Tài liệu 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 docx

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 Free Radical Group, The Heart Research Institute, Camperdown, Sydney, NSW, Australia Department of Health Sciences, University of Technology Sydney, NSW, Australia 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 95655584 Tel: +61 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 glucose or metabolic processes) to low-density lipoproteins has been previously shown to result in lipid accumulation in a murine macrophage cell line The formation of such lipid-laden cells is a hallmark of atherosclerosis 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 characterized, 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 levels were detected with glycolaldehyde-modified low-density lipoprotein than with methylglyoxal-modified low-density lipoprotein Uptake was significantly 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 Differences 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 accumulation 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 1530 FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS B E Brown et al the artery wall) in the coronary, peripheral and carotid arteries [1] Factors that may contribute to this accelerated atherosclerosis include chronic elevated glucose levels (hyperglycemia) and insulin resistance, dyslipidemias, and abnormalities of homeostasis [2] Macrovascular disease has been reported to appear in people with type diabetes at, or near the time of, first diagnosis 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] Lowdensity 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 particular relevance is the potential role of glucose (or species derived from glucose) in LDL modification [9,10] Previous studies have identified multiple potential mechanisms of LDL modification, including glycation 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) Glycoxidation consists of two related processes ) oxidation of protein-bound sugars (from glycation), and oxidation 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] 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] Elevated 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-carboxyethyl-lysines and pentosidine) being known to accumulate 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- Formation of lipid-laden cells by glycated LDL hyde and methylglyoxal, respectively) [22], providing strong evidence for the formation and subsequent reactions of these aldehydes in atherosclerotic lesions The plasma concentrations of these aldehydes are elevated in people with diabetes [23,24], although the concentrations of these materials present in the artery wall, and in atherosclerotic lesions, are unknown The role of glycation and the two facets of glycoxidation in generating modified LDLs and lipid-laden (foam) cells, in vitro or in vivo, is incompletely understood Most studies have employed conditions under which both processes have occurred, or where the nature and extent of modifications have not been quantified 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 relevance 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 cellular handling of 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 characterized [26,27] The latter may result in the accumulation of modified proteins within cells, and subsequent perturbation of cellular metabolism [16,19–21] Previously, we have characterized conditions that yield glycated, but nonoxidized, LDL [28], and have shown that such particles give rise to lipid accumulation 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 Furthermore, it is shown that both lipid and modified protein accumulate in cells, despite significant proteolytic degradation of the modified protein Results LDL characterization Glycated LDL particles were prepared using methylglyoxal, glycolaldehyde and glucose, as described FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS 1531 Formation of lipid-laden cells by glycated LDL B E Brown et al Table Lipid composition (nmol lipidỈmg)1 apo B) of native, control and glycated LDL LDL (1 mg proteinỈmL)1) was incubated with 50 lM EDTA (control LDL) or 100 mM modifying agent ± lM Cu2+, in NaCl ⁄ Pi (pH 7.4), for days at 37 °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 Native LDL LDL plus EDTA Methylglyoxal-LDL Glycolaldehyde-LDL Glucose-LDL Glucose-LDL + Cu2+ Free cholesterol Cholesteryl ester Triglyceride Phospholipid 3006 3028 2968 2940 3289 2982 935 950 870 948 982 935 2005 2080 1960 1903 2243 2146 287 300 286 285 319 279 875 911 790 761 833 764 ± ± ± ± ± ± 214 11 150 134 85 111 ± ± ± ± ± ± 209 99 167 81 138 116 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 triglyceride composition of the particles (Table 1) In contrast, significant time- and concentration-dependent glycation of apoB occurs with methylglyoxal or glycolaldehyde when compared to control or glucose-modified particles, as indicated by particle charge, aggregation, and amino acid modification [28,29] The relative electrophoretic mobility of the particles used in the current study was not significantly different to that reported previously [29], irrespective of LDL iodination (data not shown) Lipid accumulation in HMDMs Lipid accumulation was quantified after exposure of HMDMs (1 · 106 cells per well) at 37 °C, for up to 96 h, to LDL (0 or 100 lgỈmL)1) previously modified by methylglyoxal (100 mm), glycolaldehyde (100 mm) or glucose (100 mm ± lm Cu2+), or control LDL incubated with EDTA (50 lm) No change in cell viability 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 