(Received for publication, October 11, 1995; and in revised form, November 8, 1995)
From the
Native and oxidized low density lipoprotein retention within
arterial wall endothelial cell matrix (ECM) is an early event in the
pathogenesis of atherosclerosis. Previously we showed lipoprotein
lipase (LPL) addition to ECM enhanced the retention of apoB-containing
lipoproteins. In the present studies we examined whether the oxidation
of low density lipoprotein (LDL) increases its retention by
LPL-containing ECM. Except where noted, I-labeled
moderately oxidized LDL (ModOxLDL) was prepared by long term storage of
I-LDL. Without LPL,
I-ModOxLDL matrix
binding was low and nonsaturable. LPL preanchored to ECM resulted in
I-ModOxLDL binding that was saturable and 20-fold greater
than in the absence of LPL, with an association constant equal to 2.6
nM. Copper-oxidized LDL (Cu-OxLDL) was able to compete with
I-ModOxLDL, whereas a 60-fold native LDL excess had no
effect. Reconstituted apolipoprotein B from Cu-OxLDL also reduced
I-ModOxLDL to LPL, whereas liposomes derived from the
lipid extract of Cu-OxLDL had no effect on binding. These data suggest
that the increased binding of oxidized LDL to LPL
ECM may be due
to the exposure of novel apoB binding sites and not an oxidized lipid
moiety.
I-ModOxLDL binding was also not affected by
either preincubation with a 300-fold molar excess of apoE-poor HDL or
an 340-fold molar excess of Cu-OxHDL. In contrast, a 4-fold apoE-rich
HDL excess (based on protein) totally inhibited
I-ModOxLDL matrix retention. Positively charged peptides
of polyarginine mimicked the effect of apoE-rich HDL in reducing the
I-ModOxLDL retention; however, polylysine had no effect.
We postulate that the oxidation of LDL may be a mechanism that enhances
LDL retention by the ECM-bound LPL and that the protective effects of
apoE-containing HDL may in part be due to its ability to block the
retention of oxidized LDL in vivo.
Low density lipoprotein oxidation is believed to be an early
event in the initiation and progression of
atherosclerosis(1, 2, 3) . The oxidation of
LDL ()confers many biological properties on the molecule
that render the lipoprotein more
atherogenic(4, 5, 6) . Several in vitro biologic consequences of oxidized LDL (OxLOL) have been
demonstrated including, increased monocyte adhesion to endothelial
cells(7, 8, 9, 10) , monocyte
chemotaxis(11) , decreased macrophage motility and increased
cytotoxicity(12, 13, 14, 15) .
Studies imply that OxLOL promotes recruitment and retention of
monocytes/macrophages in the subintima. Oxidized LDL (OxLDL) or its
components have also been shown to stimulate the expression of several
endothelial cell proteins including vascular cell adhesion molecule-1 (16) and other adhesion molecules(17) , monocyte
chemotactic protein-1(18, 19) , macrophage
colony-stimulating factor (20) . Some of these effects may
result from the activation of transcription factors such as
NF-
B(21) . OxLDL is also avidly scavenged by macrophages (22, 23, 24) and leads to macrophage-derived
foam cell formation(3, 22) .
The process of LDL oxidation is not very well understood, but it appears to occur within the artery wall, facilitated by cellular lipoxygenase-dependent mechanisms or by nonenzymatic pathways. The phospholipid fatty acids of LDL are believed to be oxidatively modified first(25, 26) , resulting in the formation of phospholipids containing a spectrum of peroxidized fatty acids. Some of these, through chemical decomposition and/or enzymic hydrolysis, are believed to generate reactive aldehydes, which covalently modify the lysine residues of apoB and cause the surface charge to become more negative(27) . Due to the oxidative attack, apoB also undergoes cleavage, which exposes neoepitopes on the protein(28) . Although most of the biological effects of OxLDL are thought to result from oxidized lipid products, the increased scavenger receptor uptake of OxLDL is mediated by changes in surface protein charge(3) . Another important step in the process of atherogenesis is believed to be the trapping of LDL within the subendothelial space. LDL retention increases its residence time within the artery wall and therefore its susceptibility to oxidative modification. In physiological ion and salt concentrations, very little native (unmodified) LDL binds to endothelial cell-derived matrix in vitro(29) . Although lowering the salt concentration or adding divalent cations can increase LDL retention to ECM(30, 31) , this does not appear to be physiologically relevant. However, under physiological conditions, the ECM avidly binds lipoprotein lipase (LPL), which in turn markedly enhances LDL binding(29) . LPL is normally present in the artery wall and increases, due to macrophage and smooth muscle cell production, during atherogenesis(32, 33) . The increased binding of LDL to LPL-containing ECM is mediated by positively charged clusters of arginine and lysine domains of apoB(34) . Furthermore, purified or HDL-associated apolipoprotein E (apoE) is able to efficiently displace LDL from LPL-containing ECM by virtue of its positively charged domains(35) . Arginine or lysine polymers were able to mimic the LDL displacement effects of apoE(34) .
