CRLPs containing triacylglycerol enriched in saturated, monounsaturated, n6 or n3 polyunsaturated fatty acids derived from palm, olive, corn or fish oil, respectively, and macrophages der
Trang 1particles influences their uptake and induction of lipid
accumulation in macrophages
Clara De Pascale1, Michael Avella1, Javier S Perona2, Valentina Ruiz-Gutierrez2,
Caroline P D Wheeler-Jones1and Kathleen M Botham1
1 Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK
2 Instituto de la Grasa (CSIC), Seville, Spain
The first visible lesions in atherosclerosis development
are fatty streaks, which are formed when macrophages
that have invaded the artery wall take up lipid from
plasma lipoproteins in the subendothelial space and
become so engorged that they form foam cells [1] It is
known that low-density lipoprotein (LDL) has a major
role in the induction of foam cell formation, but it is
also clear that oxidation of the LDL particles, a
pro-cess that can occur within the artery wall, is nepro-cessary before extensive lipid accumulation occurs [2] Recent work in our laboratory and others, however, has pro-vided strong evidence that chylomicron remnants, the lipoproteins that carry fat and cholesterol from the diet, are also able to induce macrophages to form foam cells, and furthermore, that prior oxidation of the particles is not required for their effect [3–5]
Keywords
chylomicron remnant-like particles; fatty acid
composition; lipid accumulation;
macrophages
Correspondence
K M Botham, Department of Veterinary
Basic Sciences, Royal Veterinary College,
Royal College St, London NW1 0TU, UK
Fax: +44 207468 5204
Tel: +44 207468 5274
E-mail: kbotham@rvc.ac.uk
(Received 30 August 2006, revised 10
Octo-ber 2006, accepted 23 OctoOcto-ber 2006)
doi:10.1111/j.1742-4658.2006.05552.x
The influence of the fatty acid composition of chylomicron remnant-like particles (CRLPs) on their uptake and induction of lipid accumulation in macrophages was studied CRLPs containing triacylglycerol enriched in saturated, monounsaturated, n)6 or n)3 polyunsaturated fatty acids derived from palm, olive, corn or fish oil, respectively, and macrophages derived from the human monocyte cell line THP-1 were used Lipid accu-mulation (triacylglycerol and cholesterol) in the cells was measured after incubation with CRLPs for 5, 24 and 48 h, and uptake over 24 h was determined using CRLPs radiolabelled with [3H]triolein Total lipid accu-mulation in the macrophages was significantly greater with palm CRLPs than with the other three types of particle This was mainly due to increased triacylglycerol concentrations, whereas changes in cholesterol concentrations did not reach significance There were no significant differ-ences in lipid accumulation after incubation with olive, corn or fish CRLPs Palm and olive CRLPs were taken up by the cells at a similar rate, which was considerably faster than that observed with corn and fish CRLPs These findings demonstrate that CRLPs enriched in saturated or monoun-saturated fatty acids are taken up more rapidly by macrophages than those enriched in n)6 or n)3 polunsaturated fatty acids, and that the faster uptake rate results in greater lipid accumulation in the case of saturated fatty acid-rich particles, but not monounsaturated fatty acid-rich particles Thus, dietary saturated fatty acids carried in chylomicron remnants may enhance their propensity to induce macrophage foam cell formation
Abbreviations
CRLP, chylomicron remnant-like particle; LDL, low-density lipoprotein; LRP, LDL receptor-like protein; MUFA, monounsaturated fatty acid; PMA, phorbol 12-myristate 13-acetate; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; TC, total cholesterol; TG, triacylglycerol.
Trang 2Fat and cholesterol consumed in the diet are taken
up by intestinal cells and secreted into lymph in
chylo-microns These large triacylglycerol (TG)-rich
lipopro-teins then enter the blood via the thoracic duct and
are metabolized by lipoprotein lipase, a process that
removes some of the triacylglycerol and leaves smaller
chylomicron remnant particles, which deliver the
remaining lipid to the liver [6] A number of lines of
evidence provide a powerful case to support the
ath-erogenicity of chylomicron remnants [7,8] It has been
demonstrated that the particles enter the artery wall as
efficiently as LDL [9,10], and are retained within the
subendothelial space [7,11]; accumulation of remnants
in the plasma of apoE–⁄ – mice is associated with the
development of severe atherosclerosis [12];
remnant-like lipoproteins have been isolated from human aortic
intima and atherosclerotic plaque [13,14]; and delay in
remnant clearance from the circulation has been found
to be correlated with the development of
atheroscler-otic lesions in human patients [15,16]
In previous studies, chylomicron remnants or
chylo-micron remnant-like particles (CRLPs) have been
shown to induce extensive lipid accumulation in a
variety of macrophage types, including the murine
macrophage cell line J774 [17], mouse peritoneal
macro-phages [18], primary human monocyte-derived
macrophages [3,4] and macrophages derived from the
human monocyte cell line THP-1 [4,5,19,20] Both
cho-lesterol and TG accumulate in response to remnant
particles, and the concentrations of TG found are
much greater than those observed on exposure of the
macrophages to an equivalent amount of cholesterol in
oxidized LDL (oxLDL) [4,20] Moreover, once inside
the cells, the lipid taken up appears to be resistant to
efflux [20], as is the case with lipid originating from
oxLDL [21]
It has been known for many years that the type of
fat in the diet influences the development of
athero-sclerosis, with consumption of polyunsaturated
(PUFA) or monounsaturated (MUFA), as compared
with saturated (SFA), fatty acids decreasing the risk
[22] Our earlier work has shown that the fatty acid
composition of chylomicron remnants reflects that of
the diet [23], and modulates their removal from the
blood by the liver, with particles derived from fish
(rich in n)3 PUFAs) or corn (rich in n)6 PUFAs) oil
being taken up more rapidly than those from olive
(rich in MUFAs) or palm (rich in SFAs) oil [24–26]
The finding that variations in the fatty acid
composi-tion of remnants modify their uptake by liver cells
rai-ses the possibility that such changes also affect the
interaction of the particles with macrophages to
promote or retard foam cell formation, providing a
mechanism by which dietary fats could directly influ-ence atherosclerotic lesion development during their transport from the gut to the liver Little is known, however, about how the fatty acid composition of chylomicron remnants affects their uptake by macro-phages and induction of excessive lipid accumulation
in the cells
In this study, we have investigated the effects of chylomicron remnants of different fatty acid composi-tion on macrophage foam cell formacomposi-tion using CRLPs [4,5,19,20] and macrophages derived from the human monocyte cell line THP-1 CRLPs enriched in SFAs, MUFAs, n)6 PUFAs and n)3 PUFAs, obtained by incorporating TG from palm, olive, corn and fish oil, respectively, into the particles were incubated with THP-1 macrophages, and the concentrations of choles-terol and TG accumulated were determined In addi-tion, the effects of varying fatty acid composition
on the uptake of the CRLPs by the cells was studied using the four types of particle radiolabelled with [3H]triolein
Results
Characteristics of CRLPs The content of TG and total cholesterol (TC) and the
TG⁄ TC ratio in CRLPs containing TG from palm (palm CRLPs), olive (olive CRLPs), corn (corn CRLPs) or fish (fish CRLPs) oil are shown in Table 1 Although the TG and TC concentrations varied some-what in the different types of CRLP, with those in palm CRLPs tending to be lower, these values are dependent on the dilution of the preparations More importantly, there were no significant differences in the
TG⁄ TC ratio between palm, olive, corn and fish CRLPs Thus, although the CRLPs were added to the cell incubations according to the TG concentration, the amount of cholesterol present was also similar in experiments with the different types of particle The
Table 1 Lipid content of CRLPs derived from different oils CRLPs containing TG from palm, olive, corn or fish oils were prepared as described in Experimental procedures, and TG and TC contents were measured Data shown are the mean ± SEM from seven separate preparations.
CRLP type
TG (lmolÆmL)1)
TC (lmolÆmL)1) TG ⁄ TC
Trang 3fatty acid composition of the TG in the different types
of CRLP (Table 2) reflected that of the oils from
which they were obtained [23] Thus CRLPs prepared
with TG from palm oil or olive oil contained relatively
high concentrations of SFAs [particularly palmitic acid
(16:0)] or MUFAs [particularly oleic acid (18:1, n)9)],
respectively, whereas those containing TG from corn
or fish oil were relatively enriched in n)6 [mainly
lino-leic acid (18:2, n)6)] or n)3 PUFAs [eicosapentaenoic
acid (20:5) and docosahexanoic acid (22:6)] The
amounts of apoE in palm, olive, corn or fish CRLPs
were not signficantly different (one way analysis
of variance) as assessed by SDS⁄ PAGE [optical density
unitsÆ(lmol TG))1 (n¼ 3): palm CRLPs, 102 ± 25;
olive CRLPs, 122 ± 46; corn CRLPs, 135 ± 19; fish
CRLPs, 99 ± 36]
Assessment of the lipid peroxidation products,
4-hydroxy-2(E)-nonenal + malondialdehyde, in the
CRLPs (Table 3) showed that there were no significant
differences between the four different types of particle
(one way analysis of variance, with Tukey-Kramer’s
test post hoc), although the concentrations tended to
be more variable in the fish CRLPs, as might be
expec-ted from their relatively high content of long-chain
n)3 PUFAs such as eicosapentaenoic acid and
docosa-hexanoic acid Lipid peroxidation products after
incu-bation of the four types of CRLP with CuSO4 (10 lm)
for 6 h were significantly lower with olive CRLPs than
with the other three types, indicating that these
parti-cles are more resistant to oxidation, but there were no
significant differences between palm, corn and fish CRLPs
Effects of the fatty acid composition of CRLPs
on their induction of lipid accumulation
in THP-1 macrophages THP-1 macrophages were incubated with palm, olive, corn or fish CRLPs (0.3 lmol TGÆmL)1) for 5, 24 or
48 h, and the effects on lipid accumulation in the cells were determined Total lipid (TG + TC) concentra-tions were similar in incubaconcentra-tions with all four types of particle after 5 h, but analysis by two-way analysis of
Table 2 Fatty acid composition of TG in CRLPs CRLPs were prepared using TG from palm, olive, corn or fish oil as described in Experimen-tal procedures, and the fatty acid composition was determined by gas chromatography Data are expressed as gÆ(100 g toExperimen-tal fatty acids))1 and are the mean ± SEM from three separate preparations ND, none detected.
