© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
LDL Modified by Hypochlorous Acid Is a Potent Inhibitor of Lecithin-Cholesterol Acyltransferase Activity
From the Linus Pauling Institute (M.R.M., A.C.C., B.F.), Oregon State University, Corvallis, and Lawrence Berkeley National Laboratory (T.M.F.), Life Sciences Division, University of California at Berkeley.
Correspondence to Balz Frei, PhD, Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, OR 97331-6512. E-mail balz.frei{at}orst.edu
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Abstract |
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Abstract—Modification of low density lipoprotein (LDL) by myeloperoxidase-generated HOCl has been implicated in human atherosclerosis. Incubation of LDL with HOCl generates several reactive intermediates, primarily N-chloramines, which may react with other biomolecules. In this study, we investigated the effects of HOCl-modified LDL on the activity of lecithin-cholesterol acyltransferase (LCAT), an enzyme essential for high density lipoprotein maturation and the antiatherogenic reverse cholesterol transport pathway. We exposed human LDL (0.5 mg protein/mL) to physiological concentrations of HOCl (25 to 200 µmol/L) and characterized the resulting LDL modifications to apolipoprotein B and lipids; the modified LDL was subsequently incubated with apolipoprotein B–depleted plasma (density >1.063 g/mL fraction), which contains functional LCAT. Increasing concentrations of HOCl caused various modifications to LDL, primarily, loss of lysine residues and increases in N-chloramines and electrophoretic mobility, whereas lipid hydroperoxides were only minor products. LCAT activity was extremely sensitive to HOCl-modified LDL and was reduced by 23% and 93% by LDL preincubated with 25 and 100 µmol/L HOCl, respectively. Addition of 200 µmol/L ascorbate or N-acetyl derivatives of cysteine or methionine completely prevented LCAT inactivation by LDL preincubated with
200 µmol/L HOCl. Protecting the free thiol groups
of LCAT with 5,5'-dithio-bis-(2-nitrobenzoic acid) before exposure to
HOCl-modified LDL, which inhibits lipid hydroperoxide–mediated
inactivation of LCAT, failed to prevent the loss of enzyme activity.
Our data indicate that
N-chloramines from
HOCl-modified LDL mediate the loss of plasma LCAT activity and provide
a novel mechanism by which myeloperoxidase-generated HOCl may promote
atherogenesis.
Key Words: chloramines • HDL • LDL • lecithin-cholesterol acyltransferase • hypochlorous acid
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Introduction |
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Oxidative stress is thought to play a causal role in the pathogenesis of atherosclerosis.1 Although there are many determinants in the development of an atherosclerotic lesion, substantial in vitro evidence links oxidized forms of LDL to molecular processes relevant to atherogenesis.1 Recent in vitro and in vivo evidence suggests that myeloperoxidase (MPO) can oxidize LDL to an atherogenic form. Immunoreactive and catalytically active MPO has been found in human atherosclerotic lesions.2 Moreover, a specific marker of MPO-catalyzed oxidation, 3-chlorotyrosine, is elevated 30-fold in LDL isolated from human lesions compared with plasma-derived LDL.3 In addition, immunohistochemical studies have revealed the presence of HOCl-modified proteins in human atherosclerotic lesions4 and the colocalization of MPO and HOCl-modified proteins with monocyte/macrophages, endothelial cells, and the extracellular matrix.5 A number of studies have also shown that HOCl-modified LDL exerts various pathophysiological effects on leukocytes and vascular cells.6 7 8 9 Thus, there is strong evidence for a role of MPO in LDL oxidation and human atherogenesis.
MPO is a heme-containing enzyme, released from
activated neutrophils and monocytes, that catalyzes the
production of strong
oxidants.10 The predominant
product of this enzyme at physiological
chloride ion concentrations is
HOCl,11 an oxidant that
readily reacts with a variety of biomolecules, such as thiols,
thioethers, ascorbate, and amines, including amino
acids.12 Because of the high
reactivity of HOCl, its reactions are dependent on the relative
concentrations and reactivities of compounds in the immediate vicinity.