methylglyoxal- or glycolaldehyde-modified LDL for 0–96 h in comparison to incubation controls (LDL incubated with EDTA; Fig 1A), although these values were significantly 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) 1532 ± ± ± ± ± ± 144 82 51 92 42 29 ± ± ± ± ± ± 58 70 67 66 78 57 ± ± ± ± ± ± 73 123 67 51 53 23 In contrast to the above, significant time-dependent accumulation of cholesteryl esters in HMDMs was observed on incubation with glycolaldehyde- or methylglyoxal-modified LDL (Fig 1B) Glycolaldehyde-modified LDL induced the greatest accumulation, with this being significantly higher than for methylglyoxal-modified LDL, or control LDL, at all time points Methylglyoxal-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 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) Methylglyoxalmodified 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 cholesterol esters between HMDMs incubated with unmodified LDL and those not incubated with LDL, 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 glycolaldehyde-modified LDL The extent of endocytosis, degradation and intracellular accumulation of apoB increased over time in HMDMs exposed to control FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS B E Brown et al Fig Cellular free cholesterol (A), total cholesteryl esters (B) and percentage cholesteryl esters of total cholesterol (sum of free cholesterol 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 glycolaldehydemodified LDL (diamonds) HMDMs (1.0 · 106 cells per well) were exposed to 100 lgỈmL)1 modified LDL (1 mg proteinỈmL)1, incubated with 100 mM modifying agent or 50 lM EDTA, in NaCl ⁄ Pi, pH 7.4, for days at 37 °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 experiments, 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 LDLtreated cells, respectively, at each time point Formation of lipid-laden cells by glycated LDL 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 significantly 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 minimal, 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 glycolaldehyde- and methylglyoxal-modified LDL, and control LDL, for 96 h The use of LDL-free medium during the chase period allows the turnover of preaccumulated 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 concentrations 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-modified > methylglyoxal-modified > control (P < 0.05) With a 24 h loading period with 125I-labeled LDL prior to a 24 h chase period, a greater turnover of intracellular apoB was observed, with 35–55% of the nondegraded 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 HMDMs were exposed to methylglyoxal- or glycolaldehyde-modified LDL in the absence or presence of FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS 1533 Formation of lipid-laden cells by glycated LDL B E Brown et al Fig Time course of endocytosis (open circles), surface binding (triangles), degradation (diamonds) and intracellular accumulation (squares) of apoB from incubation control [125I]LDL (A) and [125I]LDL modified by methylglyoxal (B) or glycolaldehyde (C) in HMDMs (D) compares the data obtained at the 96 h time point on the cellular handling of apoB in HMDMs exposed to 50 lgỈmL)1 incubation control [125I]LDL (white) or 50 lgỈmL)1 [125I]LDL modified 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 lgỈmL)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 exposure to [125I]LDL were performed as described in Fig 1, after iodination of the LDL Endocytosed material is the sum of degraded 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 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 1534 Fig Turnover of accumulated apoB in HMDMs after exposure to 50 lgỈmL)1 incubation control [125I]LDL (A) or 50 lgỈmL)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 mgỈmL)1 BSA in place of serum) At the appropriate chase times, cells were lysed and processed to determine nondegraded 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 statistically elevated values (P < 0.05) compared to h chase time for nondegraded intracellular apoB and extracellular degraded apoB, respectively mAb to CD36, fucoidin or AGE–human serum albumin (HSA) for 48 h, and changes in total cellular cholesteryl 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 FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS B E Brown et al Fig Cholesteryl ester changes in HMDMs exposed to 100 lgỈmL)1 LDL modified by methylglyoxal (A) or glycolaldehyde (B) for 48 h in the absence (control) or presence of mAb to CD36 (2 lgỈmL)1), fucoidin (200 lgỈmL)1), or AGE–HSA (200 lgỈmL)1) Modified LDL was prepared and incubated with cells as described in Fig 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 primary HMDMs with glycated, but nonoxidized, LDL can give rise to time-dependent lipid loading, with this lipid accumulation occurring in parallel with the endocytosis and degradation of the protein (apoB) component 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 cellular metabolism of these species, resulting in the accumulation 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 accumulation induced by LDL modified by glycolaldehyde or Formation of lipid-laden cells by glycated LDL methylglyoxal, incubation of HMDMs with LDL modified by glucose, or glucose plus Cu2+ (with a concentration of Cu2+ similar to that detected in advanced atherosclerotic lesions [30]), did not result in significant cellular sterol accumulation This is in contrast to the results of a previous study, in which a twofold 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 a potential confounding factor Uptake of oxidized