© 1996 American Heart Association, Inc.
Articles |
A Cytotoxic Electronegative LDL Subfraction Is Present in Human Plasma
From the Laboratoire de Biochimie, Hôpital Broussais, Paris (K.D., I.M., B.V., N.M.); the Laboratoire de Biochimie Appliquée, Faculté des Sciences Pharmaceutiques et Biologiques, Châtenay-Malabry (K.D., I.M., B.C., B.V., M.A.P.-A., N.M.), France; and the University of California at Los Angeles, School of Medicine (M.E.H.).
Correspondence to Dr Karine Demuth, Laboratoire de Biochimie Appliquée (tour D4, 2ème étage), Faculté des Sciences Pharmaceutiques et Biologiques, 5 rue J.B. Clément, F-92296 Châtenay-Malabry Cedex, France.
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Abstract By using fast protein liquid chromatography, we isolated from human plasma a minor electronegative LDL subfraction designated LDL(-). After immunoaffinity chromatography against apolipoprotein (apo)(a) and apo A-I, LDL(-) represented 6.7±0.9% (mean±SD; n=18) of total LDL. Compared with the major LDL subfraction, designated LDL(+), LDL(-) contained similar amounts of thiobarbituric acid–reactive substances, conjugated dienes, and vitamin E and had a similar lipid/protein ratio and mean density. Moreover, the apo B of LDL(-) was not aggregated and its LDL receptor–binding activity was slightly increased. These results were consistent with the nonoxidized nature of LDL(-). LDL(-) showed increased contents of sialic acid (38.1±5.2 versus 28.9±3.3 nmol/mg protein; n=7; P<.01), apo C-III (1.43±0.21% versus 0.14±0.04%; n=7; P<.01), and apo E (1.64±0.26% versus 0.10±0.05%; n=7; P<.0005). Compared with LDL(+), LDL(-) displayed enhanced cytotoxic effects on cultured human umbilical vein endothelial cells, as shown by lactate dehydrogenase assay (P<.003; n=6), neutral red uptake (P<.02; n=6), and morphological studies. We also studied the relationship of LDL(-) to age and plasma lipid levels in 133 subjects. The percentage of contribution of LDL(-) to total plasma LDL correlated with age (P<.05), total cholesterol (P<.05), and LDL cholesterol (P<.003). In conclusion, this study shows that LDL(-), a circulating human plasma LDL, is an electronegative native LDL subfraction with cytotoxic effects on endothelial cells. This subfraction, which correlates positively with common atherosclerotic risk factors, might induce atherogenesis by actively contributing to alteration of the vascular endothelium.
Key Words: low-density lipoprotein • electronegativity • atherosclerotic risk factors • cytotoxicity • human umbilical vein endothelial cell
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Introduction |
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It is widely accepted that elevated LDL cholesterol levels are an important etiologic factor for atherosclerosis and that modified LDL may play an important pathogenic role in this disease.1 In vitro–modified LDL, particularly oxidized LDL, displays atherogenic biological properties, such as uptake by macrophage scavenger receptors, leading to foam cell formation, cytotoxicity, chemotaxis for circulating monocytes, inhibition of resident-macrophage mobility, vasoconstriction, and disturbances of hemostasis.2 However, although modified LDL, characterized by an increased electronegative charge,1 3 can be produced in various ways in vitro,4 in vivo circulating analogues have not always been found. As a result, we still do not fully understand how plasma LDL is modified and contributes to atheroma formation. Although oxidized LDL has been identified in atherosclerotic lesions,5 6 7 its presence in plasma is controversial.8 9 An oxidized and cytotoxic electronegative LDL subfraction was isolated from human and animal plasma by ion-exchange chromatography,4 10 11 12 but its metabolic origin was a matter of speculation. For example, it may consist of particles originating from "reverse traffic" from the arterial intima, where arterial wall cells can oxidize LDL.1 13 14 Using the same methodology, Shimano et al15 isolated from human plasma a minor, more electronegative LDL subfraction that was not oxidized; these authors did not study its cytotoxicity. In a previous paper, in which we described the fractionation of in vitro charge-modified LDL by FPLC,16 we also reported that human plasma LDL contained about 5% of a more electronegative subfraction relative to the bulk of native LDL. In the present study, we examined the physicochemical characteristics and cellular binding activity of this more electronegative native plasma LDL subfraction, designated LDL(-), to determine its oxidized or nonoxidized nature. In addition, because an increase in the negative charge of LDL may play an important role in its atherogenic properties,1 17 18 19 20 21 we examined LDL(-) cytotoxicity for cultured HUVECs as a determinant of its atherogenic potential. Finally, we examined the relationship between plasma lipid levels and the LDL(-) subfraction in 133 subjects, to assess the physiological relevance of our results.
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Methods |
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Plasma Samples
Blood samples were collected from subjects at Hôpital Broussais who were asymptomatic for renal, liver, and cardiac disease. Venous blood was collected from normolipidemic subjects into EDTA-containing Vacutainer tubes, and plasma was separated by low-speed centrifugation for 15 minutes at 4°C. Plasma samples from 140 subjects were pooled (total volume 300 mL) for each preparation of lipoprotein. Lipid parameters were determined in plasma pools, and values were within the normolipidemic range.
Isolation of LDL
LDL (d=1.019 to 1.063 g/mL) was isolated by
preparative sequential ultracentrifugation in a
Beckman L 90 ultracentrifuge (Beckman Instruments Inc). The
background density of plasma was adjusted to 1.019 g/mL with solid KBr,
and plasma was centrifuged at 45 000 rpm for 24 hours at 4°C
in a Beckman 50.2 Ti rotor. The top fraction and intermediate clear
region in each tube were removed, and the infranatants were pooled. The
background density of the infranatant was then adjusted to 1.063 g/mL
with solid KBr. Centrifugation was performed at 45 000
rpm for 24 hours at 4°C in a Beckman 70.1 Ti rotor. The supernatant
LDL was collected, mixed with a d=1.063-g/mL KBr solution
(1:2, vol/vol) containing 1 mmol/L EDTA, and washed by a final
centrifugation step as described above. LDL was
dialyzed for 24 hours at 4°C against 0.01 mol/L Tris-HCl buffer, pH
7.4, containing 1 mmol/L EDTA and stored at 4°C in the dark.