The binding
characteristics of OxLDL to ECM are largely unknown. Because the
association of OxLDL to the arterial subendothelial matrix is a
potentially pivotal step in the initiation and progression of
atherosclerosis, we explored the requirements for its binding and
potential physiologic competitors. To exclude interference in the
binding studies from enzymes typically used for enzymatic oxidation of
LDL (lipoxygenase/phospholipase), we utilized OxLDL prepared by long
term storage or by Cu oxidation. The OxLDL, used in
these studies, formed during long term storage in the absence of EDTA,
was analyzed by the standard measures for the determination of the
extent of oxidation (content of thiobarbituric acid-reactive materials,
free amino group, fluorescence (excitation; 360 nm; emission: 430 nm),
and migration on agarose gels). Because these particles were
intermediate of native and maximally oxidized LDL for all the
aforementioned assays, we used the term moderately oxidized (ModOxLDL)
to describe these LDLs. We found that ModOxLDLs bound to the LPL with
greater affinity than LDL, that the increased binding was due to
oxidative modification of the protein moiety and not lipid, and that
apoE-rich HDL and polyarginine effectively blocked the interaction
between OxLDL and LPL anchored to the ECM.
I-ModOxLDL was prepared by a
modification of Berliner et al.(10) . Briefly,
I-LDL was stored at 4 °C for greater than 45 days in
phosphate-buffered saline (0.137 M NaCl, 0.01 sodium
phosphate, pH 7.4) in the absence of EDTA. This method consistently
yielded moderately oxidized LDL, with a migration on agarose
intermediate of native and maximally copper-oxidized LDL. LDL was also
oxidized by incubation in the presence of 2-10 µM copper sulfate (Sigma). Briefly, copper sulfate was added to 1 mg
of LDL in phosphate-buffered saline, yielding a final copper
concentration of 2-10 µM, and incubated for 12 h at
37 °C. The copper sulfate was then removed utilizing Centriflo 50
concentrating cones (Amicon, Beverly, MA), the lipoproteins were then
stored in phosphate-buffered saline with 0.01% EDTA. The extent of
oxidation was determined by migration on agarose gels (Helena
Laboratories, Beaumont, TX), content of thiobarbituric acid-reactive
material(39) , loss of free amino groups(40) , and
fluorescence (excitation: 360 nm; emission: 430 nm)(23) . The
OxLDL yielded a thiobarbituric acid-reactive material value of
8-22 nmol of malondialdehyde equivalents/mg of protein, a
15-30% decrease in free amino groups (as compared with native
LDL), and a fluorescence 6-8-fold greater than native LDL.
ApoE-poor HDL was oxidized as outlined above for LDL, by incubation
with 10 µM copper. Extent of oxidation was monitored by
fluorescence (excitation: 360 nm; emission: 430
nm)(23, 41) , yielding a value 6.5-fold greater than
native HDL.
Lipid-free apoB from Cu-OXLDL was prepared essentially
as described by Parthasarathy et al.(42) . Briefly,
Cu-OxLDL (2 mg of protein, 0.3 ml) was extracted using 10 volumes of
iced-cold MeOH followed by an equivolume of CHCl. The
protein precipitate was isolated, washed, and then solubilized in
octylglucoside (6 mg/ml) by vigorous vortexing for 15 min. The solvent
fraction containing the lipids was dried, and large unilamelar vesicles
were prepared in phosphate-buffered saline by the extrusion method of
Berliner et al.(10) . Phospholipid content of the
vesicles was measured enzymatically (Wako Diagnostics, Richmond, VA).
ApoE-enriched HDL and apoE-poor HDL were isolated by Affi-Gel heparin chromatography (Bio-Rad) essentially as described by Rifici et al.(43) .
OxLDL and native LDL matrix
retention studies were performed in both the presence and absence of
LPL. Briefly, matrix was washed four times with Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) containing 3%
bovine serum albumin (BSA) (Sigma). LPL (8 µg/ml) was then added in
DMEM/BSA and allowed to incubate with the ECM for 2 h at 4 °C.
