© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Modification of Type III VLDL, Their Remnants, and VLDL From ApoE-Knockout Mice by p-Hydroxyphenylacetaldehyde, a Product of Myeloperoxidase Activity, Causes Marked Cholesteryl Ester Accumulation in Macrophages
From the Departments of Biochemistry and Medicine and the Robarts Research Institute (S.C.W., D.B.M., R.A.H., M.W.H.), University of Western Ontario, London, Ontario, Canada; and the Departments of Medicine and of Molecular Biology and Pharmacology (S.L.H. and J.W.H.), Washington University School of Medicine, St Louis, Mo.
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Abstract |
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Abstract—Very low density lipoproteins (VLDLs) from apolipoprotein (apo) E2/E2 subjects with type III hyperlipoproteinemia, VLDL remnants, and VLDL from apoE-knockout (EKO) mice are taken up poorly by macrophages. The present study examined whether VLDL modification by the reactive aldehyde p-hydroxyphenylacetaldehyde (pHA) enhances cholesteryl ester (CE) accumulation by J774A.1 macrophages. pHA is the major product derived from the oxidation of L-tyrosine by myeloperoxidase and is a component of human atherosclerotic lesions. Incubation of J774A.1 cells with native type III VLDL, their remnants, and EKO-VLDL increased cellular CE by only 3-, 5-, and 5-fold, respectively, compared with controls. In striking contrast, cells exposed to VLDL modified by purified pHA (pHA-VLDL) exhibited marked increases in cellular CE of 38-, 47-, and 35-fold, respectively (P
0.0001). Addition of the lipoprotein lipase
inhibitor tetrahydrolipstatin decreased cellular CE
accumulation induced by the 3 pHA-modified VLDL preparations by 73%,
59%, and 73%, respectively. Addition of the acyl coenzyme
A:cholesterol acyltransferase inhibitor DuP 128
to cells incubated with the pHA-modified lipoproteins decreased
cellular CE by 100%, 82%, and 95%, respectively, but had no effect
on cellular triglycerides. To examine whether the type A
scavenger receptors (SR-As) mediated the uptake of pHA-VLDL,
incubations were performed in the presence of polyinosine (poly I), a
polynucleotide known to block binding to SR-As (types I and
II), or in cells preincubated with interferon-
(IFN-), a
cytokine known to decrease expression of SR-A type I.
Coincubation of pHA-VLDL with poly I reduced cellular CE by only 38%,
44%, and 49%, respectively, whereas coincubation with IFN- reduced
CE by only 18%, 27%, and 65%, respectively. In marked contrast to
pHA-VLDL, both poly I and IFN- inhibited, by>95%, CE accumulation
induced by copper-oxidized VLDL. These results demonstrate a novel
mechanism for the conversion of type III VLDLs, their remnants, and
EKO-VLDL into atherogenic particles and suggest that macrophage
uptake of pHA-VLDL (1) requires catalytically active lipoprotein
lipase, (2) involves acyl coenzyme A:cholesterol
acyltransferase–mediated cholesterol esterification, and
(3) involves pathways distinct from the SR-A.
Key Words: foam cells • atherosclerosis • in vitro • lipoproteins • reactive aldehydes
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Introduction |
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Cholesterol-loaded macrophages, morphologically recognized as foam cells, are a characteristic feature of atherosclerotic plaques at all stages of lesion development.1 2 Unlike human LDLs,2 human hypertriglyceridemic VLDLs (HTG-VLDLs) and their remnants (VLDL-REM) possessing receptor binding–competent apoE can induce foam cell formation in cultured macrophages without the requirement for oxidation.3 4 5 6 7
The human apoE gene is polymorphic, having 3 main alleles,
2, 3, and 4, which in turn give rise to 3 protein isoforms,
E2, E3, and E4, respectively.8 9 ApoE3 and apoE4 have been
shown to be the main ligands for removal by the liver of both HTG-VLDLs
and VLDL-REM.9 Unlike apoE3 and apoE4, the apoE2 (Cys 112,
Cys 158) isoform is defective in its ability to bind to the LDL
(apoB/E) receptor.10 11 Subjects homozygous for apoE2 are
susceptible to type III hyperlipoproteinemia
(HLP).12 Subjects with type III HLP have elevated levels
of cholesteryl ester (CE) –enriched HTG-VLDLs owing to the inability
of hepatic receptors to clear these lipoproteins from the
circulation.12 13 Although subjects with type III HLP have
elevated levels of HTG-VLDLs and remnant particles, these individuals
often have normal or reduced levels of LDL and are still at an
increased risk for atherosclerosis.12 13
However, the basis for this increased risk is not fully understood.
HTG-VLDLs and VLDL-REM isolated from subjects with type IV HLP do
contain receptor binding–competent apoE and will induce CE
accumulation in cultured macrophages.3 4 In marked
contrast to type IV HTG-VLDLs, coincubation of these same
macrophages with type III HTG-VLDLs fails to induce an
appreciable increase in cellular CE content.3 4
Similar to subjects with type III HLP, apoE knockout (EKO) mice display defective remnant clearance.14 EKO mice develop atherosclerosis spontaneously,14 15 16 17 18 emphasizing the atherogenicity of REM and the importance of functional apoE in the metabolism of triglyceride (TG) -rich lipoproteins. In EKO mice, the remnant lipoproteins must therefore cause macrophage lipid accumulation by an apoE-independent mechanism. EKO mice have foam cell lesions that contain epitopes of oxidized lipoproteins and plasma that contains autoantibodies that recognize epitopes on oxidized lipoproteins.17 19 Therefore, oxidative modification of VLDLs and VLDL-REM may be responsible for lesion development in this animal model of atherosclerosis.
We have previously shown that the incubation of cultured macrophages (J774A.1 cells) with copper-oxidized (CuOx) type III HTG-VLDLs and VLDL-REM will induce foam cell formation by enhancing cellular CE accumulation to levels well above those achieved with CuOx-LDL.20 Despite the ability of transition metals such as copper to induce oxidative modification of lipoproteins in vitro, human plasma possesses very efficient antioxidant defense mechanisms to inhibit free metal ion oxidation of lipoproteins.21 22 Indeed, mass spectrometric analysis of LDL recovered from human aortas failed to demonstrate increased levels of protein markers generated subsequent to protein oxidation by free metal ions.23 Thus, the in vivo significance of free metal ions, such as copper, in lipoprotein modification is questionable.
Lipoprotein modification in vivo may occur via a pathway involving the heme protein myeloperoxidase (MPO), which is secreted by activated neutrophils and monocytes at sites of inflammation.24 25 26 27 28 MPO is a component of human atherosclerotic lesions and colocalizes with macrophages in transitional lesions.27 The concept that MPO can contribute to lipoprotein oxidation within the arterial wall is supported by (1) immunohistochemical studies, which suggest that MPO-modified proteins are present in atherosclerotic tissue,29 and (2) analytical chemistry studies that have detected elevated levels of 3-chlorotyrosine, a specific product of MPO, in human aortic atherosclerotic tissue and in LDL recovered from atherosclerotic aortic intima.28
The best-characterized product of MPO is hypochlorous acid (HOCl),
which is generated from physiological
concentrations of chloride ion (Cl-) and
H2O2.26 30 31
In addition to its ability to oxidize various protein and unsaturated
lipid moieties,32 33 34 35 36 37 38 HOCl will convert
L-tyrosine to the amphipathic aldehyde
p-hydroxyphenylacetaldehyde (pHA) in near-quantitative
yield.39 At physiological
concentrations of L-tyrosine and
Cl-, pHA is the major product of phagocyte
activation.39 pHA can covalently modify proteins via a
Schiff base reaction between the aldehyde and the -amino moiety of
lysine residues.40 pHA-modified lysine residues have been
detected by mass spectrometry in inflamed human tissues40
and human aortic atherosclerotic tissue, raising the possibility that
reactive aldehydes generated by MPO can covalently modify proteins in
vivo.41
In the present study, we tested the hypothesis that pHA modification of either HTG-VLDLs (Sf 60 to 400) isolated from subjects with type III HLP, VLDL-REM, or VLDLs isolated from EKO mice causes significant macrophage CE accumulation and foam cell formation. We now demonstrate that (1) incubation of cultured macrophages with pHA-modified type III HTG-VLDLs, type III VLDL-REM, and EKO-VLDLs results in a marked increase in macrophage CE accumulation and foam cell formation; (2) cell-secreted lipoprotein lipase (LPL) is important in the uptake process; (3) CE accumulation involves cellular acyl coenzyme A:cholesterol acyltransferase (ACAT); and (4) unlike CuOx-VLDLs, uptake of pHA-VLDLs is mediated in part by a non– class A scavenger receptor (SR-A) –mediated mechanism.
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Methods |
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Subjects
Subjects were recruited from the Outpatient Lipid and Vascular Risk Protection Clinic at the London Health Sciences Center, University Campus, London, Ontario, and the Lipid Clinic at St Michael's Hospital, Toronto, Ontario. Lipoprotein phenotypes of the type III HLP subjects were classified according to the criteria of Schaefer and Levy.42 All 12 type III HLP subjects were homozygous for the apoE2 isoform as determined by isoelectric focusing gel electrophoresis and/or DNA restriction isotyping.43 44 45 All type III HLP subjects had elevated plasma cholesterol (10.73±1.08 mmol/L, mean±SEM) and TG (7.31±0.75 mmol/L) levels due to elevated VLDL cholesterol (6.61±1.13 mmol/L) and TG (6.79±0.72 mmol/L) concentrations. These studies were approved by the University of Western Ontario Health Sciences Standing Committee on Human Research, and all subjects gave informed consent before blood sampling. EKO mice46 bred into a C57BL/6J background, strain C57BL/6J-apoetm1Unc (Jackson Laboratory, Bar Harbor, Me), were a generous gift from Dr B. Singh (University of Western Ontario, Ontario, Canada). Four- to 6-month-old EKO mice were placed on a Western-type diet (21% wt/wt fat, 0.15% wt/wt cholesterol; Harlan/Teklad) for 1 week before blood taking. The mouse experimental protocols were approved by the Animal Care Committee of the University of Western Ontario.
Lipoprotein Isolation
After a 12- to 14-hour fast, 60 to 180 mL of blood was collected
from each subject. One milliliter of blood was collected from each
overnight-fasted EKO mouse. The blood was immediately placed in tubes
containing EDTA at a final concentration of 0.15% (wt/vol). Plasma was
isolated by centrifugation (Sorvall IEC Centra-8R
centrifuge) at 2500 rpm (1000g) for 25 minutes at
4°C. Lipoproteins were collected and washed, as described
previously,47 by ultracentrifugation
in a Beckman L8 ultracentrifuge. From human plasma, the large
HTG-VLDL (Sf 60 to 400) subclass was isolated using a Beckman 55.2 Ti
rotor (1.75 hours, 40 000 rpm, 12°C) and subsequently washed using a
Beckman 70.1 Ti rotor (16 hours, 40 000 rpm, 12°C). Because of the
small volumes of plasma collected from each mouse, the entire VLDL
subclass (Sf 20 to 400) was isolated using a Beckman 50.4 Ti rotor (16
hours, 36 000 rpm, 12°C) and subsequently washed using the same
rotor and running conditions. HTG-VLDL (Sf 60 to 400) and EKO-VLDL (Sf
20 to 400) preparations were extensively dialyzed in the dark at 4°C
against a 200-fold excess of PBS (154 mmol/L NaCl, 8 mmol/L
Na2HPO4 ·
7H2O, 1.5 mmol/L
KH2PO4, and 2.7 mmol/L
KCl, pH 7.4) containing 10 µmol/L EDTA. After dialysis, the
lipoprotein samples were sterilized by passage through 0.45-µm
filters and stored at 4°C.