Purification of LDL Preparations by Immunoaffinity
Chromatography
Immunoaffinity chromatography with antibodies
against apo(a) and apo A-I was performed on LDL preparations at 4°C.
Antibodies against apo(a) (Behring) and apo A-I (Institut Pasteur) were
covalently coupled to CNBr-activated Sepharose 4B (2 mL/200
g gel) according to the manufacturer's instructions (Pharmacia). LDL
preparations were dialyzed against 0.01 mol/L Tris-HCl buffer, pH 7.4,
containing 1 mmol/L EDTA and adjusted to 0.15 mol/L NaCl, then passed
through the anti-apo(a) column (C 10/10, Pharmacia) at a flow rate
of 10 mL/h. Lipoproteins that did not bind to the immunosorbent were
then introduced into the anti–apo A-I column. The LDL collected
was dialyzed against buffer A (described below), used for FPLC
separation.
FPLC Separation
The chromatographic equipment was manufactured by
Pharmacia Biotech and consisted of an LCC-500 programmer controlling
two P-500 pumps. Two buffers were used: buffer A, 0.01 mol/L Tris-HCl,
pH 7.4, containing 1 mmol/L EDTA; buffer B, 1 mol/L NaCl in buffer A.
Buffers were degassed before use, and the system was operated at 4°C.
The LDL sample (5 mg of LDL protein) was filtered through a 0.2-µm
filter (Sartorius), introduced via a 1-mL loop into an
anion-exchange column (mono QHR 5/5), and eluted at 1 mL/min by a
linear gradient of 0% to 20% buffer B during the first 20 minutes,
followed by an elution step with 30% buffer B for 5 minutes. The
effluent was monitored by using a single-path ultraviolet monitor
at 280 nm, and 1-mL fractions were collected with a FRAC-200 fraction
collector. Three LDL subfractions, designated LDL(+)1, LDL(+)2, and
LDL(-) were collected. The sodium concentration in the
chromatographic LDL subfractions was determined with a
flame photometer (Corning 455, Ciba Corning Diagnostics).
Chromatographic LDL subfractions from 12 to 15 runs
were pooled, adjusted to 1.100 g/mL with solid KBr, and concentrated by
ultracentrifugation as described above. The
concentrated LDL subfractions were then dialyzed against buffer A
(samples for enzymatic determination of lipids were dialyzed against
EDTA-free buffer A).
Physicochemical Characteristics of the LDL(-)
Subfraction
Agarose Gel Electrophoresis
U-LDL and chromatographic LDL subfractions were all
adjusted to 0.4 mg protein per milliliter (or 0.2 mg protein per
milliliter in experiments conducted to assess the modification of LDL
subfractions by HUVECs), and 2 µL (or 4 µL) was electrophoresed for
40 minutes on Ciba-Corning Universal electrophoresis agarose gel film
using barbital buffer and the Ciba-Corning system. Films were stained
for lipid with fat red O according to Noble.22
Lipid Composition
Total and free cholesterol,
triglycerides, and phospholipids (phosphatidylcholine) were
determined in chromatographic LDL subfractions with
enzymatic test kits according to the manufacturer's recommendations
(Biotrol).
Apolipoprotein Quantification
Apos A-I, C-III, and E and Lp(a) concentrations were
determined in the chromatographic LDL subfractions by using
a noncompetitive ELISA, on a Biomeck 1000 analyzer
(Beckman).
Sialic Acid Content
Sialic acid content of chromatographic LDL
subfractions was determined according to Warren23 after
release of bound sialic acid by mild hydrolysis (15 minutes at 80°C
in 0.05 mol/L H2SO4).
Lipid-Peroxidation Markers
TBARS content of chromatographic LDL subfractions
was measured using the MDA kit from Sobioda in a modified version of
Yagi's assay with fluorimetric detection.24 Conjugated
diene and vitamin E contents were determined as previously
described.16
SDS-PAGE
SDS-PAGE analysis of LDL subfractions was performed as
previously described,16 using the Pharmacia Phast system
with Phastgel gradient 4 to 15.
Density-Gradient
Ultracentrifugation
Fractionation by density-gradient
ultracentrifugation was performed using a Beckman
SW 41 Ti rotor at 38 000 rpm for 44 hours at 4°C in a Beckman L 90
ultracentrifuge. Chromatographic LDL
subfractions were first raised to a density of 1.040 g/mL with solid
KBr. Discontinuous density gradients were made by underlayering the
following KBr solutions: 2.0 mL of d=1.019 g/mL, 2.0 mL of
d=1.024 g/mL, 3.5 mL (0.7 mg protein) of
chromatographic LDL subfractions at d=1.040
g/mL, and 4.0 mL of d=1.054 g/mL. All solutions contained 1
mmol/L EDTA. After ultracentrifugation, gradients
were collected using a density-gradient fractionator (Model 185,
Isco Inc) coupled to an LKB 2238 Uvicor SII detector, an LKB 2210
recorder, and an LKB 2212 Helirac fraction collector.
Forty-five successive fractions of 0.25 mL were isolated from each
gradient tube. To establish the density profile of each
chromatographic LDL subfraction, the protein absorbance of
LDL at 280 nm was monitored and the potassium concentration was
determined with a flame photometer (Corning).
Assays of Cellular Binding of LDL Subfractions
Cell Culture
MRC5 human fetal lung fibroblasts (Bio-Mérieux) were grown
in DMEM (GIBCO) containing 4.5 g/L glucose, 2 mmol/L glutamine, and 20
mmol/L HEPES and supplemented with 10% FCS (GIBCO), 100 U/mL
penicillin, and 100 µg/mL streptomycin, at 37°C in a humidified
atmosphere containing 5% CO2. Cells were then seeded and
grown to subconfluence in Costar 6x35-mm multiwell plates (Costar,
Polylabo) in the same medium.
