Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3469-3474
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3469-3474.)
© 1997 American Heart Association, Inc.
Endothelial Cells Prevent Accumulation of Lipid Hydroperoxides in Low-Density Lipoprotein
David M. Smalley;
Neil Hogg;
B. Kalyanaraman;
;
Kirkwood A. Pritchard, Jr
From the Department of Pathology (D.M.S., K.A.P.), Cardiovascular
Research Center (D.M.S., K.A.P.), Department of Pharmacology and Toxicology
(K.A.P.), and the Biophysics Research Institute (N.H., B.K.), Medical College
of Wisconsin, Milwaukee.
Correspondence to Kirkwood A. Pritchard, Jr, PhD, Medical College of Wisconsin, Cardiovascular Research Center, 493D, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail kpritch{at}post.its.mcw.edu
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Abstract
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Abstract A variety of cell types, including
endothelial cells,
oxidize low-density lipoprotein
(LDL). To investigate the mechanisms
by which
endothelial cells modulate LDL oxidation states,
endothelial
cell cultures were incubated with LDL (240
mg cholesterol/dL)
for 24 hours in M199 supplemented with
fetal bovine serum (FBS,
16.7%). These conditions were not toxic to
endothelial cells
over the time frame of the study.
Changes in LDL oxidation were
monitored by measuring thiobarbituric
acidreactive substances
(TBARS), lipid hydroperoxide (LOOH), and
conjugated dienes (A
234nm).
LDL medium incubated in the
absence of endothelial cells contained
higher TBARS
than did LDL medium incubated with endothelial
cells
(0.35±0.08 versus 0.23±0.08 nmol MDA/mg,
respectively). LOOHs were
higher in LDL medium incubated without
endothelial
cells than in LDL medium incubated with endothelial
cells
(6.8±4.4 versus 0.49±0.89 nmol/mg, respectively).
Conjugated
diene formation, based on changes in absorbance at
234 nm, increased to
a greater extent in LDL medium incubated
in the absence of
endothelial cells than when endothelial
cells
were present. To increase oxidative stress on the
endothelial
cell cultures, increasing concentrations of
Cu
2+ (0 to 4 µmol/L)
were added to LDL medium.
Endothelial cells prevented LOOH accumulation
until the
concentration of Cu
2+ exceeded 0.75 µmol/L.
At 1.5 and 4
µmol/L Cu
2+, endothelial cells enhanced
LOOH
formation nearly 3 and 2.5 times the LOOH values in the
corresponding
medium incubated in the absence of
endothelial cells. This loss
of protective function
however, was not permanent. Endothelial
cells,
preincubated for 24 hours with Cu
2+-containing LDL medium,
were
still able to prevent LOOH accumulation in fresh LDL medium.
Endothelial
cells prevented LOOH accumulation even when
exposed to LDL medium
that contained low concentrations of LOOHs (<22
nmol/mg).
However, endothelial cells accelerated the
accumulation of LOOHs
in LDL when exposed to LDL medium that contained
slightly higher
concentrations of preexisting LOOHs (

33 nmol/mg).
These data
indicate that endothelial cells have a
limited capacity for
preventing LOOH formation and that small increases
in LOOHs
may play a critical role in enhancing the potential of
endothelial
cells for oxidative modification of
LDL.
Key Words: LDL endothelial cells lipid peroxides
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Introduction
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Since
1984, it has been well recognized that endothelial
cells
promote oxidative modification of LDL in
culture.
1 The majority
of studies were performed
with low LDL concentrations and in
the absence of serum. In this study,
we exposed cultured human
umbilical vein endothelial
cells to high concentrations of LDL
(240 mg/dL) in the presence
of FBS (16.7%). Native LDL and oxidatively
modified LDL are important
mediators in the premature development
of
atherosclerosis (see References 2, 3
2 3 for review). The
prevailing
theory of
hypercholesterolemia-induced
atherosclerosis is that
LDL entry into the vessel wall
increases in direct response
to increases in plasma LDL
concentrations.
4 Once there, LDL
can be trapped
and then oxidatively modified.
2 Such
modifications
increase the electronegative character of the LDL
particle by
blocking the positively charged lysine residues of
apolipoprotein
B.
5 The effects of oxidatively
modified LDL on endothelial
cells, smooth muscle cells,
and monocyte function are believed
to play important roles in
accelerating atherosclerosis.