All lipoprotein samples were analyzed for protein content by a modification of the Lowry method48 ; for free fatty acids (FFAs) by using enzymatic reagents (No. 990-75401) from Wako (distributed by Immunocorp); and for TG, free cholesterol (FC), and total cholesterol (TC) by using enzymatic reagents from Boehringer Mannheim GmbH Diagnostica (for TG, No. 450032 without free glycerol; for FC, No. 310328; and for TC, No. 1442350).
Bovine Milk LPL Isolation
Bovine skim milk LPL was partially purified as described
previously.49 The LPL activity in each eluted fraction was
determined by measuring the amount of FFAs released from a
predetermined amount of a commercially obtained TG emulsion
(Intralipid, Pharmacia Inc), as described previously.20
One unit of LPL activity is defined as 1 µmol of FFA released
per mL of enzyme solution per hour.
VLDL-REM Preparation
REM-like particles of type III HTG-VLDLs were formed in vitro
under sterile conditions by incubating the HTG-VLDLs with LPL (0.2 U
per 50 µg total lipoprotein cholesterol) in the presence
of a 5% (wt/vol, final concentration) solution of fatty acid–free BSA
in PBS as described previously.20 TG hydrolysis was
allowed to proceed at 37°C for 6 hours. The reisolated VLDL-REM
preparations were dialyzed and sterilized (as stated above), and the
percent TG hydrolysis was calculated.20 The percentage of
TG hydrolyzed ranged from 15% to 30% (mean, 20±5%).
Synthesis of pHA and Modification of VLDL
pHA was synthesized by adding NaOCl (Fisher Scientific) in a 1:1
mol/mol ratio (final concentration) dropwise with constant mixing to
2 mmol/L L-tyrosine (Sigma) dissolved in ice-cold
20 mmol/L NaH2PO4
· 2H2O (pH 7.0) containing 100 µmol/L
DTPA and then warmed to 37°C for 60 minutes.40 pHA was
stored at 4°C and used in experiments within 4 days of being
synthesized. Preparations were analyzed before use by
high-performance liquid chromatography as
described previously40 and routinely found to be >95%
pure. Preparations of pHA were also analyzed for the presence
of NaOCl. There was no residual NaOCl as determined by an assay for
taurine chloramine, a product of NaOCl.50 Modification
of lipoproteins by pHA was carried out by adding filter-sterilized pHA
(1.0 mmol/L, final concentration) to the dialyzed and sterile
HTG-VLDL, VLDL-REM, and EKO-VLDL preparations at a final concentration
of 0.5 to 1 mg lipoprotein protein per mL (volumes were adjusted by
using 20 mmol/L
NaH2PO4 ·
2H2O, pH 7.0, containing 100 µmol/L DTPA)
and incubated at 37°C for 24 hours. Before addition of pHA-modified
lipoproteins to cells, samples were filtered to remove any potential
lipoprotein aggregates. pHA dose-response curves were generated with 2
separate preparations of VLDL from EKO mice. Increasing concentrations
of pHA (from 0.05 to 1.0 mmol/L) were incubated with EKO-VLDLs (1
mg of lipoprotein protein) as described above. Macrophages
incubated (as described below) with VLDLs modified with 0.5 mmol/L
and 1.0 mmol/L pHA exhibited 2.5-fold and 6-fold increases in CE,
respectively (P<0.0001 for both). All further experiments
were performed with 1 mmol/L pHA. In a time-course experiment, pHA
was incubated with EKO-VLDLs for 12, 24, and 36 hours. Compared with
24-hour incubations, 
85% of the macrophage CE accumulation
was achieved after incubation with pHA for 12 hours. No further
increase in cellular CE over the levels observed for 24 hours was
observed by extending the incubation time to 36 hours. Incubation times
for all further experiments were 24 hours.
Determination of Lysine Residues Modified by pHA
The percent of lysine residues in type III VLDLs modified by pHA
was estimated by reduction of the Schiff base, delipidation, and acid
hydrolysis of the isolated protein, and the presence of pHA-lysine was
monitored. Two separate preparations of type III VLDLs were modified in
duplicate with pHA as described above. The Schiff base adducts were
reduced by the addition of NaCNBH3 (final
concentration, 20 mmol/L) in the presence of 100 mmol/L
ammonium acetate, pH 6.8, for 1 hour at 37°C. Samples were then
delipidated with diethyl ether, and
L-[13C6]tyrosine
and
L-[13C6]pHA-lysine
were added as internal standards.40 Protein was hydrolyzed
in the presence of HBr and extracted on a C18 column, and the content
of pHA-lysine was determined by stable-isotope dilution gas
chromatography/mass spectrometry as described by Hazen
et al.40
CuOx of Lipoproteins
Dialyzed and sterile HTG-VLDL, VLDL-REM, and EKO-VLDL
preparations were oxidized in vitro, under sterile conditions, by
following a modification20 of the protocol described by
Steinbrecher et al.51
Agarose Gel Electrophoresis
End-stage modification of the lipoproteins was monitored by
measuring changes in the relative electrophoretic mobility (Rf) of the
modified lipoprotein. A 5-µg sample of each lipoprotein preparation
(native, pHA-modified, or CuOx-modified) was subjected to
electrophoresis on a 1% agarose gel.51 The mobility of
each lipoprotein sample relative to BSA (5 µg per lane) was used as a
measure of the degree of whole-particle modification. The lipoprotein
and BSA in each lane were visualized using Coomassie Brilliant Blue
R250 (Bio-Rad).