LDL Labeling
The LDL(+) subfraction, at a protein concentration of 1 mg/mL,
was labeled with 125I (sodium iodide, 13 to 17 Ci/mg,
Amersham) by using McFarlane's procedure25 as modified by
Bilheimer et al.26
Binding Assays
Before experiments were performed, receptor expression was fully
induced by preincubation of cells for 24 hours in DMEM supplemented
with 2% Ultroser G (US), a serum substitute (Sepracor). Competitive
studies between LDL subfractions for binding to the LDL receptor were
performed at 4°C according to Goldstein and Brown.27
Briefly, 125I-LDL(+) (10 µg/mL) was incubated for 2 hours
in the presence of unlabeled LDL subfractions [6 to 100 µg/mL of
LDL(+) or LDL(-)]. The medium was then removed and cells were
washed and solubilized in 0.2 mol/L NaOH. Cell-associated
radioactivity was quantifiedand cellular protein content determined.
The amount of 125I-LDL(+) bound to the cells was expressed
as a percentage of total binding in the absence of unlabeled LDL.
Percentages of cell-bound 125I-LDL(+)
(y axis) versus concentrations of unlabeled
subfractions (x axis) were plotted to obtain fitted curves,
using a nonlinear regression program (Inplot 4, Graphpad). The
concentration of each unlabeled competitor required to displace 50% of
labeled subfraction was calculated from the displacement curves.
Copper Oxidation of LDL
LDL was dialyzed against EDTA-free 0.02 mol/L phosphate buffer,
pH 7.4, containing 0.15 mol/L NaCl. LDL was adjusted to a protein
concentration of 0.4 mg/mL in dialysis buffer. CuSO4 was
added at a final concentration of 10 µmol/L per 0.4 mg protein (0.5
mmol/L stock solution in distilled water). The vials were then placed
in a water bath at 37°C for 24 hours. Oxidized LDL was dialyzed
against 0.01 mol/L Tris-HCl buffer, pH 7.4, containing 1 mmol/L
EDTA.
Cytotoxicity Determination
Cell Culture
Endothelial cells were isolated from human
umbilical cords, as follows. HUVECs were detached from the umbilical
vein by treatment for 20 to 25 minutes, at 37°C, with dispase (grade
II from Bacillus polymyxa, Boehringer Mannheim).
After centrifugation, cells were resuspended in 3 mL of
Medium 199 (Boehringer Mannheim) supplemented with 10% FCS
(Boehringer Mannheim), 1.5% HEPES, 2 mmol/L glutamine (Tech
Gen), 100 U/mL penicillin, 100 µg/mL streptomycin (Tech Gen), and 2.5
µg/mL amphotericin B (Tech Gen). The cell count was determined by
duplicate Malassez cell counts and adjusted to
106/mL with culture medium. Then,
106 cells were plated in 25 cm2 (T25)
plastic tissue-culture flasks (Falcon Plastics, Polylabo)
containing 5 mL of culture medium and incubated at 37°C in an
atmosphere of 76% N2, 19% O2,
and 5% CO2. The culture medium was changed 1 day later and
then every 2 days. When confluent, cells were trypsinized, resuspended
at the density of 100 000 to 200 000 per well in culture medium
(Ham's F-10 medium [Tech Gen] and DMEM [Tech Gen] were used for
LDH determination, and Ham's F-10 medium was used for neutral
red–uptake assay; Ham's F-10 medium was also used to assess the
modification of chromatographic LDL subfractions by
HUVECs), and plated in Nunc 4-well plates (1.9 cm2 wells)
(Nunc, Polylabo) for LDH determination or in Costar 96-well plates
(0.32 cm2 wells) for neutral red–uptake assay. Cells
were used at passage level 2. Experiments were conducted at an initial
cell density of 0.15x106 per well (in 1.0 mL) for
LDH determination and 0.03x106 per well (in 0.2 mL)
for neutral red–uptake assay. After 24 hours, cells were washed
with FCS-free medium. Chromatographic LDL subfractions
[LDL(+) and LDL(-)], U-LDL, and copper-oxidized LDL (as
cytotoxic positive control), previously dialyzed against 0.02 mol/L
phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl, were diluted to
0.2 mg/mL in FCS-free medium supplemented with glutamine and added to
the cells (in 1.0 mL for LDH determination and in 0.2 mL for neutral
red–uptake assay). The same volume of medium supplemented with
10% FCS was added to the growth-control cells. After 4, 8, 24, and
48 hours, chromatographic LDL subfraction–induced cell
injury was evaluated by the release of the cytoplasmic enzyme LDH,
neutral red uptake, and morphological studies. In three experiments,
cell-induced LDL modifications were studied by the measurement of
TBARS and vitamin E contents and by agarose gel electrophoresis.
LDH Assay
LDH activity was measured using the LDH kit from Biotrol.
Results are expressed in units per well. For each culture well, three
different samples were prepared, and LDH activity was measured in each
sample, as follows: (1) Culture supernatant (1 mL) was collected and
centrifuged at 1000 rpm for 10 minutes at 20°C. The
supernatant was used for measurement of LDH3 activity (LDH activity
released in medium by lysed cells). (2) The
centrifugation pellet was frozen at -80°C,
thawed, dissolved in 0.2 mL H2O, vortexed, and used for the
measurement of LDH2 activity (LDH activity contained in detached,
unlysed cells). (3) The cell layer was washed with phosphate buffer
containing Ca2+ and Mg2+ (Tech Gen), frozen at
-80°C, thawed, dissolved in 0.5 mL H2O, vortexed,
and centrifuged at 3000 rpm for 10 minutes at 4°C. The
supernatant (0.5 mL) was used for the measurement of LDH1 activity (LDH
activity contained in cells remaining adherent to the well at the end
of the assay period).
Three cytotoxicity indices were then calculated, as follows:
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Neutral Red–Uptake Assay
Culture medium was removed and cell layers were washed with
Ham's F-10 medium, then 100 µL of Ham's F-10 medium containing 10%
FCS and neutral red was added to each well of Costar 96-well plates
(0.32-cm2 wells); the plates were maintained for 3 hours at
37°C in a humidified atmosphere containing 5% CO2. The
culture medium was then removed, replaced by 200 µL of formol/calcium
solution (0.1 mol/L CaCl2 in 4% formol solution) for 1
minute to fix the coloration, and washed with 100 µL of FCS-free
medium. To disrupt the cells, 100 µL of acetic acid/ethanol solution
(acetic acid/ethanol/H2O [0.02:1:1, vol/vol/vol]) was
introduced in each well. After agitation, absorbance was read at 550 nm
in an ELISA plate reader (LP 400 GIBCO). Injury of the cell monolayers
was quantified by a cytotoxicity index, calculated as follows:
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Morphological Studies
At the end of the incubation periods, before removing
supernatant medium for measurement of LDH activity, the cell cultures
were examined morphologically by means of inverted light microscopy.