LDL has been previously shown to result in foam cell formation [17] The lack of cholesteryl ester accumulation resulting from LDL being incubated with glucose, in the presence or absence of Cu2+, is consistent with our previous studies using murine macrophage-like cells [29] Exposure of LDL to 100 mm glycolaldehyde has been shown previously to result in extensive modification of the Lys residues present on the apoB protein [29] Such modification has been reported to result in recognition by macrophage scavenger receptors [15,29,31] The cellular accumulation of cholesteryl esters (approximately 50% of total sterol 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 magnitude to that detected in human atherosclerotic lesions [32] It has been reported that LDL modified by 10 mm methylglyoxal for days is recognized by macrophage scavenger receptors, but results in decreased intracellular 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 cholesteryl 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 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 accumulation, 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 FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS 1535 Formation of lipid-laden cells by glycated LDL B E Brown et al study However, direct comparison between these two sets of data is not possible, as the extent of other modifications present on these in vivo-modified particles is not known We have suggested that it may be the nature 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 amino acid) modification, arising from glycation, in 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 The increased rates of endocytosis and intracellular degradation of methylglyoxal- and glycolaldehydemodified apoB protein from LDL, in HMDMs, is consistent with particle recognition by macrophage scavenger [15,29,31,33], or other receptors [35] These data are in agreement with previous, more limited, studies with glycolaldehyde-modified LDL [15,31] The cellular 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 glycolaldehyde 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 modification (or oxidation) of these particles are not known Interestingly, apoB from glycolaldehyde-modified LDL accumulated in HMDMs over time Accumulation of modified proteins has been previously implicated 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 oxidized proteins [27,37] Previous studies have shown that some proteins that contain AGEs (e.g pyrraline-modified albumin) accumulate in macrophages because of decreased cellular degradation rates and a reduced susceptibility of this glycated protein to lysosomal proteolytic enzymes [38] Thus, glycation alone appears to be sufficient to inhibit lysosomal degradation of modified proteins Interestingly, apoB from oxidized LDL has 1536 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 the cessation of 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 exposure to 10 lm Cu2+ for h [36]), consistent with poor cellular handling of the glycated apoB protein This may be partly explained by the resistance of the modified apoB to degradation by lysosomal cathepsins [40] In addition we have also shown that glycated ⁄ glycoxidized proteins can inhibit thiol-dependent lysosomal cathpesins [41], as well as other intracellular enzymes, including lactate dehydrogenase, glyceraldehyde3-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 proteolysis 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 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 ⁄ glycoxidized 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 macrophage scavenger receptor recognition for both glycated and acetylated LDL [31,46] Lys data previously reported by our group [28,29] show that that greater than 60% Lys modification is observed for methylglyoxaland glycolaldehyde-modified LDL under the conditions used in these studies, consistent with this previous conclusion To investigate the types of receptor responsible for the uptake of glycated LDL observed in the current study, cells were incubated FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS B E Brown et al with LDL glycated using glycolaldehyde or methylglyoxal, and either a mAb to CD36 [47], the SR-A inhibitor 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 endocytotic receptor, binding of AGE ligands to RAGE has been shown to activate signaling pathways [49] that potentially could have affected LDL uptake 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 formation 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 inflammation (such as atherosclerotic lesions [55]) as a result of the influx and activation of neutrophils and monocytes, can oxidize free amino acids to reactive aldehydes, including methylglyoxal [56] Both these processes might therefore be expected to give higher levels of reactive aldehydes within tissues, and particularly 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 Overall, these studies have established that LDL glycation, in the absence of significant oxidation, is sufficient 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 accumulation of modified apoB in HMDMs exposed to glycolaldehyde-modified LDL Thus, aldehyde-modified LDL may contribute to the increased atherosclerosis and Formation of lipid-laden cells by glycated LDL accumulation of glycated proteins observed in people with diabetes Experimental procedures Materials Reagents were obtained from the following sources SigmaAldrich (Castle Hill, NSW, Australia): methylglyoxal, glycolaldehyde, fatty acid-free BSA, HSA, fucoidin, trypsin [type I, Na-benzoyl-l-arginine ethyl esters,  10 000 unitsỈ(mg protein))1], EDTA, Hank’s balanced salt solution (HBSS), PenStrep (100 unitsỈmL)1 penicillin, 0.1 mgỈmL)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 CiỈmg)1 iodide) JRH Biosciences (CSL, North Ryde, NSW, Australia): RPMI-1640 medium Trace Scientific (Melbourne, 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 transition metal ions, with the exception of tissue culture reagents, for which Baxter (Old Toongabbie, NSW, Australia) sterile, endotoxin-free, water, NaCl ⁄ Pi or HBSS were used LDL modification LDL was isolated as reported previously from multiple healthy male and female donors (four males, five females, aged 22–42 years) [29] 125I-Labeling of LDL was performed, prior to other modification, using iodine monochloride [36,60] Specific activity (typically 50–100 c.p.m.