6
Moreover,
these cells have been shown to stimulate oxidative
modification
of LDL in culture.
7 8 9
The effect of LDL on endothelial cell function depends
strongly on the degree of oxidative modification, although the
definition of this parameter is variable.
Cu2+-oxidized LDL, which has a high degree of
oxidation, promotes cell injury and death.10
Oxidized LDL scavenges nitric oxide11 and
inhibits synthesis of nitric oxide synthase.12
Oxidized LDL also increases the thrombotic character of the
endothelium by increasing the production of
type 1 plasminogen activator
inhibitor.13 Minimally modified LDL
increases monocyte adherence by induction of an adhesion molecule that
is not ICAM-1.14 More recently, electronegative
LDL and MDA-lysine-modified LDL have been detected in the plasma of
individuals who are at increased risk for
atherosclerosis.15 16 17 18 However,
the effects of LDL are not restricted to oxidized forms alone, as
native LDL has also been shown to perturb endothelial
cell function in vitro in ways that are consistent with early
changes in vascular function in
hypercholesterolemia. In vitro, native LDL
increases endothelial cell ICAM-1
production,19
endocytotsis,20 and
permeability21 and induces nitric oxide synthase
to generate superoxide anion.22 Such changes in
function coincide with increased monocyte
adherence,2 vascular
permeability,23 24 and increased vascular
tone.25 26 27 28 29 These reports suggest that native LDL
and oxidized LDL likely play distinct but interdependent roles in the
progression of atherosclerosis and that
endothelial cell responses are influenced by the degree
of LDL oxidative modification.30
In this study, we have investigated the ability of
endothelial cells to affect lipid hydroperoxide(LOOH)
accumulation in LDL as a function of LOOH content. We demonstrate that
endothelial cells are very effective at limiting LOOH
accumulation in LDL and that small increases in LOOH content of
"native" LDL may represent a critical trigger for
increasing endothelial cell oxidation of LDL.
 |
Methods
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Materials
M199, FBS, heparin, HEPES, NaCl, EDTA, cholesterol
oxidase kit
(Cat. No. 352-20), potassium iodide, antibiotics, and
mycotics
were from Sigma Chemical Company. Butylated hydroxytoluene
(BHT),
thiobarbituric acid, MDA, chloroform, methanol, acetic acid,
copper
dichloride, potassium iodide, and cadmium acetate were from
Aldrich
Chemical Company. The Limulus amebocyte assay kit (Cat. No.
QCL-1000)
was from Whittaker Biochemical. Primeria culture dishes were
purchased
from Falcon Becton-Dickinson. Dulbecco's Phosphate-Buffered
Saline
(DPBS) was from Gibco BRL. Human recombinant basic fibroblastic
growth
factor (bFGF) was kindly provided by John A. Thompson
(University
of Alabama at Birmingham).
Methods
Endothelial Cell Isolation and Culture
Human umbilical vein endothelial cells were
extracted by using collagen digestion and cultured as
described.19 22 31 32 Endothelial
cell cultures were maintained in M199 media containing FBS (16.7%),
heparin (90 µg/mL), HEPES (20 mmol/L), pH 7.4,
antibiotics and mycotics, and human recombinant bFGF (10
ng/mL).19 22
Low-Density Lipoprotein Isolation
Fresh, nonfrozen human plasma (3 to 4 units) was obtained from
the Blood Center of Southeastern Wisconsin. LDL was isolated by
sequential density ultracentrifugation (d=1.019 to
1.063 g/mL) using sterile
techniques.19 22 31 32 LDL was isolated in the
absence of added antioxidants except where noted. LDL was dialyzed
against two changes of NaCl (150 mmol/L) containing EDTA
(0.27 mmol/L, 2 L) and one change of M199 (1 L).
Experiments were conducted within 5 days of isolation. In some studies,
the pooled plasma was divided into two equal portions, and BHT was
added to one portion at a final concentration of 20 µmol/L.
BHT-protected LDL was dialyzed against 2 changes of NaCl (150
mmol/L) containing EDTA (0.27 mmol/L, 2 L) and 20
µmol/L BHT and one change of M199 (1 L) without BHT. Each pool was
processed independently to prevent cross-contamination of BHT.
Cholesterol was determined with the cholesterol
oxidase kit from Sigma Chemical Company. References to LDL
concentrations in the present work are in terms of milligrams of
cholesterol. Endotoxin levels were measured with the
limulus amebocyte assay kit from Whittaker Biochemical. Endotoxin
concentrations were found to be lower than the concentrations required
to activate endothelial cells (<0.01
EU/mL).19
Thiobarbituric AcidReactive Substances
The oxidation state of LDL was determined by a modified
thiobarbituric acidreactive substances (TBARS) assay, as was
previously described.19 31 Briefly, apo
Bcontaining lipoproteins were isolated by precipitation with
phosphotungsate-MnCl2 before the addition of
thiobarbituric acid. MDA equivalents were quantified on a CytoFluor II
(PerSeptive Biosystems). Results are expressed as nanomoles of MDA
per milligram.
Iodometric Assay
LOOH was determined by the iodometric assay as described by
Girotti and coworkers.33 34 In this assay, care
was taken to minimize exposure of extracted samples to oxygen. All
solutions were purged with nitrogen before use, and the assay was
performed under dimmed lights. LOOH measurements were determined on LDL
in complete medium. Briefly, 0.001 mL of EDTA (0.5 mol/L) and
0.8 mL of chloroform:methanol (2:1) were added to 0.5 mL of sample in a
1.5-mL microfuge tube. The tubes were vortexed at high speed for 45
seconds and then centrifuged at 14,000 RPM for 5 minutes to
separate organic and aqueous phases. A 0.3-mL aliquot of the organic
phase (containing LDL lipids) was placed into a new centrifuge
tube and then evaporated to dryness under a constant stream of
nitrogen. Next, 0.02 mL of potassium iodide (1.2 g/mL) was added
to the dried samples, followed by 0.3 mL of chloroform:acetic acid
(2:3). Each tube was then purged with nitrogen, vortexed, and incubated
for 10 minutes at room temperature. Next, 0.9 mL of cadmium acetate
(0.25%) was added, and the tubes were vortexed. The tubes were
centrifuged for 5 minutes at 14,000 RPM to separate aqueous and
chloroform layers. LOOH stoichiometrically reduces iodide to
tri-iodide, which can be quantified in the aqueous layer by using the
353-nm extinction coefficient (22.5
mmol/L-1 cm-1).
Results are reported as nanomoles of LOOH per milligram.
Conjugated Diene Formation
Oxidation of polyunsaturated lipids rearranges isolated double
bonds to form conjugated dienes that absorb strongly at 234 nm.
Aliquots of LDL in complete medium were mixed with 1 mL of phosphate
buffered saline (final concentration of 5 mg/dL). Absorbances
were read in a quartz cuvette on a Beckman DU-640 UV/VIS
spectrophotometer.
Experimental Protocols
LDL was incubated under standard tissue culture conditions (5%
CO2, 95% air, 100% humidity at 37°C) in M199
containing FBS (16.7%) in Primeria 100-mm dishes, unless otherwise
noted. The dishes contained either no cells or confluent
endothelial cell monolayers. For the studies here, LDL
was added at a final concentration of 240 mg/dL.
To increase rates of oxidation during culture, confluent
endothelial cell cultures in six well cluster plates
were exposed to LDL medium containing increasing concentrations of
CuCl2 (0 to 4 µmol/L). After 24 hours, aliquots
of the Cu2+/LDL medium were removed and
analyzed for changes in LOOH. To determine whether
Cu2+/LDL exposure permanently impaired the
ability of endothelial cells to limit LOOH
accumulation, the cultures were preconditioned with LDL alone or with
LDL containing 4 µmol/L Cu2+ for 24 hours. To
control for the possibility that residual Cu2+
may have remained bound to endothelial cell proteins in
the experimental dishes, control monolayers, incubated with LDL medium,
were briefly supplemented with 4 µmol/L Cu2+.
Next, all cultures were washed four times with DPBS and then fed fresh
LDL in medium without Cu2+. After 18 hours,
aliquots were removed for LOOH measurements.
To determine whether the initial concentrations of LOOHs in LDL played
a role in modulating the ability of endothelial cells
to limit LOOH accumulation, monolayers were exposed to mildly oxidized
LDL. Fresh LDL medium was incubated under standard tissue culture
conditions for 24 hours to increase LOOH content. The next day, this
oxidized LDL and fresh LDL were mixed to yield interrelated pools
containing a range of LOOH concentrations. After mixing, aliquots were
removed for LOOH measurements. The interrelated pools were then
incubated in the absence and presence of endothelial
cells. After 24 hours, aliquots were removed and assayed for LOOH
content.
Statistics
Unless otherwise indicated, results are mean±SD. The Student's
one-tailed t test was used to determine whether data were
significantly different.
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Results
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Endothelial Cells Prevent Oxidative Modification
of LDL
To determine the effect of endothelial cells on
LDL oxidation,
LDL (240 mg/dL) was incubated in the presence and
absence of
endothelial cells. In the absence of
endothelial cells, the
concentrations of TBARS, LOOHs,
and conjugated dienes markedly
increased in comparison to starting
concentrations (Table 1

).
The presence of
endothelial cells prevented the accumulation
of TBARS,
LOOH, and conjugated dienes, a result indicating that
endothelial
cells are either preventing or removing
LOOH from LDL.
These changes in LDL oxidative modification, induced by incubation
under standard tissue culture conditions, can be attenuated by adding
BHT, a chain-breaking antioxidant, to the plasma before LDL isolation.
As can be seen in Table 2
, BHT markedly
decreased LOOH accumulation but did not eliminate it.
Modulation of LOOH Accumulation in LDL by Cu2+
It has previously been demonstrated that the oxidation of LDL by
cell culture medium depends on the content of redox-active transition
metal ions, ie, Fe3+ and
Cu2+.1 To enhance the pro-oxidant nature of the
culture medium, M199 was supplemented with increasing concentrations of
Cu2+. LOOH content increased as a function of
Cu2+ concentration in both cell and cell-free
conditions (Fig 1
). At low
Cu2+ concentrations (<0.75 µmol/L), cell-free
incubations generated higher concentrations of LOOHs than were
generated when cells were present. However, at higher
concentrations of Cu2+, the presence of cells
increased the yield of LOOHs (Fig 1
).

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Figure 1. Effect of Cu2+ on
endothelial celldependent LOOH accumulation. LDL (240
mg/dL) was incubated in M199 containing FBS (16.7%) for 24 hours with
Cu2+ in the presence (solid bars) or absence (open bars) of
endothelial cells. Samples were removed and
analyzed for LOOH content. Data represent the mean of
duplicate experiments. Bars indicate the spread of raw data points from
the mean.
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Effects of Cu2+/LDL Preconditioning on Accumulation of
LOOHs in LDL
To determine whether preconditioning endothelial
cells with Cu2+/LDL permanently altered the
ability of the cells to limit LOOH accumulation, cultures were
incubated with LDL in the presence and absence of
Cu2+ (4 µmol/L) for 24 hours. The monolayers
were washed and then incubated with fresh LDL medium without added
Cu2+. After preconditioning in
Cu2+/LDL, endothelial cells were
still able to limit LOOH formation at 18 hours in a manner that was
indistinguishable from that of endothelial cells
exposed to LDL alone (Fig 2
). These data
indicate that the ability of endothelial cells to limit
LOOH accumulation is not lost by preincubation with
Cu2+/LDL.

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Figure 2. Effect of Cu2+ on
endothelial celldependent LOOH accumulation. LDL (240
mg/dL) was incubated with M199 containing FBS (16.7%) for 18 hours in
the absence of endothelial cells (solid bars) or in the
presence of endothelial cells that had been pretreated
for 24 hours with LDL (240 mg/dL, open bars) or with LDL (240 mg/dL)
medium containing Cu2+ (4 µmol/L, hatched bars). Samples
were removed, and LOOH concentrations were determined. Data
represent mean±SD of three experiments performed in
triplicate. Comparisons were to LDL incubated without
endothelial cells (**P<.01).
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Effects of LOOH Content in LDL Medium on the Ability of
Endothelial Cells to Limit LOOH Accumulation
To determine whether the initial concentration of LOOHs in LDL
played a role in modulating the ability of endothelial
cells to limit LOOH accumulation, LDL was oxidized by incubation in the
absence of cells. This increased the LOOH content in LDL to
33
nmol/mg. Oxidized LDL was mixed with fresh LDL to yield
interrelated pools that contained a range of LOOH concentrations
(
2.5 to 33 nmol/mg; solid bars, Fig 3
). After 24 hours, the difference
between the initial and postincubation LOOH concentrations in the
presence of cells was nearly equal (hatched bars, Fig 3
).