All photographs were taken with a 35-mm camera (Olympus PM 10 AK) at
100x magnification.
Cell-Induced LDL Subfraction Modification
The modification of LDL(+) and LDL(-) subfractions by
HUVECs was stopped with 10 µL BHT (Fluka; 2 mmol/L in methanol) and
10 µL EDTA (100 mmol/L in phosphate buffer) in each culture well.
Supernatants were then collected for the measurement of TBARS and
vitamin E contents and for agarose gel electrophoresis of LDL
subfractions.
Correlation Study
Study Subjects
The 133 subjects included in the study were from
Hôpital Broussais. None had clinical signs of renal, hepatic, or
cardiac disease. They had never been treated with lipid-lowering
drugs and were not selected on the basis of their blood total
cholesterol and triglyceride levels.
Analytical Methods
Venous blood was sampled and LDL isolated as described by our
investigators.28 The percentage of contribution of
LDL(-) to total plasma LDL was determined by FPLC separation. In
this case, the LDL sample (0.25 mg of LDL protein) was introduced via a
0.5-mL loop. Total cholesterol and LDL
cholesterol were routinely determined as previously
described.28
Protein Assay
Total protein was measured using Peterson's
method,29 with bovine serum albumin as
standard.
Statistical Analysis
Data were analyzed using Student's paired or unpaired
t test. Pearson's correlation coefficients were used to
describe relations between LDL(-) percentages and quantitative
normal variables. Calculations were done on a Macintosh II SI
computer (Apple) with statistical software (Statview II, Abacus
Concepts Inc). Values of P<.05 were considered
statistically significant. All values are presented as
mean±SD.
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Purification of Total Plasma LDL by Anti-apo(a) and Anti–apo A-I Immunoaffinity Chromatography
Total U-LDL contained 2.20±0.86% Lp(a) and 0.19±0.06% apo A-I (wt/wt, mean±SD; 14 different LDL preparations), revealing its unavoidable contamination by Lp(a) (d=1.050 to 1.090 g/mL) and HDL (d=1.063 to 1.210 g/mL), which is inherent in the preparative sequential ultracentrifugation methodology. Because Lp(a) and HDL have an increased electronegative charge, we included an immunoaffinity chromatography step in our experimental procedure to remove these contaminating lipoproteins before FPLC fractionation of LDL. Anti-apo(a) and anti–apo A-I immunoaffinity chromatography removed 95±4% of Lp(a) and 89.5±4% of apo A-I (mean±SD, n=14) from LDL preparations. The low levels of Lp(a) and apo A-I in U-LDL submitted to immunoaffinity chromatography (0.11±0.07% and 0.020±0.004%, respectively) were consistent with the absence of significant contamination by Lp(a) and by particles containing apo A-I (HDL).
Isolation of LDL(-) From Total Plasma LDL by Ion-Exchange
Chromatography (FPLC)
A typical chromatogram depicting the resolution of total plasma
LDL into three subfractions is shown in Fig 1
. Two major
peaks, representing the LDL(+)1 and LDL(+)2 subfractions,
were eluted, with a theoretical NaCl concentration ranging from 0.10 to
0.20 mol/L NaCl, at retention times of 11 and 17 minutes, respectively.
LDL(+)1 averaged 38±7.5% (mean±SD; 18 different LDL preparations)
and LDL(+)2 averaged 55.3±8.1% of total LDL. Following these major
peaks, a small minor peak, corresponding to LDL(-), was eluted at
a retention time of 23 minutes and had a theoretical NaCl concentration
of 0.3 mol/L. LDL(-) was therefore defined as the most
electronegative LDL subfraction and represented 6.7±0.9%
(mean±SD, n=18) of total LDL. FPLC analytical variables such as
precision, selectivity, recovery, detection limit, linearity, and
capacity were as previously described.16 To rule out
artifactual generation of LDL(-) during immunoaffinity
chromatography, an aliquot of LDL(+)1 subfractionated
by FPLC (without preliminary immunoaffinity) was submitted to
immunoaffinity chromatography and then reinjected into
the FPLC system under the same conditions as for LDL(-)
isolation. The peak eluted at 11 minutes, indicating that retention
times were not modified by the immunoaffinity
chromatography step.
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Physicochemical Characterization of LDL(-)
The net electrical charge of chromatographic LDL
subfractions was examined by means of agarose gel electrophoresis (Fig 2). The LDL(+)1 and LDL(+)2 subfractions displayed lower
mobility than total unfractionated LDL, whereas LDL(-) displayed
higher mobility. The respective mobilities of the LDL subfractions were
not modified by immunoaffinity chromatography, again
confirming that LDL subfractions were not modified during this step.
Since the results obtained for LDL(+)1 and LDL(+)2 were similar for all
the parameters studied, the two were combined and
designated LDL(+). The chemical, physical, and biological
characteristics of LDL(-) were then compared with those of
LDL(+). The chemical composition of LDL(+) and LDL(-)
subfractions is shown in the Table. No significant
differences in total protein content, lipid composition, or the
lipid/protein ratio were observed between LDL(+) and LDL(-)
subfractions. In contrast, LDL(-) had significantly increased
contents of apo C-III, apo E, and sialic acid relative to LDL(+). No
significant differences were observed in lipid-peroxidation marker
content between LDL(+) and LDL(-) subfractions. Moreover, the
TBARS, conjugated diene, and vitamin E contents of the LDL(-)
subfraction were consistent with the properties of native LDL.
SDS-PAGE analysis showed the apo B band (550 kD) at the same
position in LDL(+) and LDL(-) subfractions and did not reveal
higher- (due to apo B self-aggregation) or lower- (due to apo B
fragmentation) molecular-weight peptides. The floating densities of
LDL(+) and LDL(-) subfractions were compared by means of
density-gradient ultracentrifugation. The two
LDL subfractions had the same main density peak of 1.033 g/mL (mean
data from two independent series of experiments, using different LDL
preparations in each series of experiments). However, while LDL(+)
showed a unimodal distribution, LDL(-) had a more
heterogeneous profile, characterized by a shoulder in the
higher-density range of the peak (between 1.035 and 1.045
g/mL).
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LDL Receptor–Binding Activity of LDL(-)
LDL(+) and LDL(-) subfractions were compared with regard to
binding activity to the LDL receptor on human MRC5 fibroblasts. The
competitive displacement of 125I-labeled LDL(+) by
unlabeled LDL(+) and LDL(-) was performed at 4°C. As shown on
the competitive binding curves (Fig 3), unlabeled
LDL(-) reduced the binding of 125I-labeled LDL(+)
more actively than the unlabeled LDL(+) subfraction. The concentrations
of unlabeled LDL subfractions required to displace 50% of
125I-labeled LDL(+) were the following: LDL(+), 12.2±3.3
µg protein per milliliter (mean±SD, n=3); LDL(-), 8.2±0.5
µg protein per milliliter.
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) or LDL(-)
(
) subfractions (5 to 100 µg/mL). Each experimental point
represents the mean amount of 125I-labeled LDL(+)
bound to cells (±SD; three different samples tested in triplicate),
expressed as a percentage of total binding in the absence of unlabeled
LDL subfractions.
Cytotoxicity of the LDL(-) Subfraction
Cytotoxicity studies were performed in serum-free medium to
avoid possible inhibition of LDL(-) properties by serum
components. Given these experimental conditions, LDL(+)-treated cells
were used as a negative cytotoxic control. This choice was motivated by
the following observations. First, confluent monolayers of adult human
venous endothelial cells can be maintained for at least
24 hours in FCS-free medium provided that protein (for instance, LDL
protein) is present.18 30 31 Second, native total LDL
(0.2 mg/mL) is not toxic after incubation times of 8 or 24 hours in
FCS-free medium.31 Third, total LDL is mainly composed of
LDL(+) particles, and we previously verified that cytotoxicity indices
of LDL(+) were similar to those of total LDL. An additional control
(HUVECs incubated in medium supplemented with FCS, ie,
growth-control cells) was used only to verify (by means of light
microscopy) the quality (purity and growth) of our cell
preparations.
LDL(-)-induced endothelial cell injury was first
examined in terms of the percentage of viable cells, using two separate
assays (Fig 4). In the LDH assay (Fig 4A), the total
cytotoxicity index of LDL(-), at a treatment dose of 0.2 mg/mL in
Ham's F-10 medium for 8 hours, was significantly higher than the total
cytotoxicity index of LDL(+). The cytotoxicity index obtained for
LDL(+)-treated cells corresponded to the percentage of spontaneous cell
death known to occur in medium with a low protein
content.18 In the neutral red–uptake assay (Fig 4B),
a significant difference in the cytotoxicity index between the LDL(+)
and LDL(-) subfractions was also obtained. In addition, since the
LDH assay reflects the injury of the total cell population, whereas
neutral red uptake reflects only the viability of cells remaining
attached to the wells at the end of the incubation period, the results
of the latter assay indicated that LDL(-) was first toxic for
adherent cells.
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To further assess the cytotoxic effect of LDL(-), morphological
studies were performed by light microscopy. As illustrated in Fig 5, growth-control HUVECs (Fig 5A) grew in confluent
monolayers with the histiotypic "cobblestone" pattern. In the
presence of LDL(+) (Fig 5B), minor shape changes, eg, cytoplasmic
contraction, were observed, but most cells remained attached to the
culture wells. These subtle morphological alterations were again
explained by the fact that growth-control cells were incubated in
FCS-supplemented culture medium, whereas LDL(+)-treated cells were
incubated with the LDL subfraction diluted in FCS-free medium. The
LDL(+) subfraction itself thus had no major detrimental effects on
cultured HUVECs. In contrast, exposure of HUVECs to LDL(-)
resulted in major morphological changes, with total disintegration of
the endothelial monolayer. The first recognizable
change was cell contraction, which was observed after incubation times
of 4 hours or more. The cells then rounded up and detached from the
bottom of the wells. After a treatment time of 8 hours (Fig 5C),
numerous cells were detached, and the remaining attached cells
exhibited marked alterations, such as disappearance of the
characteristic double refraction of the intact membranes, assumption of
a spherical shape (corresponding to a maximal cytoplasmic contraction),
and nuclear pycnosis. The concordance between these morphological and
quantitative data further established that LDL(-) was cytotoxic
for cultured HUVECs.
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To elucidate the chronological pattern of LDL(-) cytotoxicity,
LDL(+) and LDL(-) subfractions were compared with regard to the
three indices in the LDH assay (Fig 6). LDL(-)
caused significantly more cell lysis than LDL(+), and the difference in
the cell lysis index values of the two LDL subfractions correlated with
the difference in their total cytotoxicity index values. In
marked contrast, the LDL(+) and LDL(-) subfractions induced
similar degrees of cell detachment. Moreover, the LDL(-) cell
detachment index values were much lower (0.38±0.27, mean±SD, from
three wells of HUVECs, combined from six separate experiments) than
those of cell lysis and total cytotoxicity (19.85±4.22 and
20.23±4.33, respectively). LDL(-)-induced cell detachment index
values did not vary with the incubation period, whereas both cell lysis
and total cytotoxicity increased gradually with increasing exposure
times (data not shown). These data indicated that LDL(-)-induced
cell injury did not reflect simple cell detachment but rather a
specific lytic action of this subfraction. These LDH assay results,
taken together with those of the neutral red–uptake assays and
morphological observations, revealed that LDL(-) exposure injured
adherent cells, leading to cell death and subsequent detachment.
|
To determine whether cellular oxidation of LDL(-) was involved in
its cytotoxicity, the toxic effects of the LDL(-) subfraction
were compared in two different culture media in terms of the ratio of
the LDL(-) and LDL(+) total cytotoxicity indices in the LDH assay
(at a treatment dose of 0.2 mg/mL for 8 hours). Ham's F-10 medium,
known to promote cellular oxidation of LDL due to the presence of
copper ions and the absence of antioxidants,14 was used to
study LDL(-) cytotoxicity according to its susceptibility to
oxidation. In this medium, LDL(-) was 2.05±0.53-fold (mean±SD
from three wells of HUVECs, combined from six separate experiments)
more cytotoxic than LDL(+). DMEM, being copper free, was used to
investigate LDL(-) cytotoxicity independent of cellular
peroxidative processes.14 Although LDL(-) was also
significantly more cytotoxic than LDL(+) in this medium
(P<.04, by Student's paired t test, for
LDL(-) total cytotoxicity index versus LDL(+) total cytotoxicity
index, n=6), its total cytotoxicity index was only 1.30±0.23 times
higher than that of LDL(+). Moreover, the LDL(-) total
cytotoxicity index/LDL(+) total cytotoxicity index ratio was
significantly higher in Ham's F-10 than in DMEM (P<.01,
Student's unpaired t test, for Ham's F-10 versus DMEM,
n=6). These results suggested that cellular oxidation of the
LDL(-) subfraction increased its cytotoxicity. To check this
possibility, cell-induced modifications of LDL subfractions in
Ham's F-10 medium were studied for 8 hours. No difference in TBARS
content between native and cell-treated LDL was found, with either
LDL(-) or LDL(+). In contrast, the vitamin E content of LDL
subfractions fell during incubation with HUVECs [native LDL(+),
18.1±1.0 nmol/mg protein, mean±SD, n=3; cell-treated LDL(+),
16.0±1.0 nmol/mg protein; native LDL(-), 17.3±1.0 nmol/mg
protein; cell-treated LDL(-), 11.9±1.0 nmol/mg protein].
The decrease in vitamin E content was significant (P=.0003,
Student's paired t test, for native versus cell-treated
LDL, n=3), and the difference observed between native and
cell-treated LDL was greater with LDL(-) than with
LDL(+). Moreover, while no difference in vitamin E content between
native LDL(+) and LDL(-) subfractions was found, a significant
difference was observed between cell-treated LDL(+) and
LDL(-) subfractions (P<.02, Student's paired
t test, n=3). In addition, the electronegativity of the
LDL(-) subfraction was increased after incubation with HUVECs,
whereas the net electric charge of the LDL(+) subfraction was not
modified (Fig 7). These data confirmed that the
LDL(-) subfraction was more strongly modified by cells than the
LDL(+) subfraction.
|
The time dependence of LDL(-) cytotoxicity was also studied. With incubation times longer than 8 hours, no difference in cytotoxicity between LDL(+) and LDL(-) subfractions was found [mean LDL(-) total cytotoxicity index/LDL(+) total cytotoxicity index ratio at 24 hours, 1.07±0.12 (SD); mean ratio at 48 hours, 1.08±0.26; three HUVEC wells, six separate experiments]. These results were attributed to inability of FCS-free medium to sustain cell viability for periods greater than 24 hours.18 At an incubation time of 4 hours, LDL(-) was more cytotoxic than LDL(+), and the difference between the two subfractions was greater than that observed after an incubation time of 8 hours, even in DMEM [mean LDL(-) total cytotoxicity index/LDL(+) total cytotoxicity index ratio at 4 hours in DMEM, 1.70±0.27; mean ratio at 8 hours in DMEM, 1.30±0.23], suggesting that LDL(-) had early cytotoxic effects. Confirmatory data were obtained by morphological studies. After exposure times of 4 hours and 6 hours to LDL(-), HUVECs showed cytoplasmic and nuclear contraction. These morphological alterations were less marked than those observed after an exposure time of 8 hours; in contrast, no HUVEC shape changes were perceptible after an incubation time of 4 hours or 6 hours with LDL(+).
Correlation Study
The characteristics (mean±SD) of the 133 subjects studied
(114 men and 19 women) were the following: age 48±8 years; lipid
levels (mmol/L), total cholesterol 6.4±1.1;
triglycerides 1.4±0.7; HDL cholesterol
1.4±0.3; and LDL cholesterol 4.5±1.0. In this
population, the percentage of contribution of LDL(-) to total
plasma LDL was 5.4±2.2%. This value correlated positively with age
(r=.18, P<.05, Pearson's rank test). Among the
lipid parameters, the percentage concentration of
LDL(-) in total LDL correlated positively with total
cholesterol (r=.17, P<.05,
Pearson's rank test) and LDL cholesterol
(r=.26, P<.003, Pearson's rank test) (Fig 8).
|
|
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Numerous studies have demonstrated that plasma LDL is a heterogeneous collection of particles that vary in size,32 density,33 composition,34 and electrical charge35 and that some LDL forms are more atherogenic than others.36 37 Using FPLC, we isolated from normolipidemic human plasma a minor LDL subfraction, LDL(-), with a greater negative charge than the bulk of LDL. This subfraction had a low TBARS and conjugated diene content, a high vitamin E level, and no modification in lipid/protein ratio and mean density. The apo B was not aggregated, and binding activity to LDL receptors was not decreased. In addition, we verified (using human monocyte-macrophages) that scavenger receptors played no role in the cellular binding of LDL(-) (data not shown). These physicochemical and biological criteria together provide strong evidence that the LDL(-) subfraction shows little evidence of oxidation as assessed by classic parameters of lipoprotein oxidation.38 39 Although LDL(-) is a native form of LDL, it differs markedly from the bulk of plasma LDL. It was more negatively charged than LDL(+) and had significantly higher sialic acid, apo C-III, and apo E contents, a slightly higher proportion of dense particles, and greater LDL receptor–binding activity. Enrichment in sialic acid or modifications in apolipoprotein composition, which may occur via conformational changes of LDL, could account for the increased negative surface charge of LDL(-). The increased sialic acid content of LDL(-) cannot be explained by contaminating Lp(a), since we removed this lipoprotein from LDL preparations. On the other hand, as both apo E and apo C-III exist in plasma in several sialylated isoforms,40 41 42 the enrichment of LDL(-) in apos C-III and E may be one explanation for its increased sialic acid content. Indeed, the presence of apos C-III and E in the LDL(-) subfraction can be ascribed to LDL(-) itself: first, contaminating HDL was removed from LDL preparations by anti–apo A-I immunoaffinity chromatography, and second, the absence of a shoulder in the low-density range (<1.020 g/mL) of the density profiles obtained for LDL subfractions ruled out contamination by VLDL and IDL as an explanation for the enrichment of LDL(-) in these two apolipoproteins. In addition to their plausible contribution to the negative surface charge of LDL(-), changes in apolipoprotein composition could explain the greater LDL receptor–binding activity relative to LDL(+).41 43 44 45 46 Numerous studies have shown that endothelial cell injury plays a key role in the onset of atherosclerosis.47 48 49 Using both cell viability assays and morphological studies, we demonstrated that LDL(-) was cytotoxic for cultured HUVECs. Because VLDL has been reported to be toxic for cultured cells,20 we verified the absence of contaminating VLDL in our LDL preparations. The cytotoxicity of LDL(-) had three main characteristics: (1) it clearly occurred after an incubation time as short as 4 hours; (2) it induced specific lysis of adherent cells, followed by cell death and detachment; and (3) it probably involved at least two separate mechanisms, the first dependent on cellular oxidation, and the second independent of lipid peroxidation. The first mechanism of LDL(-) cytotoxicity was shown by two observations. First, LDL(-) cytotoxicity was greater in Ham's F-10 medium than in DMEM, and our studies as well as others14 31 50 51 52 have shown that cultured endothelial cells can oxidize LDL in Ham's F-10 medium but not in DMEM. Second, morphological alterations of the cells, as well as the sequence of toxic events (cell injury, death, then detachment) that we observed with the LDL(-) subfraction in Ham's F-10 medium, were similar to those reported with oxidized LDL.18 20 53 54 In addition, cytotoxicity is a well-recognized proatherogenic property of oxidized LDL.20 31 53 54 Thus, it is likely that the increased cytotoxicity of the LDL(-) subfraction in Ham's F-10 medium is related to its cellular oxidation. Indeed, although the LDL(-) subfraction was not completely oxidized by HUVECs after 8 hours in Ham's F-10 medium (ie, its TBARS content was not increased), it was much more modified than the LDL(+) subfraction [its vitamin E content was significantly lower and its electronegativity was increased relative to LDL(+)]. These results indicated that LDL(-) was more rapidly oxidizable than LDL(+). This is not surprising, since LDL(-) was enriched in sialic acid and, as we and others recently reported, desialylated LDL is more resistant to copper oxidation than native LDL.55 56 Consequently, the greater cytotoxicity of the LDL(-) subfraction relative to LDL(+) in Ham's F-10 medium could be explained at least in part by an increase in its susceptibility to oxidation. The second (peroxidation-independent) mechanism of LDL(-) cytotoxicity, which was revealed in DMEM, remains to be elucidated.
As regards the physiological relevance of our in vitro findings, whether or not the LDL(-) subfraction also displays cytotoxic effects in vivo despite its small percentage of contribution to total plasma LDL must be discussed. Different LDL subfractions, representing smaller percentages of total LDL than our LDL(-) subfraction, have already been proposed as markers for atherosclerosis.11 12 15 57 58 Moreover, three elements are in favor of the increased atherogenicity of the LDL(-) subfraction in vivo. First, the most atherogenic LDL being modified LDL,13 the LDL(-) subfraction should be more atherogenic than the bulk of native plasma LDL because of its increased susceptibility to cellular modification. Second, we showed that the LDL(-) subfraction was enriched in apo E relative to LDL(+). It has been demonstrated that an increased apo E content in LDL favors binding to arterial proteoglycans and likely promotes sequestration in the artery wall.59 60 In addition, it is well established that, in vivo, LDL oxidation (and subsequent cytotoxicity) mainly occurs locally within the intima of the artery.13 Thus, the local cellular environment might promote an increase in LDL(-) concentration above that present in plasma and consequently favor the detrimental effects of LDL(-) in vivo. Third, we report here that the percentage of contribution of LDL(-) to total LDL in a general population correlated positively with common atherosclerotic risk factors such as age, total cholesterol, and LDL cholesterol.
In conclusion, the LDL(-) subfraction, a circulating human plasma LDL, is an electronegative native subfraction of total plasma LDL that is cytotoxic for endothelial cells. This LDL(-) subfraction might conceivably contribute to the atherogenicity of total plasma LDL. These findings warrant further investigations to determine the metabolic origin of this LDL(-) subfraction, the identity of its toxic components, and the mechanisms by which it damages cultured cells. To firmly establish its clinical significance, we are currently examining the relationships between the LDL(-) subfraction and plasma lipids, as well as correlations with clinical parameters, in a prospective study.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This work was supported in part by US Public Health Service grants RO1 HL 50379 and HL 30568 (to Dr Haberland). We thank J.C. Mazière and his team for their helpful contribution to the binding studies.
Received July 27, 1995; accepted January 19, 1996.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Steinberg D, Parthasarathy S, Carew TE, Khoo JG, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915-924. [Medline] [Order article via Infotrieve]
2. Steinbrecher UP, Zhang H, Lougheed M. Role of oxidatively modified LDL in atherosclerosis. Free Radic Biol Med. 1990;9:155-168. [Medline] [Order article via Infotrieve]
3. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983;52:223-261. [Medline] [Order article via Infotrieve]
4.
Avogaro P, Bittolon-Bon G, Cazzolato G.
Presence of a modified low density lipoprotein in humans.
Arteriosclerosis. 1988;8:79-87.
5.
Haberland ME, Fong D, Cheng L.
Malondialdehyde-altered protein occurs in atheroma of
Watanabe heritable hyperlipidemic rabbits.
Science. 1988;241:215-218.
6.
Palinski W, Rosenfeld ME, Ylä-Herttuala S,
Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg
D, Witztum JL. Low density lipoprotein undergoes oxidative
modification in vivo. Proc Natl Acad Sci U S A. 1989;86:1372-1376.
7. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.
8. Esterbauer H, Jürgens G, Quehenberger O, Koller E. Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J Lipid Res. 1987;28:495-509. [Abstract]
9.
Frei B, Stocker R, Ames BN. Antioxidant
defenses and lipid peroxidation in human blood plasma.
Proc Natl Acad Sci U S A. 1988;85:9748-9752.
10. Avogaro P, Cazzolato G, Bittolon-Bon G. Some questions concerning a small, more electronegative LDL circulating in human plasma. Atherosclerosis. 1991;91:163-171. [Medline] [Order article via Infotrieve]
11. Cazzolato G, Avogaro P, Bittolon-Bon G. Characterization of a more electronegatively charged LDL subfraction by ion exchange HPLC. Free Radic Biol Med. 1991;11:247-253. [Medline] [Order article via Infotrieve]
12. Hodis HN, Kramsch DM, Avogaro P, Bittolon-Bon G, Cazzolato G, Hwang J, Peterson H, Sevanian A. Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL-). J Lipid Res. 1994;35:669-677. [Abstract]
13.
Leake DS. Oxidized low density lipoproteins and
atherogenesis. Br Heart J. 1993;69:476-478.
14.
Steinbrecher UP, Parthasarathy S, Leake DS, Witztum JL,
Steinberg D. Modifications of low density lipoprotein by
endothelial cells involves lipid peroxidation and
degradation of low density lipoprotein phospholipids.
Proc Natl Acad Sci U S A. 1984;81:3883-3887.
15. Shimano H, Yamada N, Ishibashi S, Mokuno H, Mori N, Gotoda T, Harada K, Akanuma Y, Murase T, Yazaki Y, Takaku F. Oxidation-labile subfraction of human plasma low density lipoprotein isolated by ion-exchange chromatography. J Lipid Res. 1991;32:763-773. [Abstract]
16. Vedie B, Myara I, Pech MA, Mazière JC, Caprani A, Moatti N. Fractionation of charge-modified low density lipoproteins by fast protein liquid chromatography. J Lipid Res. 1991;32:1359-1369. [Abstract]
17.
Goldstein JL, Ho YK, Basu SK, Brown MS. Binding
site on macrophages that mediates uptake and degradation of
acetylated low density lipoprotein, producing massive
cholesterol deposition. Proc Natl Acad Sci
U S A. 1979;76:333-337.
18. Henriksen T, Evensen SA, Carlander B. Injury to human endothelial cells in culture induced by low density lipoproteins. Scand J Clin Lab Invest. 1979;39:361-368. [Medline] [Order article via Infotrieve]
19.
Henriksen T, Mahoney EM, Steinberg D. Enhanced
macrophage degradation of low density lipoprotein previously
incubated with cultured endothelial cells: recognition
by receptors for acetylated low density lipoprotein.
Proc Natl Acad Sci U S A. 1981;78:6499-6503.
20.
Hessler JR, Morel DW, Lewis J, Chisolm GM.
Lipoprotein oxidation and lipoprotein-induced cytotoxicity.
Arteriosclerosis. 1983;3:215-222.
21.
Fogelman AM, Schechter I, Seager J, Hokom M, Childs JS,
Edwards PA. Malondialdehyde alteration of low density
lipoproteins leads to cholesterol ester accumulation in
human monocyte-derived macrophages. Proc Natl
Acad Sci U S A. 1980;77:2214-2218.
22. Noble RP. Electrophoretic separation of plasma lipoproteins in agarose gel. J Lipid Res. 1968;9:693-700. [Abstract]
23.
Warren L. The thiobarbituric assay of sialic
acids. J Biol Chem. 1959;234:1971-1975.
24. Yagi KA. A simple fluorimetric assay for lipoperoxide in blood plasma. Biochem Res. 1976;15:212-216.
25. McFarlane AS. Efficient trace-labelling of proteins with iodine. Nature. 1958;182:53-56. [Medline] [Order article via Infotrieve]
26. Bilheimer DW, Eisenberg S, Levy RI. The metabolism of very low density lipoproteins, I: preliminary in vitro and in vivo observations. Biochim Biophys Acta. 1972;260:212-221. [Medline] [Order article via Infotrieve]
27.
Goldstein JL, Brown MS. Binding and degradation
of low density lipoproteins by cultured human fibroblasts.
J Biol Chem. 1974;249:5153-5162.
28.
Chappey B, Myara I, Giral P, Kerharo G, Plainfosse MC,
Levenson J, Simon A, Moatti N, and the PCVMETRA Group. Evaluation of
the sialic acid content of LDL as a marker of coronary
calcification and extracoronary
atherosclerosis in asymptomatic
hypercholesterolemic subjects. Arterioscler
Thromb Vasc Biol. 1995;15:334-339.
29. Peterson GL. A simplification of the protein assay method of Lowry et al which is more generally applicable. Anal Biochem. 1977;83:346-356. [Medline] [Order article via Infotrieve]
30. Horio T, Kohno M, Yasunari K, Murakawa K, Yokokawa K, Ikeda M, Fukui T, Takeda T. Stimulation of endothelin-1 release by low density and very low density lipoproteins in cultured human endothelial cells. Atherosclerosis. 1993;101:185-190. [Medline] [Order article via Infotrieve]
31.
Triau JE, Meydani SN, Schaefer EJ. Oxidized low
density lipoprotein stimulates prostacyclin production by adult
human vascular endothelial cells.
Arteriosclerosis. 1988;8:810-818.
32. Shen MS, Krauss RM, Lindgren FT, Forte TM. Heterogeneity of serum low density lipoproteins in normal human subjects. J Lipid Res. 1981;22:236-244. [Abstract]
33. Nelson CA, Morris MD. The ultracentrifugal heterogeneity of serum low density lipoproteins in normal humans. Biochem Med. 1977;18:1-9.[Medline] [Order article via Infotrieve]
34. Kirchhausen TG, Fless G, Scanu AM. The structure of plasma low density lipoproteins: experimental facts and interpretation—a minireview. Lipids. 1980;15:464-467. [Medline] [Order article via Infotrieve]
35. Sundaram SG, Shakir KMM, Margolis S. Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins. Anal Biochem. 1978;88:425-433. [Medline] [Order article via Infotrieve]
36. Rudel LL, Parks JS, Johnson FL, Babiak J. Low density lipoproteins in atherosclerosis. J Lipid Res. 1986;27:465-474. [Medline] [Order article via Infotrieve]
37. Krauss RM. Heterogeneity of plasma low density lipoproteins and atherosclerosis risk. Curr Opin Lipidol. 1994;5:339-349.[Medline] [Order article via Infotrieve]
38. