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. Brown
1
, Imran Rashid
1
, David M. van Reyk
2
and Michael J. Davies
1,3
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
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
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:

(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
125
I-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.
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
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].
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. N
e
-carboxymethyl-lysines and N
e
-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]. N
e
-carboxymethyl-lysine and
N
e
-carboxyethyl-lysine can arise from reaction of Lys
residues with reactive aldehydes (glyoxal⁄ glycolalde-
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 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 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 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].
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
B. E. Brown et al. Formation of lipid-laden cells by glycated LDL
FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS 1531

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
previously [29], irrespective of LDL iodination (data
not shown).
Lipid accumulation in HMDMs
Lipid accumulation was quantified after exposure of
HMDMs (1 · 10
6
cells per well) at 37 °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 ±1lm Cu
2+
), 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 (± Cu
2+
)-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).
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.

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 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
125
I-labeled LDL,
with the levels of cell surface, endocytosed, degraded
and intracellular accumulated apoB being determined
by radioactive counting.
125
I-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
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 Cu
2+
, in NaCl ⁄ P
i
(pH 7.4), for 7 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 Free cholesterol Cholesteryl ester Triglyceride Phospholipid
Native LDL 3006 ± 214 935 ± 209 2005 ± 144 287 ± 58 875 ± 73
LDL plus EDTA 3028 ± 11 950 ± 99 2080 ± 82 300 ± 70 911 ± 123
Methylglyoxal-LDL 2968 ± 150 870 ± 167 1960 ± 51 286 ± 67 790 ± 67
Glycolaldehyde-LDL 2940 ± 134 948 ± 81 1903 ± 92 285 ± 66 761 ± 51
Glucose-LDL 3289 ± 85 982 ± 138 2243 ± 42 319 ± 78 833 ± 53
Glucose-LDL + Cu
2+
2982 ± 111 935 ± 116 2146 ± 29 279 ± 57 764 ± 23
Formation of lipid-laden cells by glycated LDL B. E. Brown et al.
1532 FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS
LDL (Fig. 2A), methylglyoxal-modified LDL (Fig. 2B),
glycolaldehyde-modified LDL (Fig. 2C) and LDL
modified by glucose ± Cu
2+
(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 (± Cu
2+
)-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-
fied > methylglyoxal-modified > control (P<0.05).
With a 24 h loading period with
125
I-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
HMDMs were exposed to methylglyoxal- or glycol-
aldehyde-modified LDL in the absence or presence of
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 · 10
6
cells per well) were
exposed to 100 lgÆmL
)1
modified LDL (1 mg proteinÆmL
)1

, incuba-
ted with 100 m
M modifying agent or 50 lM EDTA, in NaCl ⁄ P
i
,
pH 7.4, for 7 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 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.
B. E. Brown et al. Formation of lipid-laden cells by glycated LDL
FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS 1533
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.
Fig. 3. Turnover of accumulated apoB in HMDMs after exposure to
50 lgÆmL
)1

incubation control [
125
I]LDL (A) or 50 lgÆmL
)1
[
125
I]LDL
modified by methylglyoxal (B) or glycolaldehyde (C) for 96 h. The
preparation and cellular exposure to [
125
I]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
(squares) of apoB from incubation control [
125
I]LDL (A) and
[

125
I]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 lgÆmL
)1
incubation control [
125
I]LDL (white) or 50 lgÆmL
)1
[
125
I]LDL modi-
fied by methylglyoxal (black), glycolaldehyde (horizontal stripes) or
glucose in the absence (dots) or presence (vertical stripes) of Cu
2+
.
HMDMs (1.0 · 10
6
cells per well) were exposed to 50 lgÆmL
)1
modified [
125
I]LDL for up to 96 h (with fresh medium and [
125
I]LDL
added at 48 h) before analyses. The preparation and cellular expo-
sure to [
125
I]LDL were performed as described in Fig. 1, after iodi-
nation 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 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.
Formation of lipid-laden cells by glycated LDL B. E. Brown et al.
1534 FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS
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
methylglyoxal, incubation of HMDMs with LDL
modified by glucose, or glucose plus Cu
2+