Thiols and methionine residues are manyfold more reactive with HOCl
than are other amino acids and
amines.12 At neutral pH,
reagent or MPO-generated HOCl preferentially oxidizes the apoB moiety
of LDL.13 14
Neither LDL-associated lipids nor antioxidants (eg,
-tocopherol and ß-carotene) appear to be major targets
of HOCl.14 15 Of
the various amino acid residues in apoB modified by HOCl, lysine
residues quantitatively represent the major
target.13 14
The reaction of HOCl with the 
-amino group of lysine
residues results in the formation of
N-chloramines (reaction 1).
Lysine chloramines can decompose to form aldehydes (reaction 2) and/or
react directly with free thiols and/or methionine
residues.16 The reactions are
as follows: reaction 1,
R-CH2-NH2+HOCl
R-CH2-NHCl+H2O;
reaction 2,
R-CH2-NHCl+H2OR-CH=O+NH4++Cl-.
It has been suggested that lysine chloramine–derived aldehydes participate in HOCl-induced cross-linking of apoB and aggregation of LDL,14 which result in the conversion of LDL to a ligand for the scavenger receptors of macrophages.13 17 However, it is important to note that lysine chloramines are more likely to react with free thiols and/or methionine residues16 than to decompose to aldehydes. The specificity of the former reactions suggests that biomolecules possessing biologically active cysteine and/or methionine residues may be inactivated by HOCl-modified LDL. Thus, unlike lipoxygenase-dependent or metal ion–dependent modifications of LDL, which involve the derivatization of lysine residues by lipid hydroperoxide breakdown products,1 HOCl directly modifies apoB lysine residues to N-chloramines, which may enhance the atherogenicity of LDL in a number of ways.
Lecithin-cholesterol acyltransferase (LCAT), an enzyme essential for HDL maturation and the antiatherogenic reverse cholesterol transport pathway,18 has 2 free cysteine residues that can modulate enzymatic activity.19 It has been demonstrated that LCAT loses activity when exposed to copper-oxidized LDL or lipoxygenase-generated hydroperoxides.20 21 22 The mechanism of this inactivation is thought to involve adduct formation between aldehydic lipid hydroperoxide breakdown products and the free cysteine residues of LCAT.22 23 Considering the lability of LCAT when it is exposed to thiol-specific reagents, we hypothesized that HOCl-modified LDL may impair LCAT function through the facile reaction of N-chloramines with free thiols.
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Methods |
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Materials
ApoA-I was isolated from human plasma and purified as previously described.24 Egg L-
-phosphatidylcholine
was purchased from Avanti Polar Lipids, and
[4-14C]cholesterol was
obtained from NEN Products. Human leukocyte MPO and HOCl were
obtained from Calbiochem and Aldrich, respectively.
7-Fluorobenzo-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) was from
Molecular Probes. PD-10 gel filtration columns were from Pharmacia
Biotech. All other reagents were from Sigma Chemical Co. All solvents
were high-performance liquid chromatography
(HPLC) grade. PBS was composed of 10 mmol/L sodium phosphate
buffer, 140 mmol/L NaCl, and 100 µmol/L diethylenetriamine
pentaacetic acid, pH 7.4.
Lipoprotein Isolation
Blood was obtained with informed consent from fasted
volunteers. Heparin was used to prevent coagulation, and plasma was
separated from cellular blood components by low-speed
centrifugation
(1200g, 4°C, 20 minutes).
Lipoproteins (LDL, density [d]=1.019 to 1.063 g/mL; HDL, d=1.063 to
1.21 g/mL) were rapidly isolated from plasma by preparative sequential
ultracentrifugation with use of an Optima-TL
ultracentrifuge and a TLA-100.4 rotor (Beckman Instruments).