Fatty acid
CRLPs
Table 3 Concentrations of malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE) in CRLPs MDA and 4-HNE concentrations in CRLPs containing TG from palm, olive, corn or fish oil (10 l M ) were determined before (0 h) and after (6 h) incubation with CuSO4 (10 l M ) for 6 h Data shown are the mean ± SEM from three separate preparations *P < 0.05, **P < 0.01 versus olive CRLPs (one-way analysis of variance, with Tukey-Kramer’s test multiple comparison test post hoc).
CRLP type
4-HNE + MDA [pmolÆ(nmol TG))1]
Trang 4variance showed that there was significantly more
accumulation in cells exposed to palm CRLPs than in
those exposed to olive, corn or fish CRLPs, with
signi-ficant differences after 24 and 48 h (Fig 1) Similar
analysis of the TG and TC content of the cells showed
that TG followed a similar pattern to that found for
total lipid (Fig 2A), with palm CRLPs causing greater
accumulation than corn (significant at 24 and 48 h) or
olive or fish (significant at 48 h) CRLPs However,
although there were similar trends in TC
concentra-tions, because of the lower cellular concentraconcentra-tions,
these changes did not reach significance
Effects of the fatty acid composition of CRLPs
on their uptake by THP-1 macrophages
The uptake of CRLPs by THP-1 macrophages was
assessed using particles labelled in TG with [3H]triolein
There were no significant differences (one-way analysis
of variance) between the specific activities of the
four different types of CRLP used [dpmÆ(nmol TG))1
(n¼ 4): palm, 2601 ± 326; olive, 2041 ± 154; corn,
2290 ± 303; fish, 1764 ± 140) Cells were incubated
with radiolabelled palm, olive, corn or fish CRLPs for
periods up to 24 h, and the radioactivity associated
with the cells was measured (Fig 3) Palm and olive
CRLPs were taken up by the cells significantly more
rapidly than corn and fish CRLPs (P < 0.01) with
sig-nificant differences at 13 h (P < 0.01) and 24 h
(P < 0.01) Corn and fish CRLPs were taken up at
very similar rates, and there were also no significant
differences in the rates of uptake of palm and olive
CRLPs
Fig 2 THP-1 macrophages were incubated with CRLPs contain-ing TG from palm, olive, corn or fish oil, and the TG (A) and TC (B) accumulated in the cells was measured after 5, 24 and
48 h Data are the mean from three separate experiments, and error bars show the SEM *P < 0.05, **P < 0.01 versus palm CRLPs.
Fig 1 THP-1 macrophages were incubated with CRLPs containing
TG from palm, olive, corn or fish oil, and the total lipid (TG + TC)
accumulated in the cells was measured after 5, 24 and 48 h Data
are the mean from three separate experiments, and error bars
show the SEM *P < 0.05, **P < 0.01 versus palm CRLPs.
Fig 3 THP-1 macrophages were incubated with CRLPs contain-ing TG from palm, olive, corn or fish oil radiolabelled with [ 3 H]triolein, and the amount of radioactivity associated with the cells after the time periods up to 24 h was measured Each point is the mean from three separate experiments, and error bars show the SEM.
Trang 5In previous work, we have used human
monocyte-derived macrophages and macrophages monocyte-derived from
the human monocyte cell line THP-1 to investigate the
uptake of chylomicron remnants and their induction of
lipid accumulation in the cells [4,5,7,19,20] As
chylo-micron remnants uncontaminated with lipoproteins of
a similar density such as chylomicrons and
very-low-density lipoprotein cannot be obtained easily from
human blood, for these studies we used model CRLPs
We have reported previously [4] that these particles
have a diameter of about 130 nm, which is within the
range reported for human chylomicron remnants (50–
150 nm [27]), and that the lipid composition in terms
of the percentage lipid mass of the various lipid classes
and TG⁄ TC molar ratio is also similar to that of
physiological remnants [6] Although there is some
overlap in size between small chylomicrons and
chylo-micron remnants, the TG⁄ TC molar ratio in
chylomicron-like particles would be expected to be
considerably higher (30–40 : 1 [6]) than that in our
CRLPs (9–12 : 1, Table 1) Thus, the CRLPs used
resemble physiological remnants in size, density and
lipid composition and also contain human apoE They
differ from physiological chylomicron remnants,
how-ever, in that they lack apoB48 Howhow-ever, CRLPs of
this type without apoB48 have been shown to behave
like the physiological lipoprotein in their clearance
from the blood and metabolism in vivo, and in their
effects on cultured cells in vitro [28–30] In addition, in
earlier work we have demonstrated that chylomicron
remnants prepared in rats in vivo cause comparable
lipid accumulation in J774 macrophages to that
observed in the present study with CRLPs and THP-1
macrophages [17] As the composition of CRLPs can
be easily manipulated, they provide a suitable and
con-venient model for the present investigation
We have demonstrated previously that the fatty acid
composition of chylomicron remnants reflects that of
the fatty meal from which they were derived Thus,
remnants from rats given an oral dose of palm, olive,
corn or fish oil are enriched in SFAs, MUFAs, n)6
PUFAs and n)3 PUFAs [23], as well as containing a
range of other fatty acids, and these enrichments
influ-ence the uptake and metabolism of the particles by the
liver [24–26,31] The CRLPs used in our previous work
with macrophages contained a single TG molecular
species, trilinolein, but to mimic the physiological
situ-ation more closely, for the current investigsitu-ation of the
effects of the fatty acid composition of the particles on
macrophage foam cell formation, we used CRLPs
con-taining TG derived from natural dietary oils Thus, the
particles were enriched in the particular type of fatty acid predominating in the oil, but also contained a complex mixture of other fatty acids, as occurs in vivo
As might be expected, the fatty acid composition of CRLPs containing TG from palm, olive, corn or fish oil (Table 2) was similar to both that of the parent oils and that of rat physiological remnants derived from them [23], so that the CRLPs were enriched in SFAs, MUFAs, n)6 PUFAs and n)3 PUFAs, respectively Earlier work in our laboratory and others has estab-lished that chylomicron remnants are taken up by macro-phages and induce the extensive lipid accumulation associated with foam cell formation [3,4,7,17,18,32,33] However, although the degree of unsaturation of diet-ary fatty acids is believed to influence atherosclerotic plaque development [22], it is not known whether the propensity of the remnants to induce foam cell forma-tion is affected by their fatty acid composiforma-tion The results presented here indicate that CRLPs enriched in SFAs are taken up more rapidly by macrophages than those high in n)6 or n)3 PUFAs and cause greater lipid accumulation in the cells (Figs 1–3) The latter effect was mainly due to increased concentrations of
TG, although the TC content showed similar trends which did not reach significance Interestingly, although the rate of uptake of the olive particles was comparable to that of palm CRLPs (Fig 3), the amount of lipid accumulated in the cells was signifi-cantly lower As the radiolabel in these experiments was in the oleate moiety of triolein, these findings sug-gest that TG taken up from CRLPs containing high concentrations of oleate may be metabolized more readily than TG from the other types of CRLP, caus-ing an increase in the release of the radiolabel as the free fatty acid for oxidation or in its transfer to phos-pholipid, thus lowering the lipid accumulation (TG + TC) despite the relatively high rate of uptake This conclusion is supported by findings of Peres and coworkers [34,35] with 14C-labelled fatty acids, show-ing that oleate is oxidized more rapidly than palmitate
in macrophages and that 7.5 times more oleate than palmitate is transferred from these cells to lymphocytes
in coculture, indicating that macrophage metabolism
of oleate is more active than that of palmitate
Although it has been established in extensive studies that LDL has a major role in foam cell formation, it is also clear that oxidation of the particles greatly enhan-ces their effects [2] In striking contrast, chylomicron remnants have been shown to cause extensive lipid accumulation in macrophages without prior oxidation
of the particles [3,4,7] Furthermore, work in our laboratory has shown that protection of CRLPs from oxidation by incorporation of antioxidants such as
Trang 6lycopene or probucol into the particles enhances,
rather than inhibits, their uptake and induction of lipid
accumulation in THP-1 macrophages [5,19] These
findings suggest that, in sharp contrast with LDL,
oxi-dation of chylomicron remnants decreases their uptake
by the cells It is possible therefore that the differential
rates of uptake of CRLPs of different fatty acid
com-positions by macrophages observed in the present
study is due to differences in the oxidative state of the
particles Evaluation of the lipid peroxidation
prod-ucts, 4-hydroxy-2(E)-nonenal and malondialdehyde, in
the preparations indicates that there were no
signifi-cant differences between the four types of particle
tes-ted at the start of the experiments Moreover, in
general, there was no obvious correlation between the
resistance to oxidation of the various particle types
during incubation and the rate of their uptake by the
THP-1 macrophages, suggesting that other factors are
mainly responsible for the different uptake rates
observed The olive CRLPs, however, were
signifi-cantly more resistant to oxidation than the other types,
and this may provide part of the explanation for their
more rapid uptake compared with corn and fish
CRLPs, as the latter particles may become oxidized
during the incubation period
It is known that the liver takes up chylomicron
rem-nants mainly via the LDL receptor and the LDL
receptor-like protein (LRP) [36], which both recognize
apoE In contrast, the exact mechanisms by which
chylomicron remnants are taken up by macrophages
have not been definitively established However, the
LDL receptor and the LRP are both expressed by
these cells, and a number of studies have demonstrated
that they play an important role [7,18,33], although
there may be other entry routes including phagocytosis
and scavenger receptors such as scavenger receptor A
(SR-A) and CD36 [7,36], which are known to play a
part in the uptake of oxidized or chemically modified
LDL [37] Factors that influence the binding of apoE
to its receptors therefore are likely to affect the uptake
of chylomicron remnants by macrophages It has been
demonstrated that ApoE does not bind to the LDL
receptor family in its lipid-free state The
receptor-binding region is in an amphipathic a-helix which is
part of a four-helix bundle structure in the N-terminal
domain, and interaction with lipid is necessary to
induce a conformational change that promotes high
affinity for the receptors [38] Because of the
import-ance of apoE conformation for receptor binding, it is
thought that not all apoE molecules on a particular
remnant particle are able to act as ligands In addition,
apoE has been shown to adopt different conformations
when complexed to different lipids [39], and it has been
suggested that the lipid composition of lipoproteins can alter the conformation of the protein [40] Thus, the differential uptake of CRLPs of different fatty acid composition by macrophages demonstrated here may
be explained by effects on uptake via the LDL recep-tor and the LRP due to differences in both the confor-mation of apoE and the number of apoE molecules able to bind to the receptors
A receptor for apoB48 has been identified in endo-thelial cells and has also been reported to be expressed
in macrophages, and it has been suggested that it may
be involved in the uptake of chylomicron remnants by macrophages [41] However, antibodies to apoB48 have been shown not to inhibit the uptake of chylomi-cron remnants by rat macrophages [42], and Elsegood
et al [33] were unable to detect binding of chylo-micron remnants to a protein with a molecular mass corresponding to the apoB48 receptor in THP-1 macro-phages Furthermore, we have found that the expression of the apoB48 receptor is very low in the THP-1 macrophages used in this study, although we were able to demonstrate strong expression in human umbilical vein endothelial cells using the same system (F Bejta and K M Botham, unpublished results) It
is unlikely therefore that the lack of apoB48 in our CRLPs has any significant effect on the results obtained
In summary, this study shows that the fatty acid composition of chylomicron remnants influences their uptake and induction of lipid accumulation in macro-phages, with those enriched in SFAs or MUFAs being taken up more rapidly than those enriched in n)6 or n)3 PUFAs The faster uptake rate results in greater lipid accumulation in the case of SFAs, but not MUFA-rich particles, possibly because of increased intracellular metabolism of TG in the latter case These findings suggest that dietary SFAs carried in chylo-micron remnants may enhance their ability to induce macrophage foam cell formation, thus increasing their atherogenicity
Experimental procedures
Fetal bovine serum (heat inactivated), penicillin, strepto-mycin and 2-mercaptoethanol were obtain from Gibco (Paisley, UK) RPMI 1640, Trypan blue, fatty acid-free albumin (BSA), phospholipids, cholesterol, cholesteryl oleate, phorbol 12-myristate 13-acetate (PMA) and Menhaden fish oil were supplied by Sigma (Poole, Dorset, UK) Palm (KTC Edibles Ltd, Wednesbury, UK), extra virgin olive (Bertolli; Unilever Foods UK, Crawley, Sur-rey, UK) and corn (Mazola; Bestfoods UK Ltd, Esher, UK) oil were purchased from domestic suppliers The
Trang 7radioisotope [3H]triolein was supplied by Perkin–Elmer
(Beaconsfield, UK)
Preparation of CRLPs
TGs for CRLP preparation were isolated from palm,
olive, corn and fish oils as follows: 1.5 mL of each oil was
added to 30 mL chloroform⁄ methanol (2 : 1, v ⁄ v) and
0.88% KCl (40% total volume), mixed and left at 4C
overnight The upper aqueous phase was then removed,
and TGs were isolated from the chloroform phase by
TLC (hexane⁄ diethyl ether ⁄ formic acid (80 : 20 : 2, v ⁄ v ⁄ v)
The band corresponding to TG was collected, resuspended
in chloroform, and centrifuged twice at 1200 g for 20 min
(4C) with an MSE Mistral 3000i centrifuge (MSE,
London, UK) with 43122-105 swingout rotor to remove
the silica gel The chloroform supernatant was collected
after each centrifugation and kept under argon at )20 C
until required
CRLPs were prepared by sonication (power setting
22–24 lm, 20 min, at 56C) of a lipid mixture containing
70% TG extracted from oils as above, 2% cholesterol, 5%
cholesteryl ester and 25% phospholipids in 0.9% (w⁄ v) NaCl
in Tricine buffer (20 mm, pH 7.4) The resulting emulsion
was brought to a density of 1.21 gÆmL)1with KBr, layered
under a stepwise density gradient as described previously [43],
and centrifuged at 17 000 g for 20 min at 20C with a
Beck-man Optima L-80 centrifuge (BeckBeck-man Coulter, High
Wgcombe, UK) with SW40Ti swingout rotor The upper
layer of grossly emulsified lipids was then removed and
replaced with an equal volume of NaCl solution (d¼
1.020 gÆmL)1), and tubes were centrifuged at 70 000 g for 1 h
(20C) with a Beckman Optima L-80 centrifuge with SW40Ti
swingout rotor Lipid particles harvested from the top layer
were incubated with d > 1.020 gÆmL)1 fraction of human
plasma (National Blood Transfusion Service, North London
Centre, London, UK) prepared by ultracentrifugation and
dialysed before use as described previously [27] at 37C with
shaking for 4 h (1 volume of particles : 2 volumes plasma)
CRLPs were then isolated by ultracentrifugation at d¼
1.006 gÆmL)1(120 000 g for 12 h at 4C), harvested from the
top layer, purified by a second centrifugation at the same
den-sity with a Beckman Optima L-80 centrifuge with SW40Ti
swingout rotor (202 000 g for 4 h at 4C) and stored at 4 C
under argon until required All preparations were used within
1 week SDS⁄ PAGE analysis showed that all four types of
particles contained similar amounts of apoE For preparation
of CRLPs labelled with [3H]triolein, the radioisotope
(1.85 MBq) was added to the lipid mixture before sonication
The level of oxidation of the CRLPs containing TG from
the four different oils was determined by measuring the
concentrations of 4-hydroxy-2(E)-nonenal and
malondialde-hyde using the commercially available Bioxytech LPO-586
assay (Oxis International Inc, Portland, OR, USA),
follow-ing the manufacturer’s instructions
Culture of THP-1 cells and experimental protocol
THP-1 monocytes were maintained in suspension in RPMI 1640 culture medium containing 10% fetal bovine serum, 100 UÆmL)1 penicillin, 100 mgÆmL)1 streptomycin and 50 lm 2-mercaptoethanol (culture medium) at a density
of (3–9)· 105
cellsÆmL)1at 37C in 5% air ⁄ 95% CO2 The cells were induced to differentiate into macrophages by incubation with PMA (200 ngÆmL)1) for 72 h After this time, the cells adhering to the culture dishes were washed with warm NaCl⁄ Pi(3· 2 mL) to remove any undifferenti-ated cells and traces of PMA The viability of the THP-1 macrophages, as assessed by Trypan blue exclusion, was
> 95% in all experiments
For lipid-accumulation studies, CRLPs (0.3 lmol triacyl-glycerolÆmL)1) were incubated with macrophages for 5, 24
or 48 h The cells were then washed with NaCl⁄ Pi (3· 3 mL), harvested in 0.7 mL NaCl ⁄ Pi, disrupted by sonication for 5 s (22–24 lm, power setting), and a sample was taken for protein determination Lipids were extracted with chloroform⁄ methanol (2 : 1, v ⁄ v) and 0.88% KCl (40% total volume) For uptake studies, THP-1 macroph-ages were incubated with CRLPs labelled with [3H]triolein (0.3 lmol TGÆmL)1) for times up to 24 h After the incuba-tion period, cells were washed with NaCl⁄ Pi (4· 3 mL), and resuspended in 0.5 mL NaOH (0.5 m) Radioactivity was determined by liquid-scintillation counting using Hydrofluor as the scintillant
Analytical methods
TC (cholesterol + cholesteryl ester) and TG in CRLPs and
in cells was determined by enzymatic analysis using com-mercial enzymatic reagent kits (Alpha Laboratories, East-leigh, UK) For analysis of the fatty acid composition of
TG in the CRLPs, TG were transmethylated using sodium methoxide in methanol (0.5%), and the resulting fatty acid methyl esters were analysed by gas chromatography, using
a model 5890 series II gas chromatograph (Hewlett-Packard
Co, Avondale, PA, USA) equipped with a flame ionization detector and a capillary silica column Supelcowax 10 (Sul-pelco Co, Bellefonte, PA, USA) of 60 m length and 0.25 mm internal diameter The relative apoE content of the different types of CRLP was assessed using SDS⁄ PAGE The gels were stained with Coomassie blue and the bands were quantified by absorbance volume analy-sis Cell protein content was measured by the method of Lowry et al [44] with BSA as standard
Statistical analysis
Data were analysed by two-way analysis of variance fol-lowed by Bonferroni’s multiple comparison test except where indicated otherwise
Trang 8This work was supported by a grant from the British
Heart Foundation (PG⁄ 04 ⁄ 021 ⁄ 16758)
References
1 Libby P, Geng YJ, Aikawa M, Schoenbeck U, Mach F,
Clinton SK, Sukhova GK & Lee RT (1996)
Macro-phages and atherosclerotic plaque stability Curr Opin
Lipidol 7, 330–335
2 Albertini R, Moratti R & DeLuca G (2002) Oxidation
of low-density lipoprotein in atherosclerosis from basic
biochemistry to clinical studies Curr Mol Med 2,
579–592
3 Yu KC & Mamo JCL (2000) Chylomicron
remnant-induced foam cell formation and cytotoxicity: a possible
mechanism for cell death in atherosclerosis Clin Sci 98,
183–192
4 Batt KV, Avella M, Moore EH, Jackson B, Suckling KE
& Botham KM (2004) Differential effects of low density
lipoprotein and chylomicron remnants on lipid
accumu-lation in human macrophages Exp Biol Med 229, 528–
537
5 Moore EH, Napolitano M, Avella M, Bejta F, Suckling
KE, Bravo E & Botham KM (2004) Protection of
chylomicron remnants from oxidation by incorporation
of probucol into the particles enhances their uptake by
human macrophages and increases lipid accumulation in
the cells Eur J Biochem 271, 2417–2427
6 Redgrave TG (1983) Formation and metabolism of
chylomicrons Int Rev Physiol 28, 103–130
7 Botham KM, Bravo E, Elliott J & Wheeler-Jones CPD
(2005) Direct interaction of dietary lipids carried in
chylomicron remnants with cells of the artery wall:
implications for atherosclerosis development Curr
Pharmaceut Design 11, 3681–3695
8 Wilhelm MG & Cooper AD (2003) Induction of
athero-sclerosis by human chylomicron remnants: a hypothesis
J Atheroscler Thromb 10, 132–139
9 Mamo JC & Wheeler JR (1994) Chylomicrons or
their remnants penetrate the rabbit thoracic aorta as
efficiently as do smaller macromolecules, including
low-density lipoprotein, high-low-density lipoprotein, and
albu-min Coron Artery Dis 5, 695–705
10 Grieve DJ, Avella MA, Elliott J & Botham KM (1998)
Influence of chylomicron remnants on endothelial cell
function in the isolated perfused rat aorta
Atherosclero-sis 139, 273–281
11 Proctor SD, Vine DF & Mamo JCL (2002) Arterial
retention of apolipoprotein B48- and B100-containing
lipoproteins in atherosclerosis Curr Opin Lipidol 13,
461–470
12 Boren J, Gustafsson M, Skalen K, Flood C &
Innerar-ity TL (2000) Role of extracellular retention of low
density lipoproteins in atherosclerosis Curr Opin Lipidol
11, 451–456
13 Ghung BH, Talis G, Yalamoori V, Anantharamaiah GM
& Segrest JP (1994) Liposome-like particles isolated from human atherosclerotic plaques are structurally similar to surface remnants of triglyceride-rich lipoproteins Arterioscler Thromb 14, 622–635
14 Pal S, Semorine K, Watts GF & Mamo J (2003) Identi-fication of lipoproteins of intestinal origin in human atherosclerotic plaque Clin Chem Lab Med 41, 792– 795
15 Patsch JR, Miesenboch G, Hopferwieser T, Muhlberger V, Knapp E, Dunn JK, Gotto AM & Patsch W (1992) Relation of triglyceride metabolism and cardiovascular disease Arterioscler Thromb 12, 1336–1345
16 Benlian P, De Gennes PL, Foubert L, Zhang H, Gagne
SE & Hayden M (1996) Premature atherosclerosis in patients with familial chylomicronemia caused by muta-tions in the lipoprotein lipase gene N Engl J Med 335, 848–854
17 Napolitano M, Rivabene R, Avella M, Botham KM & Bravo E (2001) The internal redox balance of the cells influences the metabolism of lipids of dietary origin by J774 macrophages: implications for foam cell formation
J Vasc Res 38, 350–360
18 Fujioka Y, Cooper AD & Fong LG (1998) Multiple processes are involved in the uptake of chylomicron remnants by mouse peritoneal macrophages J Lipid Res
39, 2339–2349
19 Moore EH, Napolitano M, Prosperi A, Avella M, Suck-ling KE, Bravo E & Botham KM (2003) Incorporation
of lycopene into chylomicron remnant-like particles enhances their induction of lipid accumulation in macrophages Biochem Biophys Res Commun 312, 1216– 1219
20 Moore EH, Bejta F, Avella M, Suckling KE & Botham
KM (2005) Efflux of lipid from macrophages after induction of lipid accumulation by chylomicron rem-nants Biochim Biophys Acta 1735, 20–29
21 Yancey PG & Jerome WG (2001) Lysosomal cholesterol derived from mildly oxidized low density lipoprotein is resistant to efflux J Lipid Res 42, 317–327
22 Moreno JJ & Mitjavila MT (2003) The degree of unsa-turation of dietary fatty acids and the development of atherosclerosis (review) J Nutr Biochem 14, 182–195
23 Lambert MS, Botham KM & Mayes PA (1996) Modifi-cation of the composition of dietary oils and fats upon incorporation into chylomicron remnants Br J Nutr 76, 435–445
24 Lambert MS, Botham KM & Mayes PA (1995) Variations in composition of dietary fats affect hepatic uptake and metabolism of chylomicron remnants Biochem J 310, 845–852
Trang 925 Bravo E, Ortu G, Cantafora A, Lambert MS, Avella M,
Mayes PA & Botham KM (1995) Comparison of the
hepatic uptake and processing of cholesterol from
chylo-microns of different fatty acid composition in the rat in
vivo Biochim Biophys Acta 1258, 328–336
26 Lambert MS, Avella MA, Berhane Y, Shervill E &
Botham KM (2001) The fatty acid composition of
chylo-micron remnants influences their binding and
internali-sation by isolated hepatocytes Eur J Biochem 268,
3983–3992
27 Rye KA, Clay MA & Barter PJ (1999) Overview of
plasma lipid transport In Plasma Lipids and their Role
in Disease(Barter PJ & Rye KA, eds), pp 1–16
Har-wood Academic Publishers, Sydney
28 Redgrave TG, Ly HL, Quintao CR, Ramberg CF &
Boston R (1993) Clearance from plasma of
triacylgly-cerol and cholesteryl ester after intravenous injection
of chylomicron-like lipid emulsions in rats and man
Biochem J 290, 843–847
29 Maranhao RC, Fers MC, Martins MT, Mesquita CH,
Toffoletto O, Vinagre CGC, Gianinni SD & Pileggi F
(1996) Plasma kinetics of a chylomicron-like emulsion
in patients with coronary artery disease Atherosclerosis
126, 15–25
30 Diard P, Malewiak M-I, Lagrange D & Griglio S (1994)
Hepatic lipase may act as a ligand in the uptake of
arti-ficial chylomicron remnant-like particles by isolated rat
hepatocytes Biochem J 299, 889–894
31 Lambert MS, Avella MA, Botham KM & Mayes PA
(2000) The type of dietary fat alters the hepatic uptake
and biliary excretion of cholesterol from chylomicron
remnants Br J Nutr 83, 431–438
32 Ellsworth JL, Fong LG, Kraemer FB & Cooper AD
(1990) Differences in the processing of chylomicron
remnants and b-VLDL by macrophages J Lipid Res 31,
1399–1411
33 Elsegood CL, Pal S, Roach PD & Mamo JCL (2001)
The binding and uptake of chylomicron remnants by
primary and THP-1 human macrophages: determination
of binding proteins Clin Sci 101, 111–119
34 Peres CM, Homem de Bittencourt PI, Costa M, Curi R
& Williams JF (1997) Evidence for thr transfer in cul-ture of [14C]-labelled fatty acids from macrophages to lymphocytes Biochem Mol Biol Int 43, 1137–1144
35 Peres CM, Procopio J, Costa M & Curi R (1999) Thio-glycolate-elicited rat macrophages exhibit alterations in incorporation and oxidation of fatty acids Lipids 34, 1193–1197
36 Yu KC & Cooper AD (2001) Postprandial lipoproteins and atherosclerosis Front Biosci 6, D332–D354
37 Linton MF & Fazio S (2001) Class A scavenger recep-tors, macrophages and atherosclerosis Curr Opin Lipi-dol 12, 489–495
38 Saito H, Lund-Katz S & Phillips MC (2004) Contribu-tions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins Prog Lipid Res 43, 350–380
39 Hatters DM, Budagunta MS, Voss JC & Weisgraber KH (2005) Modulation of apolipoprotein E structure by domain interaction: differences in bound and lipid-free forms J Biol Chem 280, 34288–34295
40 Narayanaswami V & Ryan RO (2000) Molecular basis
of exchangeable apolipoprotein function Biochim Bio-phys Acta 1483, 15–36
41 Gianturco SH & Bradley WA (1999) Pathophysiology
of triglyceride-rich lipoproteins in atherothrombosis: cel-lular aspects Clin Cardiol 22 (Suppl II), II–7–II-14
42 Nestel PJ, Billington T & Bazelmans J (1985) Meta-bolism of human plasma triacylglycerol-rich lipoproteins
in rodent macrophages: capacity for interaction at beta-VLDL receptor Biochim Biophys Acta 837, 314–324
43 Napolitano M, Batt KV, Avella M, Bravo E & Botham
KM (2001) Lipid synthesis in macrophages derived from the human cell line THP-1: modulation of the effects of native and oxidized chylomicron remnant-like particles
by oestrogen Clin Sci 101, 403–413
44 Lowry OH, Rosebrough NJ, Farr AL & Randall RJ (1951) Protein measurment with Folin phenol reagent
J Biol Chem 193, 265–275