Ỉng)1 apoB protein) was determined by c-counting (Cobra II; Packard, Downers Grove, IL, USA) Acetylation of LDL was performed as reported previously [29] Modification of LDL was performed as described previously [28] Briefly, sterile LDL (1 mg proteinỈmL)1) was incubated with 100 mm glycolaldehyde, methylglyoxal or glucose (± lm CuSO4) in Chelex-treated NaCl ⁄ Pi at 37 °C for 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 electrophoretic mobility [29] LDL lipid composition (total cholesterol, free cholesterol, triglycerides and phospholipids) was determined using a Roche Diagnostics ⁄ Hitachi 902 FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS 1537 Formation of lipid-laden cells by glycated LDL B E Brown et al autoanalyzer (Roche Diagnostics GmbH, Mannheim, Germany) [61,62] Cholesteryl ester concentrations were calculated as the difference between total and free cholesterol concentrations Isolation and culture of HMDMs 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 : in HBSS, and 30 mL samples were underlaid with 15 mL of Lymphoprep and centrifuged using a Beckman (Palo Alto, CA, USA) GS–6KR centrifuge with a GH3Ỉ8 rotor (2060 g, 40 min, 22 °C) Peripheral mononuclear cells were isolated from the interface, washed, and resuspended in 30 mL The cells were then loaded into a Beckman Avanti J–20XPI centrifuge equipped with a JE 5.0 elutriation rotor (770 g, flow rate mLỈmin)1) The flow rate was increased by mLỈmin)1 every 10 min, and the monocyte cell fractions collected with flow rates of 15, 16, 17, 18 and, finally, 40 mLỈmin)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 cellsỈmL)1 in RPMI-1640, no serum), added to 12-well plates (1 mL per well; Costar, Corning, NY, USA), and left to adhere for 1–2 h Cells were then washed, and RPMI medium [containing 10% heat-inactivated human serum, mm glutamine and 1% (v ⁄ v) PenStrep] was added; this was followed by incubation (5% CO2, 37 °C) for 9–11 days, with the medium being changed every days, to give matured HMDMs Cellular cholesterol and cholesteryl ester analysis HMDMs were exposed to or 100 lgỈmL)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] Cellular apoB accumulation and turnover HMDMs were incubated with modified [125I]LDL (50 lg proteinỈmL)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 mgỈmL)1 BSA 1538 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, °C) to remove surface-bound ligand [36] This medium was retained to quantify cell surfacebound apoB Triton X-100 (1 mL, 0.1% v ⁄ v) was then added (30 min, °C) Of the resulting lysate, 0.5 mL was used to measure total intracellular radioactivity BSA (0.1 mL, 30 mgỈmL)1) and trichloroacetic acid (1 mL, m) were added to the remaining lysate, and medium samples; this was followed by incubation (20 min, °C) and centrifugation using a Sorvall (Sorvall Instruments, Newtown, CT, USA) RT600B centrifuge and a H1000B rotor (10 min, 1500 g, °C) to precipitate proteins The supernatant (1 mL) was added to AgNO3 (0.25 mL, 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 extracellular 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, respectively Receptor blocking HMDMs were exposed to or 100 lgỈmL)1 control or modified LDL for 48 h in medium containing 10% lipoprotein-deficient serum with 200 lgỈmL)1 fucoidin [48], lgỈmL)1 mAb to CD36 [47], or 200 lgỈmL)1 AGE–HSA [48] AGE–HSA was prepared by incubation of 20 mgỈmL)1 HSA with m glucose for weeks at 37 °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 quantified by HPLC, as described above Protein assay Protein concentrations were quantified using the bicinchoninic acid assay (Pierce, Rockford, IL, USA) with 60 of incubation at 60 °C, using BSA as a standard Data analysis 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 Bonferroni’s post hoc analysis, with P < 0.05 taken as significant FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS B E Brown et al Acknowledgements This work was supported by grants from the 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RT (1998) Presence of DOPA and amino acid hydroperoxides in proteins modified with advanced glycation end products (AGEs): amino acid oxidation products as a possible sources of oxidative stress induced by AGE proteins Biochem J 330, 233–239 FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS 1541 ... resulting in the accumulation 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... although the concentrations of these materials present in the artery wall, and in atherosclerotic lesions, are unknown The role of glycation and the two facets of glycoxidation in generating modified... Klein RL & Virella G (1996) Modification of lipoproteins in diabetes Diabetes Metab Rev 12, 69–90 10 Tomkin GH & Owens D (2001) Abnormalities in apo B-containing lipoproteins in diabetes and atherosclerosis