(with a con-
centration of Cu
2+
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 a potential confounding 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
Cu
2+
, 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 modifica-
tion 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 mag-
nitude to that detected in human atherosclerotic
lesions [32].
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
Fig. 4. Cholesteryl ester changes in HMDMs exposed to
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 lgÆ mL
)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.
B. E. Brown et al. Formation of lipid-laden cells by glycated LDL
FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS 1535
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
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 intracellu-

lar degradation of methylglyoxal- and glycolaldehyde-
modified 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, 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 Cu
2+
) 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.
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
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 expo-
sure to 10 lm Cu
2+
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 lactate dehydrogenase, 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.
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 studies, consistent with this
previous conclusion. To investigate the types of recep-
tor responsible for the uptake of glycated LDL
observed in the current study, cells were incubated
Formation of lipid-laden cells by glycated LDL B. E. Brown et al.
1536 FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS
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.
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, including methylglyoxal [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.
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

accumulation of glycated proteins observed in people
with diabetes.
Experimental procedures
Materials
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, N
a
-benzoyl-l-arginine ethyl esters,  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 ⁄ P
i
, (pH 7.4). BDH
(Merck, Kilsyth, VIC, Australia): glucose. Bio-Rad (Regents
Park, NSW, Australia): Chelex-100 resin. ICN (Seven Hills,
NSW, Australia): CuSO
4
. Amersham Biosciences (Castle
Hill, NSW, Australia): PD10 columns and Na
125
I(‡ 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 ⁄ P
i
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].
125
I-Labeling of LDL was per-
formed, prior to other modification, using iodine mono-
chloride [36,60]. Specific activity (typically 50–100
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 CuSO
4
) in Chelex-treated NaCl ⁄ P
i
at 37 °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
B. E. Brown et al. Formation of lipid-laden cells by glycated LDL
FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS 1537
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
Monocytes were isolated by countercurrent elutriation
[63,64], using HBSS (with phenol red and 0.01% EDTA,
but without Ca
2+
and Mg
2+
). 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 °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 J–20XPI centrifuge
equipped with a JE 5.0 elutriation rotor (770 g, flow rate
9mLÆmin
)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 · 10
6
cellsÆmL
)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, 4 mm glutamine and 1% (v⁄ v) PenStrep] was
added; this was followed by incubation (5% CO
2
,37°C)
for 9–11 days, with the medium being changed every

3 days, to give matured HMDMs.
Cellular cholesterol and cholesteryl ester analysis
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].
Cellular apoB accumulation and turnover
HMDMs were incubated with modified [
125
I]LDL (50 lg
proteinÆmL
)1
) as described above. Cell medium (0.5 mL)
and cells (after being washed twice with cold NaCl ⁄ P
i
)
were sampled at the indicated times. For turnover studies,
the [
125
I]LDL-containing medium was removed after the
accumulation phase. The cells were then washed with
warm NaCl ⁄ P
i

, and medium containing 1 mgÆmL
)1
BSA
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 ⁄ P
i
. 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 °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 °C). Of the resulting lysate, 0.5 mL was
used to measure total intracellular 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 °C)
and centrifugation using a Sorvall (Sorvall Instruments,
Newtown, CT, USA) RT600B centrifuge and a H1000B
rotor (10 min, 1500 g,4°C) to precipitate proteins. The
supernatant (1 mL) was added to AgNO
3
(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 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
HMDMs were exposed to 0 or 100 lgÆmL
)1
control or
modified LDL for 48 h in medium containing 10% lipopro-
tein-deficient serum with 200 lgÆmL
)1
fucoidin [48],
2 lgÆmL
)1
mAb to CD36 [47], or 200 lgÆmL
)1
AGE–HSA
[48]. AGE–HSA was prepared by incubation of
20 mgÆmL
)1
HSA with 1 m glucose for 4 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 quanti-
fied by HPLC, as described above.
Protein assay
Protein concentrations were quantified using the bicinchoni-
nic acid assay (Pierce, Rockford, IL, USA) with 60 min 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 Bon-
ferroni’s post hoc analysis, with P < 0.05 taken as signifi-
cant.
Formation of lipid-laden cells by glycated LDL B. E. Brown et al.
1538 FEBS Journal 274 (2007) 1530–1541 ª 2007 The Authors Journal compilation ª 2007 FEBS
Acknowledgements
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.
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