Endothelial cells prevented LOOH accumulation in pools
1, 2, and 3 (open bars, Fig 3
). Interestingly,
endothelial cells were unable to reduce LOOHs below
what was initially present. In contrast to data for pools 1 to 3,
endothelial cells incubated with pool 4 increased
rather than prevented LOOH formation. These data show that
endothelial cells attenuated increases in LOOH
accumulation as long as the initial LOOH concentrations did not exceed
a critical value, in this case somewhere between 22 and 33
nmol/mg. If the starting concentration of LOOHs was higher than
this critical value, then endothelial cells promoted
LOOH accumulation.

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Figure 3. Effect of LDL-LOOH content on
endothelial celldependent LDL oxidation. LDL (240
mg/dL) was preincubated with M199 containing FBS (16.7%) for 24 hours
in the absence of endothelial cells. The preincubated
LDL, which contained an initial LOOH concentration of 33 nmol/mg, was
diluted with fresh LDL medium to yield interrelated pools containing
initial LOOH concentrations of 2.5, 11, 22, and 33 nmol/mg (solid
bars). The interrelated pools were then incubated without
endothelial cells (hatched bars) and with
endothelial cells (open bars). Samples were removed
after 24 hours, and LOOH content was determined. Data represent
mean±SD (n=3). Comparisons were LOOH content in LDL medium incubated
without endothelial cells versus LDL medium incubated
with endothelial cells (*P<.05,
**P<.01).
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Discussion
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This report shows that endothelial cells play an
important role
in limiting LOOH accumulation in LDL. Furthermore, when
the
LOOH content of LDL exceeds a critical value,
endothelial cells
accelerate LOOH accumulation. The
culture conditions that were
used in the present study are in
contrast to early studies using
low LDL concentrations and low or no
serum.
1 35 It is well
recognized that
hypercholesterolemic concentrations of LDL (>160
mg/dL)
are toxic to endothelial cells in the
absence of serum. Consequently,
serum was used as a supplement in all
the experiments reported
here. Serum, in cell-free conditions, markedly
reduced the rate
of LDL oxidation (data not shown) but did not prevent
it (see
Table 1

). It has been previously noted that serum was able to
limit
the ability of cells to oxidize LDL when LDL is present in
lower
concentrations.
36 37 As a result, most
oxidation studies have
been performed in the absence of serum. The
mechanism by which
serum prevents oxidation is unknown but may include
the presence
of antioxidants, chelation of transition metal ions in
cell
culture media, or the presence of HDL.
38
In this study, cultured human umbilical vein
endothelial cells were incubated with a LDL
cholesterol concentration (240 mg/dL) that is
associated with hypercholesterolemia. In
agreement with previous reports, essentially no LDL oxidation was
observed in LDL medium that was exposed to endothelial
cells.19 22 The culture conditions were not found
to be cytotoxic, probably because M199, in combination with high
concentrations of serum (16.7%), is well known for inhibiting
oxidation. Under these conditions, the ratio of LDL to
endothelial cells could be increased to approximately 4
to 6 mg/106 cells, which improved our
ability to determine whether the endothelial cell
cultures induced subtle changes in LDL oxidation states. Paradoxically,
LDL that was incubated with endothelial cells for 24
hours contained lower amounts of peroxides than did LDL incubated in
cell-free conditions. Thus, endothelial cells were
preventing the accumulation of LOOHs and/or increasing the removal of
LOOHs from LDL particles. The fact that cell-free LDL oxidation was
inhibited by BHT indicates that LOOH accumulation occurred by a
propagatory mechanism.
These data suggest that endothelial cells have one or
more mechanisms to limit LDL oxidation. Endothelial
cells may prevent LOOH accumulation by the generation of nitric
oxide39 or the release of
thiols40 or may remove LOOHs by
metabolism of phospholipid hydroperoxide by glutathione
peroxidase.41 42 Such protective mechanisms may
be overwhelmed, however, if the rate of LDL oxidation is faster than
the ability of the cells to limit LOOH accumulation. For example, in
Fig 3
, endothelial cells were able to hold the
accumulation of LOOHs in LDL constant as long as initial concentrations
of LOOH were low. When the initial LOOH concentrations were slightly
higher, oxidative modification of LDL by endothelial
cells increased by what appeared to be an autocatalytic mechanism.
These findings may have important implications for atherogenesis in
that circulating levels of electronegative LDL16
and MDA-modified LDL18 have been detected in
plasma and increased levels of "catalytic" metal ions were found in
atherosclerotic "gruel"43 and mechanically
injured vessels.44
Endothelial celldependent LDL oxidation has an
absolute requirement for transition metal ions in the cell culture
medium.1 45 Most media, including the M199 used
here, are often formulated with Fe3+ salts that,
in combination with reducing agents such as ascorbate and cysteine,
represent a low-level oxidative stress. When the transition
metal content of M199 was increased by addition of
Cu2+, endothelial cell activity
switched from inhibiting LOOH accumulation to promoting LDL oxidation.
Endothelial cells began to promote LDL oxidation at a
Cu2+ concentration of 0.75 µmol/L and above.
Below 0.75 µmol/L Cu2+,
endothelial cells were still able to limit LOOH
accumulation. This suggests that endothelial cells have
both mechanisms for LOOH removal and mechanisms for lipid oxidation.
The outcome in cell function depends strongly on the
Cu2+ concentration in the cell culture
medium.
Two possibilities for how Cu2+ modulates the
behavior of endothelial cells are that
Cu2+ permanently alters the ability of
endothelial cells to limit LOOH accumulation and that
Cu2+ partially oxidizes LDL lipid to form LOOH
and that this LOOH-loaded LDL is then more susceptible to cellular
oxidation. To test the first of these hypotheses,
endothelial cells were pretreated with
Cu2+/LDL or LDL alone for 24 hours. We found that
when the cells that had been pretreated with
Cu2+/LDL were fed fresh LDL media without
Cu2+, they behaved in a manner that was identical
to that of control cells treated with LDL alone. To test the second
hypothesisthat LOOH-loaded LDL is more susceptible to oxidationLDL
was preoxidized by incubation for 24 hours in the absence of cells.
Endothelial cells promoted LDL oxidation only when they
were presented with LDL that contained 33 nmol LOOH/mg.
However, if cells were presented with LDL containing lower
LOOHs (2.5 to 22 nmol LOOH/mg), accumulation of LOOH could still be
prevented.
Endothelial cell metabolism of oxidized
lipids is gaining acceptance as an important antiatherogenic
mechanism. To date, endothelial cells have been shown
to metabolize individual phospholipid and cholesterol ester
hydroperoxides46 47 and, under certain
conditions, to reduce the oxidation status of
LDL.40 LOOH in LDL may be removed and metabolized
by a phospholipase A2/peroxidase pathway.42 45 46
In contrast, endothelial cell oxidation of LDL has been
rigorously examined. The kinetics of transition metal iondependent
LDL oxidation have been shown to depend strongly on the starting LOOH
content of LDL.33 Reducing agents generated by
the endothelium can reduce transition metal ions, that
will, in turn, ultimately accelerate
Cu2+-dependent LDL
oxidation.40 48
The physiological consequences of these
observations are that endothelial cells may
represent a mechanistic barrier against the buildup of LOOHs in
both plasma and interstitial LDL. In the presence of an
additional oxidative stress, however, endothelial cells
may promote LDL oxidation, which could accelerate foam cell formation.
It is intriguing to consider that the additional oxidative stress may
come from the endothelium itself after chronic exposure
to high LDL concentrations. We have previously demonstrated that high
concentrations of LDL stimulate endothelial cells to
generate superoxide anion by uncoupled endothelial
nitric oxide synthase activity.22 LDL induction
of nitric oxide synthase superoxide anion was also found to increase
the formation of peroxynitrite, a well-recognized oxidant that is
capable of lipid peroxidation,49 DNA
damage,50 and nitration of LDL
proteins.51 By this mechanism, LDL may play an
important role in inducing endothelial cells to promote
LDL oxidation. This possibility remains to be determined.
 |
Acknowledgments
|
|---|
This work was supported in part by National Institutes of Health
grant
48251 (to K.A.P.), HL47250 (to B.K.), and GM55792 (to N.H.)
and
American Heart Association, Wisconsin Affiliate grant 95-GB-56
(D.M.S.).
The authors thank Dr John A. Thompson (University of Alabama
at
Birmingham, Birmingham, Ala) for providing recombinant human
bFGF,
the Birthing Center at Waukesha Memorial Hospital for
umbilical cords,
and the Blood Center of Southeastern Wisconsin
for human plasma. We
also thank Michelle Curtis and Carmen Torres
for their technical
assistance.
Received March 20, 1997;
accepted June 11, 1997.
 |
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