Following the preincubation with LPL, the matrix was then washed three
times with DMEM/BSA to remove any unbound LPL. Next, I-ModOxLDL or
I-LDL was added to the ECM
alone or in the presence of various unlabeled lipoproteins, liposomes,
reconstituted oxidized apoB, or polypeptides and incubated for 1 h at
37 °C. The medium was then removed, and the matrix was washed three
times with DMEM/BSA. Finally, the retained
I-lipoproteins
were released by the addition of 50 units of heparin (Sigma) and
quantitated as described previously (29) .
Figure 1:
Retention
of I-ModOxLDL and
I-LDL with LPL bound to
ECM. A,
I-ModOxLDL binding curve and Scatchard
analysis. ECM was prepared from confluent porcine aortic endothelial
cells as described under ``Materials and Methods.'' The ECM
was preincubated in media with or without LPL (8 µg/ml) for 2 h at
4 °C. Unbound LPL was removed by washing, and then
I-ModOxLDL was added at the indicated concentrations and
allowed to interact with the ECM for 1 h at 37 °C. The ECM was
again washed, and the bound
I-ModOxLDL was released by
the addition of heparin (50 units/ml) and analyzed as described under
``Materials and Methods.'' Scatchard analysis of the
I-ModOxLDL binding, in the presence of LPL, is shown in
the inset. The lines represent the calculated best
fit of the data. B,
I-LDL binding curve to LPL
bound to ECM. LPL was bound to ECM as described above and then
incubated with the indicated concentrations of
I-LDL. The
bound
I-LDL was released by the addition of heparin. Data
are the mean ± S.E. from triplicate
wells.
Figure 2:
Agarose gel electrophoresis of
Cu-oxidized LDL. LDL was oxidized to varying degrees
by the addition of copper sulfate as described under ``Materials
and Methods.'' Lane 1, native LDL; lane 2,
I-ModOxLDL (prepared from long term storage); lanes
3-7, LDL incubated for 12 h at 37 °C, in the presence of
2, 4, 6, 8, and 10 µM copper sulfate,
respectively.
Figure 3:
Effect of native or vortexed LDL on I-ModOxLDL binding to LPL
ECM. LPL was bound to ECM
as described in Fig. 1and then incubated with
I-ModOxLDL (3.125 µg/ml) in DMEM/BSA alone (Control) or DMEM/BSA containing LDL (200 µg/ml) or the
same LDL that was previously vortexed. Plates were washed with buffer,
and heparin-releasable radioactivity was determined as described in Fig. 1. Data represent mean ± S.E. of an experiment
performed in triplicate.
Figure 4:
Competition of I-ModOxLDL by
Cu
-oxidized LDL. A, LPL was bound to ECM as
described in Fig. 1and then incubated with
I-ModOxLDL in the presence or absence of various
concentrations of Cu-OxLDL that were oxidized to a varying extent as
described in Fig. 2. The legend indicates the concentration of
copper used for oxidation. B,
I-ModOxLDL (12.5
µg/ml) was incubated in the presence or absence of 12.5 µg/ml
of Cu-OxLDL or the reconstituted apoB or lipid extract from the
Cu-OxLDL. Plates were washed with buffer, and heparin-releasable
radioactivity was determined as described in Fig. 1. Data
represent the mean ± S.E. of an experiment performed in
triplicate.
Figure 5:
Effect of Cu-OxLDL on I-LDL
binding to LPL
ECM. LPL was bound to ECM as described in Fig. 1and then incubated with
I-LDL (12.5
µg/ml) in DMEM/BSA alone (Control) or DMEM/BSA containing
Cu-OxLDL at either 50 or 100 µg/ml. The Cu-OxLDL was generated as
described under ``Materials and Methods'' in the presence of
8 µM copper sulfate. Plates were washed with buffer, and
heparin-releasable radioactivity was determined as described in Fig. 1. Data represent the mean ± S.E. of an experiment
performed in triplicate.
Figure 6:
Effect of preincubation with apoE-poor HDL
on I-ModOxLDL binding to LPL
ECM.