Standard methods25 were used,
but corrections were made for the higher gravitational forces generated
by the Optima ultracentrifuge and the shorter path length of
the TLA-100.4 rotor. Isolated lipoproteins were desalted by gel
filtration with the use of PD-10 columns equilibrated with PBS. The
protein content of isolated lipoproteins was determined by using the
Lowry Micro Method Kit (Sigma P5656). The d>1.063 g/mL fraction
of plasma (containing LCAT, HDL, and non-apoB plasma proteins) and
lipoprotein-depleted plasma were isolated after a single
ultracentrifugation step and dialyzed into
PBS.
LDL Incubations With HOCl
HOCl was standardized at 292 nm [=350
(mol/L)-1 ·
cm-1] as
previously described.26 Bolus
HOCl was added with gentle mixing to LDL (0.5 mg protein/mL) in PBS;
incubations were carried out for 30 minutes at 37°C. Final HOCl
concentrations ranged from 25 to 200 µmol/L, corresponding to
HOCl:apoB molar ratios from 25:1 to 200:1. Modified LDL was kept on ice
(for <1 hour) until characterized and used in incubations containing
LCAT.
Characterization of LDL Protein
Modifications
Unmodified lysine residues were assessed by
fluorescamine
fluorescence,27 and
loss of tryptophan residues was assessed directly by
fluorescence (excitation 280 nm, emission 335
nm).13 Loss of cysteine
residues was monitored by fluorescence after derivatization
with ABD-F.16 All
fluorometric measurements were performed by using a Hitachi F-2500
Fluorescence Spectrophotometer (Hitachi Instruments).
N-Chloramines were measured by
the thionitrobenzoic acid
assay.28 Changes in LDL
charge characteristics, reflecting modification of lysine residues,
were assessed by agarose gel
electrophoresis.16
Determination of LDL Lipid Peroxidation
Thiobarbituric acid–reactive substances (TBARS) in
LDL were measured by the method of Kosugi et
al.29 Cholesteryl ester
hydroperoxide content of LDL was determined, as previously
described,30 by use of a
specific and sensitive method with HPLC separation and post-column
chemiluminescence detection. Vitamin E was assessed by HPLC with
electrochemical detection as previously
described.30
Incubations of HOCl-Modified LDL With
LCAT
Control and HOCl-modified LDLs were added to the
d>1.063 g/mL fraction of human plasma (containing HDL, LCAT, and
plasma proteins) and incubated at 37°C for 30 minutes. Preliminary
studies demonstrated that LCAT inactivation was complete within 15
minutes, and incubations lasting >30 minutes did not result in
additional loss of LCAT activity. The reconstituted plasma incubations
contained 50 µg of LDL protein and represented a 3-fold
dilution of plasma with an LDL protein concentration of 1 mg/mL. In
some experiments, ascorbate or the
N-acetyl derivatives of
cysteine, histidine, lysine, methionine, tryptophan, and tyrosine
(final concentration 200 µmol/L) were preincubated at 37°C for 30
minutes with HOCl-modified LDL. In other experiments, the d>1.063 g/mL
fraction of plasma was incubated at 37°C for 30 minutes in the
presence or absence of the reversible thiol-blocking reagent
5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB, 1.7 mmol/L); excess
DTNB was subsequently removed by dialysis in PBS. LCAT activity was
completely inhibited after this procedure and could be restored by the
addition of 5 mmol/L ß-mercaptoethanol.
Assessment of LCAT Activity
LCAT activity was measured by the exogenous "common
substrate" (ie, proteoliposome) method of Chen and
Albers.31 This method uses an
excess of
[14C]cholesterol-labeled
proteoliposome substrate composed of human apoA-I:egg-yolk
phosphatidylcholine:unesterified cholesterol at a molar
ratio of 0.8:250:12.5. The assay is dependent on the amount of active
LCAT and independent of endogenous plasma substrates and
cofactors. In addition to the proteoliposome substrate, LCAT reaction
mixtures contained 20 mmol/L Tris HCl (pH 8.0), 0.15 mol/L NaCl,
0.27 mmol/L EDTA, 0.5% human serum albumin, and 5
mmol/L ß-mercaptoethanol. Aliquots (40 µL) of reconstituted plasma
(ie, mixtures of control or HOCl-modified LDL with the d>1.063 g/mL
fraction) were added to start the reaction; incubations were carried
out for 30 minutes at 37°C. The reaction was terminated by the
addition of ethanol (final concentration 50%). After hexane
extraction, the labeled reaction product
([14C]cholesteryl ester) was separated
from the reaction substrate
([14C]cholesterol) by
thin-layer chromatography. The radioactivity associated
with the labeled substrate and product was quantified by liquid
scintillation counting. Results are expressed either as percent
esterification of
[14C]cholesterol per 30
minutes or as a percentage of control LCAT
activity.
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Results |
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Characterization of HOCl-Modified LDL
As previously reported by us,16 treatment of LDL with increasing concentrations of HOCl (25 to 200 µmol/L) resulted in dose-dependent oxidation of apoB cysteine, tryptophan, and lysine residues (Table
).
Because apoB contains approximately 4 cysteine, 37 tryptophan, and 356
lysine residues,32 these data
suggest that thiols are more sensitive to modification by HOCl than are
tryptophan residues and that tryptophan residues are more sensitive
than are lysine residues. A small increase in the electrophoretic
mobility of LDL was also observed (1.5-fold at 200 µmol/L HOCl,
Table)
and is likely the result of HOCl-induced lysine modification. As
expected, the decrease in apoB lysine residues and the increase in LDL
electrophoretic mobility reflect the incremental increases in formation
of N-chloramines
(Table).
Similar modifications were observed by using
MPO/H2O2/Cl-
to generate HOCl instead of adding reagent HOCl (data not
shown).
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Effects of HOCl-Modified LDL on LCAT
Activity
Incubation of HOCl-modified LDL with the d>1.063 g/mL
fraction of human plasma (containing HDL, LCAT, and plasma proteins)
resulted in dose-dependent inactivation of LCAT activity
(Figure 1), as determined by the exogenous "common
substrate" (ie, proteoliposome)
method.31 Whereas native LDL
had no effect on LCAT activity, LDL modified with 100 µmol/L HOCl
reduced LCAT activity by almost 100%
(Figure 1). Similar results were obtained with LDL modified
by
MPO/H2O2/Cl-
(data not shown). It should be noted that 100 µmol/L HOCl is within
the physiological range of HOCl, ie, 200
µmol/L.33 34 A
similar inactivation of LCAT activity was observed when HOCl-treated
HDL was added to lipoprotein-deficient plasma (d>1.21 g/mL, data not
shown). Because the method used to measure LCAT activity in these
experiments is thought to be independent of endogenous
substrates, it is unlikely that oxidative modification of
HDL-associated apoA-I can account for the loss of enzymatic activity
observed. In contrast to HOCl-modified lipoproteins, HOCl-modified BSA
had very little effect on LCAT activity. In 2 independent experiments,
incubation of the d>1.063 g/mL fraction of plasma with 0.5 mg/mL BSA
treated with 25 to 200 µmol/L HOCl resulted in a maximal decrease in
LCAT activity by only 13%. These data suggest that the reactive
component(s) responsible for inactivation of LCAT is specific to
lipoproteins and/or that there is a specific interaction between LCAT
and the lipoproteins that does not occur with
BSA.
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Role of
N-Chloramines in LCAT
Inactivation by HOCl-Modified LDL
To determine the reactive species associated with
HOCl-modified LDL and the possible amino acid target(s) on LCAT,
ascorbate and several
N-acetylated amino
acids were incubated with HOCl-modified LDL before its addition to the
LCAT-containing samples
(Figure 2). The sulfur-containing amino acids cysteine and
methionine (200 µmol/L each) significantly inhibited inactivation of
LCAT by HOCl-modified LDL
(Figure 2A). In contrast, the
N-acetyl derivatives of
tryptophan, lysine, histidine, and tyrosine (200 µmol/L each) did not
exert any significant effect on inactivation of LCAT by HOCl-modified
LDL
(Figure 2B). These data mimic the reactivity of model
N-chloramines, such as
N-acetyl-lysine chloramine,
toward these amino acids.16
Preincubation of HOCl-modified LDL with 200 µmol/L ascorbate, which
we have previously shown to reduce
N-chloramines back to their
parent amines,16 also
significantly inhibited the inactivation of LCAT
(Figure 2A). Higher concentrations of ascorbate (400
µmol/L) completely prevented enzyme inactivation (data not shown).
These data suggest that LDL-associated
N-chloramines are the reactive
species involved in inactivation of LCAT and that the likely target(s)
on LCAT are cysteine and/or methionine residues.
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Role of Lipid Hydroperoxides in LCAT
Inactivation by HOCl-Modified LDL
Recent studies have reported that oxidized
lipids,20 in particular,
lipid
hydroperoxides,21 22
can inhibit LCAT activity. Thus, it is possible that lipid
hydroperoxides rather than, or in addition to,
N-chloramines are responsible
for the inactivation of LCAT by HOCl-modified LDL. Treatment of LDL
with increasing concentrations of HOCl resulted in a small
dose-dependent increase in lipid hydroperoxides, which was inhibited by
the lipid-soluble antioxidant butylated hydroxytoluene (BHT), as shown
in
Figure 3A. In contrast, treatment of LDL with HOCl did not
cause an increase in TBARS from background levels (
2 nmol/mg LDL
protein, data not shown). Furthermore, no significant loss of vitamin E
was observed in LDL exposed to concentrations of HOCl of up to 200
µmol/L (data not shown), consistent with previous
reports.13 14
HOCl-modified LDL containing BHT, and thus containing decreased levels
of lipid hydroperoxides
(Figure 3A), was equally potent in inhibiting LCAT activity
as was HOCl-modified LDL not containing BHT
(Figure 3B). These data suggest that LDL-associated lipid
hydroperoxides are not involved in the inactivation of LCAT by
HOCl-modified LDL.
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Involvement of Thiols in LCAT Inactivation by
HOCl-Modified LDL
Because LCAT contains 2 free cysteine residues (Cys31
and Cys184) located in proximity to its active
site,19 it is possible that
LDL-associated N-chloramines
are inactivating the enzyme by oxidizing the thiols to sulfenic or
sulfinic acids. Therefore, the LCAT-containing samples were
preincubated with the reversible thiol-specific reagent DTNB to
sterically block the active site of LCAT and prevent its free cysteine
residues from reacting with HOCl-modified LDL. However, treatment of
LCAT with DTNB failed to inhibit inactivation of the enzyme by
HOCl-modified LDL
(Figure 4). These data suggest that inactivation of LCAT by
HOCl-modified LDL is thiol independent and may, therefore, involve
oxidation of the methionine residues of
LCAT.
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Discussion |
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The present study demonstrates that HOCl-modified LDL inhibits plasma LCAT activity. The HOCl concentrations required for LDL modification to completely inhibit LCAT activity (ie,
100 to 150
µmol/L) are well within the range considered
physiologically relevant (ie, the levels
generated extracellularly over a 30-minute period by
5x106
neutrophils/mL).33 34
Inactivation of LCAT by HOCl-modified LDL appears to be independent of
lipid hydroperoxides and, at least in part, dependent on
N-chloramines, inasmuch as
ascorbate completely inhibited inactivation of the enzyme. Although
HOCl could potentially inactivate LCAT directly, its
reactivity is less selective than that of
N-chloramines, which, as we
have previously shown, react predominantly with thiols, methionine, and
ascorbate.16 Also, the
association of LCAT with oxidatively modified lipoproteins may enhance
inactivation of the enzyme by LDL-associated
N-chloramines (see
below).
Interestingly, inactivation of LCAT by LDL-associated
chloramines does not appear to involve thiol modification but, rather,
may involve oxidation of methionine residues. There are several lines
of evidence in support of this notion: (1) reversible protection of the
thiol groups of LCAT with DTNB did not inhibit inactivation of the
enzyme by HOCl-modified LDL; (2) preincubation of HOCl-modified LDL
with N-acetyl-methionine
protected against subsequent inactivation of LCAT; (3) methionine
residues are the only other major protein-associated targets, apart
from thiol residues, that are readily oxidized by
N-chloramines16 ;
and (4) preliminary experiments have shown that HOCl-modified LDL
mediates inactivation of the methionine-dependent protein
1-antiproteinase by an
N-chloramine–dependent
mechanism (see below).
Our data suggest that HOCl-modified LDL inhibits LCAT activity by a mechanism different from that proposed for LCAT inactivation by copper-oxidized LDL.20 22 In contrast to copper-induced oxidation of LDL, the HOCl concentrations used in the present study did not affect LDL-associated TBARS or vitamin E levels. Use of a sensitive HPLC-postcolumn chemiluminescence method30 demonstrated that HOCl induced the formation of small amounts of lipid hydroperoxides. However, the levels of lipid hydroperoxides formed were not associated with a measurable increase in LCAT inactivation. It should be noted that copper-oxidized LDL containing 4 to 7 nmol TBARS/mg protein also contains substantial levels of lipid hydroperoxides (9 to 25 nmol/mg LDL protein; M.R. McCall, B. Frei, unpublished data, 2000) but reduces LCAT activity by only 50%.4
The mechanism by which HOCl-modified LDL inhibits LCAT
activity likely involves
N-chloramines, which account
for 30% of the HOCl added to LDL. The
N-acetyl derivatives of
cysteine and methionine, as well as ascorbate, effectively protected
against inactivation of LCAT by HOCl-modified LDL. We have shown
previously that both ascorbate and these sulfur-containing amino acids
are effective scavengers of
N-chloramines.16
In contrast, the N-acetyl
derivatives of tryptophan, lysine, histidine, and tyrosine, which
cannot scavenge
N-chloramines,16
did not protect against LCAT inactivation.
Inactivation of LCAT by lipid hydroperoxides isolated from copper-oxidized LDL is thought to involve the free cysteine residues of LCAT.22 Although N-chloramines react readily with thiol groups,16 reversible blocking of the free cysteine residues of LCAT with DTNB did not protect against loss of enzyme activity by HOCl-modified LDL. Oxidation of the thiol groups to higher oxidation states, such as sulfenic or sulfinic acids, may not cause sufficient steric hindrance to inhibit the enzyme compared with derivatization of the thiols with DTNB.19
Thus, we hypothesize that LDL-associated N-chloramines alter the enzymatic activity of LCAT by oxidizing methionine residues and thereby modifying protein conformation. Although N-acetyl histidine did not protect against inactivation of LCAT by HOCl-modified LDL, we cannot rule out the possibility that N-chloramines directly affect the amino acids in the catalytic site of LCAT (ie, aspartate, histidine, and serine18 ). However, it is of particular interest to note that lipid hydroperoxides35 and N-chloramines16 can oxidize methionine residues. This suggests that LDL-associated protein modifications induced by HOCl and LDL-associated lipid modifications induced by copper or lipoxygenase ultimately contribute to LCAT inactivation via this common mechanism.
A number of proteins have been shown to be
inactivated via
N-chloramine–dependent
oxidation of essential methionine residues to methionine
sulfoxide.36 In plasma, these
include the protease inhibitors
1-antiproteinase37 38
and
2-macroglobulin,39 40
as well as the fifth component of human
complement.41 Oxidation of
the methionine residues was associated with conformational changes in
some of the
proteins.39 40 We
have found that HOCl-modified LDL also inactivates
1-antiproteinase with an
IC50 of 58 µmol/L HOCl used to modify LDL (0.5
mg protein/mL). This inactivation is presumably due to oxidation of the
reactive site methionine residue in
1-antiproteinase42
by LDL-associated
N-chloramines, inasmuch as
preincubation of the HOCl-modified LDL with ascorbate almost completely
abrogated inactivation of the protease inhibitor (A.C.
