s balanced salt solution (HBSS), PenStrep (100 unitsÆmL)1 penicillin, 0.1 mgÆmL)1 streptomycin), and Dulbecco’s NaCl ⁄ Pi, (pH 7.4). BDH (Merck, Kilsyth, VIC, Australia): glucose. Bio-Rad (Regents Park, NSW, Australia): Chelex-100 resin. ICN (Seven Hills, NSW, Australia): CuSO4. Amersham Biosciences (Castle Hill, NSW, Australia): PD10 columns and Na125I (‡ 15 CiÆmg)1 iodide). JRH Biosciences (CSL, North Ryde, NSW, Australia): RPMI-1640 medium. Trace Scientific (Mel- bourne, VC, Australia): glutamine. Australian Red Cross, Clarence St Blood Bank: human serum. Axis-Shield (Oslo, Norway): Lymphoprep. BD Biosciences-Pharmingen (San Diego, CA, USA): purified mouse anti-(human CD36) mAb. All other chemicals were of analytical grade, and all solvents were of HPLC grade.

Solutions were prepared with nanopure water (Milli Q system, Millipore-Waters, Lane Cove, NSW, Australia) treated with washed Chelex-100 resin to remove trace trans- ition metal ions, with the exception of tissue culture rea- gents, for which Baxter (Old Toongabbie, NSW, Australia) sterile, endotoxin-free, water, NaCl ⁄ Pi or HBSS were used.

LDL modification

including methylglyoxal

[36,60]. activity Specific (typically

The aldehyde concentrations utilized in this study are higher than those reported for plasma from both healthy controls and people with diabetes [23,24,50,51]. These plasma values (up to 0.5 mm [51]) are, however, potentially misleading, as they represent only the (small) fraction of these highly reactive species that has not undergone reaction with plasma proteins, a process that is known to be extremely rapid and efficient [52]. The true flux of these compounds is therefore likely to be considerably higher. Irrespective of this, it is clear that the levels of these aldehydes are elevated in people with diabetes [24]. Furthermore, the levels of these aldehydes may be substantially greater in the artery wall than in plasma, as a result of cell-mediated forma- tion of these species, with the major route to such aldehydes being via the intracellular decomposition of triose phosphates [53], the concentrations of which are markedly elevated in hyperglycemia [54]. It has also been shown that the heme enzyme myeloperoxidase, which is present at elevated levels at sites of inflamma- tion (such as atherosclerotic lesions [55]) as a result of the influx and activation of neutrophils and mono- cytes, can oxidize free amino acids to reactive alde- hydes, [56]. Both these processes might therefore be expected to give higher levels of reactive aldehydes within tissues, and partic- ularly at sites of inflammation, than would be present in plasma. Subendothelial entrapment of LDL [57–59] may also result in more extensive LDL modification than observed in the circulation, as a result of longer exposure times.

LDL was isolated as reported previously from multiple healthy male and female donors (four males, five females, [29]. 125I-Labeling of LDL was per- aged 22–42 years) formed, prior to other modification, using iodine mono- 50–100 chloride c.p.m.Æng)1 apoB protein) was determined by c-counting (Cobra II; Packard, Downers Grove, IL, USA). Acetylation of LDL was performed as reported previously [29]. Modifi- cation of LDL was performed as described previously [28]. Briefly, sterile LDL (1 mg proteinÆmL)1) was incubated with 100 mm glycolaldehyde, methylglyoxal or glucose (± 1 lm CuSO4) in Chelex-treated NaCl ⁄ Pi at 37 (cid:2)C for 7 days. Incubation controls contained 50 lm EDTA in place of glucose or aldehyde. Excess reagents were removed by elution of the LDL through PD10 columns before use. Modification was confirmed by changes in relative elec- trophoretic mobility [29]. LDL lipid composition (total cho- lesterol, free cholesterol, triglycerides and phospholipids) was determined using a Roche Diagnostics ⁄ Hitachi 902

Overall, these studies have established that LDL gly- cation, in the absence of significant oxidation, is suffi- cient to induce lipid loading in primary human macrophages, primarily via the scavenger receptors SR-A and CD36. The accumulation of lipid in these macrophages is accompanied by increased endocytosis and degradation of apoB, with the difference in the rates of the latter two processes resulting in accumula- tion of modified apoB in HMDMs exposed to glycolal- dehyde-modified LDL. Thus, aldehyde-modified LDL may contribute to the increased atherosclerosis and

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B. E. Brown et al.

Formation of lipid-laden cells by glycated LDL

autoanalyzer (Roche Diagnostics GmbH, Mannheim, Ger- many) [61,62]. Cholesteryl ester concentrations were calcu- lated as the difference between total and free cholesterol concentrations.

Isolation and culture of HMDMs

intracellular

J–20XPI

was added; this was followed by incubation for 0–24 h. At the indicated times, medium (0.5 mL) was collected, and the cells were washed with cold NaCl ⁄ Pi. For both the accumulation and turnover studies, after the medium was collected, trypsin (1 mL, 0.01% w ⁄ v) was added to the wells (60 min, 4 (cid:2)C) to remove surface-bound ligand [36]. This medium was retained to quantify cell surface- bound apoB. Triton X-100 (1 mL, 0.1% v ⁄ v) was then added (30 min, 4 (cid:2)C). Of the resulting lysate, 0.5 mL was used to measure total radioactivity. BSA (0.1 mL, 30 mgÆmL)1) and trichloroacetic acid (1 mL, 3 m) were added to the remaining lysate, and medium samples; this was followed by incubation (20 min, 4 (cid:2)C) and centrifugation using a Sorvall (Sorvall Instruments, Newtown, CT, USA) RT600B centrifuge and a H1000B rotor (10 min, 1500 g, 4 (cid:2)C) to precipitate proteins. The (0.25 mL, supernatant (1 mL) was added to AgNO3 0.7 m) and respun to precipitate free iodide. One milliliter of the iodide-free, trichloroacetic acid-soluble, supernatant from the medium or lysate was counted to quantify extra- cellular and intracellular degraded apoB, respectively [36]. The medium and cell protein pellets were washed (3 · 5% w ⁄ v trichloroacetic acid), and then counted to determine extracellular and intracellular nondegraded apoB, respect- ively.

Receptor blocking

Monocytes were isolated by countercurrent elutriation [63,64], using HBSS (with phenol red and 0.01% EDTA, but without Ca2+ and Mg2+). White cell concentrates were diluted 1 : 2 in HBSS, and 30 mL samples were underlaid with 15 mL of Lymphoprep and centrifuged using a Beckman (Palo Alto, CA, USA) GS–6KR centri- fuge with a GH3Æ8 rotor (2060 g, 40 min, 22 (cid:2)C). Periph- eral mononuclear cells were isolated from the interface, washed, and resuspended in 30 mL. The cells were then loaded into a Beckman Avanti centrifuge equipped with a JE 5.0 elutriation rotor (770 g, flow rate 9 mLÆmin)1). The flow rate was increased by 1 mLÆmin)1 every 10 min, and the monocyte cell fractions collected with flow rates of 15, 16, 17, 18 and, finally, 40 mLÆmin)1 were collected and combined. The presence of monocytes was confirmed by cytospinning and staining (Diff Quik, Narrabeen, NSW, Australia). Cells were diluted (1.0 · 106 cellsÆmL)1 in RPMI-1640, no serum), added to 12-well plates (1 mL per well; Costar, Corning, NY, USA), and to adhere for 1–2 h. Cells were then washed, and left RPMI medium [containing 10% heat-inactivated human serum, 4 mm glutamine and 1% (v ⁄ v) PenStrep] was added; this was followed by incubation (5% CO2, 37 (cid:2)C) for 9–11 days, with the medium being changed every 3 days, to give matured HMDMs. incubation prepared by

Cellular cholesterol and cholesteryl ester analysis

HMDMs were exposed to 0 or 100 lgÆmL)1 control or modified LDL for 48 h in medium containing 10% lipopro- serum with 200 lgÆmL)1 fucoidin [48], tein-deficient 2 lgÆmL)1 mAb to CD36 [47], or 200 lgÆmL)1 AGE–HSA [48]. AGE–HSA was of 20 mgÆmL)1 HSA with 1 m glucose for 4 weeks at 37 (cid:2)C, followed by dialysis to remove unreacted glucose [65]. At the end of 48 h, cell medium samples were collected, and the cells were washed and lysed in water. Cell viability was determined by assaying lactate dehydrogenase release, and cellular cholesterol and cholesteryl ester content was quanti- fied by HPLC, as described above.

Protein assay

HMDMs were exposed to 0 or 100 lgÆmL)1 modified LDL for 0–96 h in medium containing 10% lipoprotein-deficient serum (prepared as reported previously [29]). Fresh LDL and medium were added at 48 h. Cell medium samples were collected at the stated times, and the cells were washed and lysed in water. Cell viability was determined by assaying lactate dehydrogenase release [29]. Cellular cholesterol and cholesteryl ester content was quantified using HPLC, as described previously [29].

Protein concentrations were quantified using the bicinchoni- nic acid assay (Pierce, Rockford, IL, USA) with 60 min of incubation at 60 (cid:2)C, using BSA as a standard.

Cellular apoB accumulation and turnover

Data analysis

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Data are expressed as mean ± SEM from three or more separate experiments with triplicate samples. One-way or two-way analysis of variance (anova) was used with Bon- ferroni’s post hoc analysis, with P < 0.05 taken as signifi- cant. HMDMs were incubated with modified [125I]LDL (50 lg proteinÆmL)1) as described above. Cell medium (0.5 mL) and cells (after being washed twice with cold NaCl ⁄ Pi) were sampled at the indicated times. For turnover studies, the [125I]LDL-containing medium was removed after the accumulation phase. The cells were then washed with warm NaCl ⁄ Pi, and medium containing 1 mgÆmL)1 BSA

B. E. Brown et al.

Formation of lipid-laden cells by glycated LDL

14 Zeng J & Davies MJ (2005) Evidence for the formation

Acknowledgements

of adducts and S-(carboxymethyl) cysteine on reaction of alpha-dicarbonyl compounds with thiol groups on amino acids, peptides, and proteins. Chem Res Toxicol 18, 1232–1241.

15 Kawamura M, Heinecke JW & Chait A (1994) Pathophy- siological concentrations of glucose promote oxidative modification of low density lipoprotein by a superoxide- dependent pathway. J Clin Invest 94, 771–778.

This work was supported by grants from the Diabetes Australia Research Trust and the Australian Research Council. B. E. Brown and I. Rashid gratefully acknow- ledge receipt of Australian Postgraduate Awards administered through the University of Sydney. The authors thank Professor Roger T. Dean and Associate Professor Wendy Jessup for helpful discussions, and Mr Pat Pisansarakit for the isolation of HMDMs.

16 Baynes JW & Thorpe SR (1999) Role of oxidative stress in diabetic complications ) a new perspective on an old paradigm. Diabetes 48, 1–9.

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