I-ModOxLDL was incubated with approximately a 300-fold
molar excess (16-fold excess based on phospholipid) of apoE-poor HDL
for 24 h at 37 °C, or the same concentration of HDL was added to
the
I-ModOxLDL just prior to being added to the ECM. LPL
was bound to ECM as described in Fig. 1and then incubated with
the indicated concentrations of
I-ModOxLDL in DMEM/BSA
alone or DMEM/BSA containing apoE-poor HDL. Plates were washed with
buffer, and heparin-releasable radioactivity was determined as
described in Fig. 1. Data represent the mean ± S.E. of an
experiment performed in triplicate.
Figure 7:
Competition of I-ModOxLDL by
Cu
-oxidized apoE-poor HDL. LPL was bound to ECM as
described in Fig. 1and then incubated with
I-ModOxLDL alone (Control) or in the presence of
the indicated concentrations of HDL that was oxidized as described
under ``Materials and Methods.'' Plates were washed with
buffer, and heparin-releasable radioactivity was determined as
described in Fig. 1. Data represent the mean ± S.E. of an
experiment performed in triplicate.
Figure 8:
Effect of apoE-rich or apoE-poor HDL on I-ModOxLDL binding to LPL
ECM. LPL was bound to ECM
as described in Fig. 1and then incubated with
I-ModOxLDL (3.125 µg/ml) in DMEM/BSA containing
apoE-rich (A) or apoE-poor (B) HDL at the indicated
concentrations. Plates were washed with buffer, and heparin-releasable
radioactivity was determined as described in Fig. 1. Data
represent mean ± S.E. of an experiment performed in triplicate.
In panel B, Control denotes
I-ModOxLDL
(3.125 µg/ml) in DMEM/BSA alone.
Figure 9:
Effect of polyarginine or polylysine on I-ModOxLDL binding to LPL
ECM. A, LPL was
bound to ECM as described in Fig. 1and then incubated with
I-ModOxLDL (3.125 µg/ml) in DMEM/BSA alone (Control) or DMEM/BSA containing heparin (50 units/ml),
polylysine, or polyarginine at the indicated concentrations. B, displacement curve
I-ModOxLDL by
polyarginine. LPL was bound to ECM as described in Fig. 1and
then incubated with
I-ModOxLDL (3.125 µg/ml) in
DMEM/BSA containing polyarginine at the indicated concentrations.
Plates were washed with buffer, and heparin-releasable radioactivity
was determined as described in Fig. 1. Data represent the mean
± S.E. of an experiment performed in
triplicate.
The enhanced binding of native LDL to ECM is facilitated
through an LPL bridge formed between matrix glycosaminoglycans and the
lipoprotein(35) . The retention of LDL did not require an
enzymatically active protein, since inactivated LPL (overnight
incubation at 25 °C) adsorbed to a microfilter plate was able to
enhance LDL retention in an LPL concentration-dependent manner. ()However, the binding of LPL to the cell proteoglycans does
require the enzyme to be active (46) . We previously observed
that apoE-rich HDL and apoE mimetics (polyarginine or polylysine) were
effective competitors of native LDL bound to LPL
ECM and that this
interaction did not release LPL from the matrix(34) . In the
subendothelium of atherosclerotic plaques epitopes of oxidized apoB
have been found(47, 48, 49) , suggesting
either their localized subendothelium production or rapid sequestration
from plasma. In this regard, in the current studies, we investigated
the comparative binding affinity of native LDL and ModOxLDL to ECM
alone and in the presence of LPL and the ability of apoE-rich and
apoE-poor HDL and apoE mimetics to reduce this retention.
In the
current studies, an experimental model of the subendothelial matrix was
utilized to investigate the binding characteristics of I-ModOxLDL to the ECM. We found the binding of OxLDL to
ECM alone was nonsaturating and of low affinity. In contrast, LPL
greatly enhanced the saturable and high affinity binding of this
modified lipoprotein. Compared with native LDL, OxLDL-bound ECM
anchored LPL with a 4-fold greater affinity. The increased affinity was
caused exclusively by the modification of LDL apoB and not the lipid.
The affinity of OxLDL binding to LPL bound to ECM was dependent on the
extent of LDL oxidation. Neither native LDL, aggregated LDL, nor
apoE-poor HDL could effectively compete with
I-ModOxLDL
for ECM-bound LPL; in contrast, apoE-rich HDL efficiently blocked this
interaction. Polyarginine mimicked the effects observed with apoE-rich
HDL; however, polylysine was unexpectedly ineffective in competing with
I-ModOxLDL for ECM-bound LPL.
Four separate
experiments suggest that the binding interaction between LPL bound to
matrix and OxLDL is mediated by modified apoB rather than oxidized
lipid. First, the isolated, reconstituted apoprotein moiety of Cu-OxLDL
decreased the interaction of I-ModOxLDL with
LPL
ECM, whereas the lipid portion had no effect. Preincubation
with excess apoE-poor HDL, to remove any oxidized lipids, did not
diminish matrix binding. Oxidized apoE-poor HDL also did not compete
with OxLDL for binding. Last, both apoE-rich HDL and polyarginine
effectively competed with OxLDL. Prior studies (31) have also
shown that lyso-PC, a component of oxidized LDL, did not interfere with
the retention of LDL with LPL-containing matrix. Collectively these
data suggest that it is the oxidation of apoB that causes the enhanced
binding.
The experiments involving polyarginine and polylysine may
help explain an apparent discrepancy between the present studies and
our previous studies with nonoxidatively modified LDL. Native LDL
modified by acetylation (modification of lysine residues) (50) and cyclohexanedione (modification of arginine residues) (51) do not block LDL binding to LPL-matrix as efficiently as
native LDL(34) . However, in the present study, we show that
oxidation of LDL, which has been shown to modify the lysine residues,
greatly enhances its association with LPL and can completely block LDL
retention. Why does OxLDL bind with higher affinity to LPL-containing
ECM, and why is polylysine ineffective in blocking OxLDL retention?
This apparent paradox may be explained with the process of LDL
oxidation. Oxidation of apoB leads to its
fragmentation(28, 52, 53) , which may expose
novel sites and also confer conformational changes. This, in turn, may
result in a higher affinity binding of OxLDL to LPLECM. Oxidation
also modifies the lysine residues on apoB. The lysine modification
would decrease its role in the binding to LPL, which may account for
the inability of polylysine to inhibit the OxLDL binding. Further
experiments are necessary to delineate the precise mechanisms of these
effects.
The ability of apoE-rich HDL to compete with oxidized LDL increases our understanding of the importance of this lipoprotein fraction and in particular apoE. Studies in apoE transgenic and apoE-deficient mice demonstrate the antiatherogenic properties of apoE. Mice, which are normally atherosclerosis-resistant, become very prone to atherosclerosis when they are made apoE-deficient by gene targeting (54, 55) . Interestingly, these mice present with both circulating oxidized LDL and antibodies to epitopes of oxidized apoB(49) . Recently, Shimano et al. created transgenic mice overexpressing apoE in the arterial wall(56) . When these mice were fed an atherogenic diet, significant decreases in fatty streak formation as compared with nontransgenic controls was observed. Therefore, in these models, the excess of apoE within the lesions may impede the retention of OxLDL and thereby inhibit the progression of atherosclerosis.
Epitopes of oxidized apoB have been demonstrated in the extracellular matrix of lesion areas in animals and humans using antibodies to malondialdehyde OxLDL(47, 48, 49, 57) . Furthermore, OxLDL has been shown in the plasma of subjects with severe atherosclerosis and diabetes(28, 58, 59, 60, 61) . Based on the studies presented here, it is possible that when circulating OxLDL infiltrates the artery wall it is captured by matrix-bound LPL. Alternatively, native LDL may first bind to the matrix-bound LPL, and then be oxidized by cells within the arterial wall, thereby resulting in higher affinity binding. In this regard, it has been shown in vitro that the binding of LDL to matrix proteoglycans increases the lipoprotein's susceptibility to oxidation (62) . In either scenario, OxLDL may be selectively trapped within the artery wall by such interactions as those presented here. The accumulation of native and CuOxLDL have been studied in vivo in balloon catheter-deendothelialized rabbit aorta(63) . The accumulation of native and OxLDL in areas of regenerating endothelium were similar. However, in areas that were still de-endothelialized, OxLDL was retained 2.9-fold more than native LDL, despite its more rapid clearance from plasma. Additionally, the extent of lysine modification of ModOxLDL in these studies is sufficient to be recognized by the scavenger receptor (64). Thus, oxidation may enhance retention in the arterial wall by both increased binding to LPL and uptake through the scavenger receptor, markedly increasing its ability to promote various steps in the pathogenesis of atherosclerosis.
In conclusion, we have demonstrated that oxidized LDL has a higher affinity to LPL anchored to subendothelial matrix than native LDL and that the increased retention is due to the oxidation of apoB, not the lipids. Furthermore, unlike native LDL, it is mainly the arginine residues, not the lysines, that play an important role in this retention. And finally, apoE-rich HDL has been shown to be as effective in blocking the retention of OxLDL, strengthening its role as an antiatherogenic agent.