Carr, B. Frei, unpublished data, 2001).
The data of the present study indicate that the
reaction of HOCl-modified LDL with LCAT is specific to lipoproteins.
For example, HOCl-modified albumin did not inhibit LCAT, in
contrast to HOCl-modified HDL. Because it is unlikely that
N-chloramines formed on LDL
transfer to HDL, where they react with LCAT, our data suggest that LCAT
and LDL interact directly in the reconstituted plasma system. On the
basis of binding affinities, Kosek et
al43 estimated that 20%
of LCAT is bound to LDL in plasma. Indirect evidence for an association
of LCAT with HOCl-modified LDL was obtained when reagent HOCl-mediated
was compared with HOCl-modified LDL-mediated inactivation of LCAT in
whole plasma. Direct addition of 100 and 200 µmol/L HOCl to plasma
did not cause measurable inhibition of LCAT activity, whereas addition
of LDL modified with 100 or 200 µmol/L HOCl caused 9% and 19%
inactivation of enzyme activity, respectively (M.R. McCall, A.C. Carr,
B. Frei, unpublished data, 2000). The degree of LCAT
inactivation by HOCl-modified LDL in whole plasma was less than that in
reconstituted plasma, suggesting some protection by low molecular
weight antioxidants, such as ascorbate. Thus, it would appear that a
localized microenvironment within the artery wall in which antioxidant
defenses have been depleted is the most likely site for LCAT
inactivation by HOCl-modified LDL to occur in vivo.
HDL has antiatherogenic properties that are due to multiple functions in the reverse cholesterol transport pathway.18 It facilitates the efflux and net transfer of excess cholesterol from atherosclerotic foam cells; it provides the activator (ie, apoA-I) and substrates (ie, cholesterol and phospholipids) for LCAT, the enzyme that maintains the concentration gradient along which foam cell–derived cholesterol flows; and it facilitates the transport of foam cell–derived cholesterol to the liver for reutilization or catabolism.18 HOCl-modified HDL impairs cholesterol efflux from macrophages,44 and we have now shown that HOCl-modified LDL is a potent inhibitor of LCAT activity. Thus, HOCl-modified lipoproteins (HDL and LDL) may enhance atherosclerosis in part by impairing the antiatherogenic reverse cholesterol transport pathway.
Although the exact role of LCAT in human atherosclerosis and coronary artery disease remains to be established, a few studies have shown that LCAT activity is significantly reduced (24% to 50% of control individuals) in patients with coronary artery disease and in patients after myocardial infarction.45 Furthermore, there is evidence that some cases of human LCAT deficiency are associated with premature atherosclerosis and coronary artery disease.46
In summary, we have shown that inactivation of LCAT by HOCl-modified LDL is independent of lipid hydroperoxides but is, at least in part, dependent on N-chloramines. The small molecule antioxidant ascorbate, which eliminates N-chloramines, completely inhibited inactivation of the enzyme. Inhibition of LCAT activity by HOCl-modified LDL does not appear to involve thiol modification but may involve oxidation of methionine residues. The latter may result in conformational changes and subsequent LCAT inactivation, suggesting a novel additional mechanism by which MPO-derived HOCl may accelerate atherosclerosis.
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Acknowledgments |
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This work was supported by grants from the American Heart Association, Northwest Affiliate (9920420Z to A.C.C.) and the National Institutes of Health (HL-49954, HL-56170, and HL-60886 to B.F. and HL-18574 to T.M.F.).
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Footnotes |
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1 Both authors contributed equally to this study.
Received January 19, 2001; accepted March 20, 2001.
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References |
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