Cell Culture
J774A.1 cells, a murine macrophage–like cell line that
secretes LPL but not apoE,52 53 were used in this study.
J774A.1 cells obtained from the American Type Culture Collection
(Manassas, Va) were maintained in culture and set up for experiments as
outlined previously.4 20 For each lipoprotein preparation,
between 50 and 300 µg of total lipoprotein cholesterol
per mL of medium was added to duplicate wells of cells and incubated
for 16 hours at 37°C. The LPL inhibitor
tetrahydrolipstatin (THL, Orlistat, provided by Hoffmann-LaRoche
Pharmaceuticals Ltd) was used at a concentration of 1.0 µmol/L.
At this concentration, THL inhibited the activity of 0.25 U of bovine
milk LPL by >95% (data not shown). Previous studies demonstrated that
J774A.1 macrophages secrete 0.25 U of LPL activity in 24
hours.4 The THL stock solution was made up in dimethyl
sulfoxide (DMSO) and then diluted with Dulbecco's modified Eagle's
medium (DMEM) plus lipoprotein-deficient serum (LPDS) before being
added to the cells. Control dishes received an equal volume (not
exceeding 10 µL per well) of DMSO alone. In experiments in which the
ACAT inhibitor DuP 128 (provided by DuPont Merck
Pharmaceutical Co, Wilmington, Del) was used, 10 µmol/L (final
concentration) was added in a volume of 5 µL DMSO per mL medium. The
polynucleotide polyinosine (poly I, Sigma) was dissolved in
sterile deionized water and used in cell culture experiments at a
concentration of 100 µg/mL medium. We have shown that this
concentration completely blocks macrophage uptake of
CuOx-LDLs,54 and others have shown that this concentration
completely blocks macrophage uptake of acetylated
LDLs.55 56 Control dishes received poly I in the absence
of any lipoprotein. Recombinant murine interferon-gamma (IFN-,
Gibco) was diluted in DMEM plus LPDS and added at a concentration of 50
U/mL medium. This concentration of IFN- will inhibit J774A.1 uptake
of CuOx-LDL by >95% (S.C.W. et al, unpublished data, 1997). Cells
receiving IFN- were exposed to the cytokine for a total of
40 hours. For the first 24 hours, cells were pretreated with the
cytokine alone. For the remaining 16 hours, fresh DMEM plus
LPDS and fresh IFN- (with or without lipoproteins) were added. Stock
IFN- was kept at -80°C, with fresh dilutions in DMEM plus LPDS
made for each experiment. The working-solution of IFN- was not used
when it was >48 hours old.
Analysis of Cellular Lipid Mass and Rate of
Cholesterol Esterification
The cell-lipoprotein incubations were terminated by 2 washes
with Tris buffer (0.15 mol/L NaCl, 50 mmol/L Tris, and 0.2%
wt/vol fatty acid–free BSA, pH 7.4) and 2 additional washes with Tris
buffer without fatty acid–free BSA. Cell lipids were extracted in situ
with two 30-minute incubations in 1.0 mL of hexane/isopropanol, 3:2
vol/vol. The solvents from each extraction were pooled for
analysis. To each dish, 1.0 mL of 0.1N NaOH was added and
incubated overnight at room temperature to digest the cells. Cell
protein was determined by a modification of the Lowry
method.48 Cellular TC, FC, and TG masses were determined
spectrophotometrically by a modification of a method described
previously,57 using enzymatic reagents from
Boehringer Mannheim (see above) and a Vmax kinetic 96-multiwell
microplate reader (Molecular Devices). In brief, each
hexane/isopropanol sample was evaporated to dryness under
N2 and resuspended in 1.2 mL of a
chloroform/Triton X-100 mixture (0.5% Triton X-100 vol/vol), and the
solvent was evaporated again under N2 and finally
resolubilized in 300 µL of deionized water (final sample
concentration, 2% Triton X-100). Two 50-µL aliquots of each sample
were then pipetted into individual wells of a 96-multiwell, flat-bottom
microtiter plate (Nunc, Gibco) and assayed for TC mass at 490 nm. Two
more 50-µL aliquots of each sample were also pipetted into individual
wells of a second 96-multiwell microtiter plate and assayed for FC mass
at 490 nm. CE mass was calculated by taking the difference between the
TC and FC mass values. To determine the TG mass of each sample, 75 µL
of each sample was first diluted with 50 µL of a 2% (vol/vol) Triton
X-100 solution (in deionized water), and then two 50-µL aliquots were
pipetted into individual wells of a third 96-multiwell microtiter plate
and assayed for TG mass at 490 nm. Cholesterol and TG
standards (range between 1 and 20 µg per well) used to generate
standard curves were processed in an identical fashion to that of the
experimental samples. Cellular lipid results are reported as micrograms
of cellular lipid (CE or TG) per milligram of cell protein. The
incorporation of [14C]oleic acid into CE was
determined as described previously.3 Values are reported
as millimoles of [14C]cholesteryl oleate per
milligram of cell protein.
Statistical Analysis
In each experiment, duplicate cell-culture wells were used for
each specific lipoprotein preparation, with the resulting values
combined to give a mean value. Mean values from separate experiments
were then used to calculate a group mean±SEM for each condition. The n
referred to in each experiment indicates the number of different
patients' samples or EKO mouse samples used to determine each
experimental parameter. Statistical significance between
control and experimental group mean values was assessed by a Student's
t test. A 2-tailed P0.05 was considered
statistically significant.
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Results |
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Lipoprotein Composition After pHA Modification or CuOx Modification of VLDL Preparations
The lipoprotein compositions used in the cell studies are summarized in Table 1
. The process of in
vitro REM formation caused not only a 20% reduction
(P=0.04) in the TG-CE ratio of type III VLDL-REM but also a
4-fold (P=0.02) increase in the FFA-protein ratio of the
newly formed REM (Table 1). pHA modification of type III
HTG-VLDL, type III VLDL-REM, and EKO-VLDL did not cause a significant
change in the composition of the lipoproteins (Table 1). In
contrast, CuOx increased by 69% the TG-CE ratio of type III HTG-VLDL
(P=0.0009, Table 1) and decreased by 73% and 85%,
respectively, the TC-protein and CE-protein ratios of EKO-VLDL
(P0.02, Table 1). CuOx had no significant effect on
the lipoprotein composition of type III VLDL-REM.
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Electrophoretic Mobility and pHA-Lysine Content of pHA-Modified
VLDL Preparations
As determined by electrophoresis on agarose gels, pHA modification
only modestly increased the Rf of type III HTG-VLDL, type III VLDL-REM,
and EKO-VLDL by 17%, 6%, and 10%, respectively (P0.04,
Table 2). In marked contrast, CuOx of
VLDL preparations caused a dramatic shift in the Rf of these
lipoproteins (92%, 45%, and 41% for type III HTG-VLDL, type III
VLDL-REM, and EKO-VLDL, respectively, P0.001; Table 2). In all cases, the increase in Rf induced by CuOx was
significantly greater than that induced by pHA modification
(P0.002, Table 2). The process of in vitro REM
formation also resulted in a significant increase in the Rf of native
type III VLDL-REM (Table 2). This observation has been
previously reported by our laboratory for type III
VLDL-REM20 and by others for LDL58 59
and is the result of lipoprotein modification by FFAs generated by
lipid hydrolysis (see FFA-protein ratios in Table 1
). Because
the Rf of VLDL reflects the surface charge of the lipoproteins, these
results suggest that only a small fraction of protein lysine residues
are modified by pHA. The percent of total lysine residues modified by
pHA was estimated for 2 type III VLDL samples. As determined by
stable-isotope dilution gas chromatography/mass
spectrometry, 0.045± 0.0007 moles of pHA-lysine per mole of lysine
were detected, indicating that 4.5% of total lysines were
modified.
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pHA Modification of VLDLs Significantly Increases Their Ability to
Induce CE Accumulation in Cultured Macrophages
Incubation of J774A.1 cells with native type III HTG-VLDL, type
III VLDL-REM, or EKO-VLDL induced only a modest increase in cellular CE
content (up to a 3-, 5-, and 5-fold increase, respectively;
P0.001) compared with control cells (Figure 1A). In contrast to their native
counterparts, incubation of cells with pHA type III HTG-VLDL, pHA type
III VLDL-REM, or pHA EKO-VLDL produced marked increases in cellular CE
content (up to a 38-, 47-, and 35-fold increase, respectively;
P0.0001) compared with control cells (Figure 1A).
The difference in cellular CE content induced by native versus pHA-VLDL
was statistically significant at each of the concentrations of
lipoproteins tested (P0.0001, Figure 1A).
Incubation of cells with either native or pHA type III HTG-VLDL or
pHA-type III VLDL-REM resulted in similar elevations in cellular TG (as
high as a 12-fold and 10-fold increase, respectively) compared with
control cells (P0.0001, Figure 1B). Incubation of
cells with the TG-poor native EKO-VLDL or pHA EKO-VLDL caused no
significant change in cellular TG content (Figure 1B).
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), type III VLDL-REM (
), and EKO-VLDL (
) or pHA-type
III HTG-VLDL (
), pHA-type III VLDL-REM (
), and pHA-EKO-VLDL (
)
(50 to 300 µg lipoprotein cholesterol per mL medium; n=3
for native and pHA-modified type III HTG-VLDL and VLDL-REM; n=4 for
native and pHA-modified EKO-VLDL). Control cell lipid values are
represented by 
. The values are expressed as mean±SEM
for each incubation condition. *Significantly different at
P
Uptake of pHA-Modified VLDL Is Blocked by Addition of the LPL
Inhibitor THL
We next examined whether TG hydrolysis is a necessary first step
in the uptake of pHA-VLDL. Coincubation of native type III HTG-VLDL and
of type III VLDL-REM with the LPL inhibitor THL decreased
the already low cellular CE content by 73.4% and 27.7%, respectively,
with only the former being statistically significant
(P=0.001, Figure 2A). In
addition, coincubation of native type III HTG-VLDL and of type III
VLDL-REM with THL decreased the elevated cellular TG content by 97%
and 95%, respectively (both P=0.001, Figure 2B and 2D). Coincubation of THL and either pHA type III HTG-VLDL or pHA type
III VLDL-REM resulted in a 73% and 59% reduction, respectively, in
cellular CE content (P0.001). THL inhibited the increase
in cellular TG content (97% and 93%, respectively;
P0.001) induced by these pHA-VLDL preparations.
Coincubation of THL with either native EKO-VLDL or pHA EKO-VLDL caused
a 100% and 72.9% decrease, respectively, in cellular CE content
(P0.001, Figure 2A) but had no significant effect
on cellular TG content, which remained at baseline levels (Figure 2B).
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In contrast to pHA-VLDL, THL had no significant effect on the increase
in either cellular CE or TG content induced by CuOx HTG-VLDL or CuOx
VLDL-REM (Figure 2C and 2D). In addition, CuOx HTG-VLDL and CuOx
VLDL-REM caused significantly less cellular TG loading compared with
that in cells incubated with either their native or pHA-modified
counterparts in the absence of THL (P0.006, Figure 2D).
CE Accumulation Induced by pHA-Modified VLDL Involves ACAT
Activity
The esterification of cholesterol by ACAT is an
important reaction in intracellular cholesterol
metabolism. Lipoprotein CE, taken up by receptor-mediated
processes, is hydrolyzed to FC in the lysosomes by an acid CE
hydrolase60 and subsequently reesterified to CE by ACAT in
the endoplasmic reticulum.61 Because lipoprotein uptake by
cells does not necessarily stimulate cholesterol
esterification,62 63 64 65 66 we examined whether increased
cholesterol esterification paralleled the uptake of
pHA-VLDL and whether the ACAT inhibitor DuP 128 would
inhibit both cholesterol esterification and CE
accumulation. As shown in Figure 3, incubation of cells with pHA type III HTG-VLDL, pHA type III VLDL-REM,
or pHA EKO-VLDL significantly increased the incorporation of
[14C]oleate into cellular CE
(P0.001) compared with their unmodified counterparts.
Addition of DuP 128 to cells exposed to pHA-modified lipoproteins
completely inhibited these increases in cholesterol
esterification, such that the values obtained were less than those
observed for non–pHA-modified lipoproteins. The values for the various
lipoproteins plus DuP 128 were not statistically different from those
for control cells plus DuP 128. The reductions in
cholesterol esterification after the addition of DuP 128 to
cells incubated with pHA-modified lipoproteins were statistically
significant (P<0.001) compared with their unmodified
counterparts (Figure 3). DuP 128 did not affect the
incorporation of [14C]oleate into cellular TG
(data not shown). As shown in Figure 4A, coincubation of cells with DuP 128 and pHA-VLDL completely inhibited
the increases observed in cellular CE mass induced by the pHA-VLDL
preparations (P0.0001). The reductions observed were
similar in magnitude to reductions in cholesterol
esterification. DuP 128 did not affect the ability of native or pHA
type III HTG-VLDL and pHA type III VLDL-REM to induce cellular TG
accumulation (Figure 4B).
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Uptake of pHA-Modified VLDL Is Only Partially Inhibited by Poly I
and IFN-
To examine whether the SR-A was responsible for the cellular
uptake of pHA-VLDL, we performed coincubation experiments with either
poly I, a polynucleotide that blocks binding to the SR-A
(types I and II),55 56 67 or IFN-, a cytokine
that decreases expression of the SR-A (type I).68 pHA-VLDL
preparations were incubated in the presence of poly I at concentrations
that completely blocked the ability of CuOx LDL to increase cellular
CE. Coincubation of pHA type III HTG-VLDL, pHA type III VLDL-REM, and
pHA EKO-VLDL with poly I inhibited the increase in cellular CE
accumulation by only 38%, 44%, and 49%, respectively
(P0.001, Figure 5A). In
contrast, coincubation of CuOx HTG-VLDL, CuOx VLDL-REM, and CuOx
EKO-VLDL with poly I inhibited the increase in cellular CE
accumulations by 100%, 94%, and 99%, respectively
(P0.001, Figure 5C). In all cases, coincubation of
native VLDL with poly I had no effect on either cellular CE or TG
content (Figure 5A through 5D).
|
IFN- was preincubated with macrophages at concentrations
that completely inhibit the cellular CE accumulation induced by CuOx
LDL. Incubation of pHA type III HTG-VLDL, pHA type III VLDL-REM, and
pHA EKO-VLDL with IFN-–treated cells decreased cellular CE by only
18%, 27%, and 65%, respectively (P0.001, Figure 6A). In contrast, IFN- inhibited the
increase in cellular CE accumulations after incubation with CuOx
HTG-VLDL, CuOx VLDL-REM, and CuOx EKO-VLDL by 89%, 89%, and 99%,
respectively (P0.001, Figure 6C).
|
Incubation of macrophages with pHA-VLDL in the presence
of poly I or after pretreatment of cells with IFN- had no
significant effect on cellular TG content (Figures 5B and 6B). In contrast, coincubation of CuOx HTG-VLDL and CuOx
VLDL-REM with either poly I or IFN- inhibited cellular TG
accumulations by >89% (P0.001, Figures 5D and 6D). In all cases, coincubation of native VLDL with IFN- had
no effect on either cellular CE or TG content (Figure 6A through
6D).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
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A number of studies suggest that oxidation or chemical modification of lipoproteins is an important step in promoting atherogenesis.69 70 71 Many studies emphasize that reactive aldehyde products formed during lipid peroxidation are possible mediators in converting lipoproteins to atherogenic particles.24 69 72 73 74 75 76 77 Recent studies suggest that alternative sources of aldehydes exist at sites of vascular disease and inflammation and that these reactive aldehydes are formed during MPO-mediated oxidation of amino acids.39 40 In the present study, we tested the hypothesis that type III HTG-VLDL, type III VLDL-REM, and VLDL from EKO mice could be modified by pHA, a reactive aldehyde derived from the action of MPO on L-tyrosine, and that incubation of these modified lipoproteins with cultured macrophages would lead to foam cell formation. Our findings demonstrate for the first time that pHA is capable of modifying VLDL and VLDL remnants, a process that induces significant CE accumulation in cultured macrophages. Cellular uptake of pHA-modified VLDL (1) requires catalytically active LPL, (2) involves ACAT-mediated cholesterol esterification, and (3) unlike CuOx VLDL, is only partially mediated by the SR-A.
We have previously shown that the CE and TG components of an HTG-VLDL particle are taken up by J774A.1 macrophages in a 2-step process.4 The first step requires the interaction between VLDL and cell-secreted LPL. During this interaction, the TG core of VLDL is hydrolyzed extracellularly by LPL, with the resulting FFAs being subsequently taken up by the macrophage and reesterified into TGs within the cell. As lipolysis proceeds, the receptor-binding epitopes of apoE on VLDL become exposed, allowing the CE-rich VLDL-REM to be taken up by macrophages via a receptor-mediated process involving apoE.4 Because the apoE2 isoform associated with type III HTG-VLDL is receptor binding–defective and EKO-VLDL is devoid of apoE altogether, these CE-rich VLDL preparations, when incubated with macrophages, are not readily taken up, and only small increases in cellular CE contents are observed. However, incubation of cells with type III HTG-VLDL will cause a significant increase in cellular TG content, indicating that extracellular lipolysis is unaffected by the apoE2 isoform. In contrast, exposure of J774A.1 cells to HTG-VLDL, which contains receptor binding–competent apoE, leads to increases in both cellular CE and TG content, both of which can be blocked by inhibition of the catalytic function of LPL.4
Macrophage accumulation of both the CE and TG components of pHA-VLDL requires catalytically active LPL. Thus, the idea of a 2-step mechanism also appears to be applicable to pHA type III HTG-VLDL. Coincubation of pHA type III HTG-VLDL with THL, a catalytic inhibitor of LPL, completely inhibited (97%) cellular TG accumulation and caused a significant, concomitant reduction (73%) in cellular CE content. One interpretation of these results is that the amphipathic properties of pHA39 result in its accumulation and modification of the lipoprotein at the aqueous-hydrophobic interface. In the absence of lipolysis, the sites, which undergo pHA modification, may be inaccessible for recognition by cellular receptors. Alternatively, incubation with pHA may result in the formation of pHA adducts of apolipoproteins that then require a conformational change, mediated by the hydrolysis of VLDL-TG, to be accessible to cell surface receptors. We observed that pHA type III VLDL-REMs were less sensitive to inhibition of macrophage CE accumulation by THL compared with pHA type III VLDL. This observation suggests that these REMs contain a significant number of TG-depleted particles that readily form adducts in the presence of pHA and are recognized directly by cellular receptors in the absence of further lipolysis. Therefore, in vivo, the action of LPL-mediated hydrolysis of pHA HTG-VLDL could have the net effect of exposing pHA-modified epitopes so that they can be recognized by cell surface receptors. In the case of EKO-VLDL, which has a smaller core TG content, the ability of THL to inhibit the uptake of pHA EKO-VLDL was surprising. It is possible that phospholipid hydrolysis, mediated by LPL, is required for enhanced exposure of pHA-protein adducts, which mediate receptor recognition.
In contrast to pHA-VLDL, THL failed to inhibit either cellular CE or TG accumulation induced by either CuOx HTG-VLDL or CuOx type III VLDL-REM. This finding suggests that VLDL and VLDL-REM modified by pHA are processed by cells differently than are lipoproteins oxidized by copper and that macrophage uptake of CuOx-VLDL preparations does not involve a mechanism requiring LPL activity.
Foam cells are formed when macrophages internalize lipoproteins, an event that results in the stimulation of the intracellular cholesterol esterification enzyme ACAT.78 The observation that an ACAT inhibitor, DuP 128, could block both pHA-VLDL–induced CE mass accumulation and esterification of oleate to cholesterol, indicates that pHA-VLDL CE is not trapped within a lysosomal compartment of the cell, as has been proposed for CuOx LDL.79 These results suggest that CEs that accumulate in pHA-VLDL–treated macrophages require active ACAT for acylation of FC derived from VLDL CE hydrolysis.
It has been proposed that the SR-A is directly responsible for the
uptake of chemically modified VLDL (acetylated
VLDL)80 and CuOx ß-VLDL,81 as
macrophage uptake was blocked by poly I. These 2 forms of
lipoprotein modification, like pHA,40 result in covalent
modification of apolipoprotein lysine residues via Schiff base
reactions involving the -amino group. Poly I binds competitively to
the SR-A (types I and II), thus preventing binding and uptake of SR-A
ligands.55 56 67 IFN-, a potent T lymphocyte–produced
cytokine, has been shown to downregulate macrophage
SR-A (type I) expression.68 Our studies with poly I
confirm that the SR-A and/or other poly I–sensitive macrophage
receptors67 82 are directly involved in the uptake of CuOx
lipoproteins. In contrast, SR-A apparently does not mediate the uptake
of the majority of pHA-modified lipoproteins. Similarly, IFN-
treatment of macrophages almost completely blocked the uptake
of CuOx-VLDL preparations, whereas the CE accumulations caused by pHA
type III HTG-VLDL and pHA type III VLDL-REM were inhibited by only
26%. Also, IFN- treatment failed to completely block CE
accumulation induced by pHA EKO-VLDL. Collectively, these experiments
demonstrate that most of the cellular uptake of pHA-VLDL is likely to
involve mechanisms distinct from the SR-A.
The receptors responsible for macrophage-mediated uptake of
pHA-VLDL are unknown at present. Together with the LPL inhibition
experiments, our studies with poly I and IFN- suggest that the cell
surface processes responsible for the uptake of pHA-VLDL are different
from those mediating the cellular uptake of CuOx lipoproteins.
Recently, Suzuki et al83 examined lesion development in
SR-A/apoE double-knockout mice. Lesion formation, which had occurred as
a result of apoE deficiency, was only partially prevented by also
knocking out the SR-A; SR-A/apoE double-knockout mice showed a 58%
reduction in lesion area compared with EKO mice.83
Consistent with our findings, the authors of this study
concluded that other receptors besides the SR-A must participate in
lesion development in EKO mice.83
The level of lysine modification we observed in pHA-VLDL and pHA-VLDL REM was almost an order of magnitude lower than other forms of modified lipoproteins. LDL modification by acetylation84 or malondialdehyde85 requires that >40% and 26%, respectively, of lysines be modified to confer SR recognition on the lipoprotein. LDL oxidized with FA oxidation products,86 copper,51 or hypochlorite (HOCl)87 88 also exhibited high levels of modified lysine residues (25%, 30%, and 68%, respectively). The low level of modification we observed in VLDL and VLDL remnants modified with 1 mmol/L pHA is achieved under conditions that might be expected in vivo, ie, concentrations of HOCl that are within the range as those attained by maximally activated neutrophils at circulating cell concentrations89 and physiological concentrations of L-tyrosine.39 These observations suggest that the extent of pHA modification we observe in vitro may be physiologically relevant.
The present study focussed on the modification of VLDL by pHA, the
major product of L-tyrosine oxidation by HOCl. Direct
oxidation of LDL by HOCl has been previously implicated in LDL-induced
foam cell formation.29 87 88 Clearly, if HOCl is being
generated in vivo, it will execute a series of competitive reactions,
including ones with proteins as well as with low-molecular-weight
components of the extracellular fluid. It is difficult to predict which
reactions would predominate. However, Hazen et al40 41
have demonstrated that pHA-lysine is formed in vivo at sites of
inflammation and atherosclerotic tissue. Although we did not directly
investigate lipoprotein oxidation by HOCl, our results with pHA
modification of VLDL and VLDL REM demonstrate some similarities, but
also some important differences, compared with HOCl-oxidized
LDL.29 87 88 The lipid composition of pHA-modified VLDL
was unaltered relative to native lipoproteins (Table 1),
suggesting that particle modification was not due to lipid-derived
aldehydes, a property that was also observed for HOCl-oxidized
LDL.87 In marked contrast to HOCl-modified LDL, pHA
modification of VLDL does not result in lipoprotein aggregation. In
addition, modification of only 4.5% of VLDL lysine residues was
required to significantly enhance macrophage CE accumulation,
whereas modification of 68% of lysine residues in HOCl-modified LDL is
required for macrophage CE loading.87
Consistent with these findings, the Rf of pHA-modified VLDL in
agarose was increased only slightly (Table 2), whereas
HOCl-modified LDL substantially increased its mobility.87
Thus, pHA modification represents an alternate pathway for VLDL
and VLDL REM modification that is mechanistically distinct from HOCl
modification of LDL. Importantly, we have also demonstrated that
pHA-VLDL uptake by macrophages is clearly different from the
uptake of CuOx lipoproteins and that the majority of uptake does not
involve the macrophage SR-A type I/II. The mechanism for
interaction of HOCl-modified LDL with macrophages has not been
described but is likely to reflect phagocytosis of the aggregated
lipoproteins.88
In conclusion, our results clearly demonstrate that human type III HTG-VLDLs, their REMs, and VLDL from EKO mice are modified by pHA in vitro and that pHA-VLDL is capable of enhancing macrophage cellular CE accumulation well above that of their native counterparts. Findings from recent studies have demonstrated that MPO-generated pHA covalently modifies proteins in vitro40 and, in vivo, pHA-modified proteins are found at sites of inflammation40 and in atherosclerotic lesions.41 Other in vivo studies have found (1) colocalization of catalytically active MPO with macrophages,27 (2) MPO-specific products,28 29 and (3) MPO-modified proteins in human atherosclerotic lesions.28 29 Collectively, these results are consistent with the hypothesis that pHA modification of VLDL and VLDL-REM takes place within the vascular wall of patients with type III HLP and in EKO mice.
We hypothesize that in the arterial intima, HTG-VLDL and/or partially catabolized HTG-VLDL interact with macrophage-secreted LPL, thereby trapping the lipoprotein within the extracellular matrix and to the macrophage cell surface. MPO-derived pHA would subsequently modify VLDL apolipoproteins, and after further TG hydrolysis by LPL, these pHA adducts would be accessible to cell surface receptors. This form of lipoprotein modification would allow for rapid particle uptake via both SR-A and non–SR-A–mediated mechanisms, resulting in CE accumulation and foam cell formation. Thus, pHA modification may represent a potential mechanism to explain atherosclerosis-associated foam cell formation in patients with type III HLP. Modification by pHA may also occur with lipoproteins, present in the arterial intima, associated with other forms of dyslipidemia.41 It is not known whether the nature of the particle modification or the mechanisms for macrophage uptake are similar for different lipoproteins.
|
Acknowledgments |
|---|
This work was supported by a Medical Research Council of Canada Grant (MT 8014) to M.W.H. S.C.W. is a recipient of a Medical Research Council of Canada Studentship. D.B.M. is a recipient of a Heart and Stroke Foundation of Canada Research Fellowship. M.W.H. and R.A.H. are Career Investigators of the Heart and Stroke Foundation of Ontario. S.L.H. is a Howard Hughes Medical Institute Postdoctoral Research Fellow. J.W.H. is an Established Investigator of the American Heart Association. We are grateful to Sandra Kleinstiver and Cynthia Sawyez for their expert technical assistance.
|
Footnotes |
|---|
A preliminary report of this article was presented at the 70th Scientific Sessions of the American Heart Association, Orlando, Fla, November 9–12, 1997, and has been published in abstract form (Circulation. 1997;96[suppl I]:I-921).
Received May 27, 1998; accepted September 25, 1998.
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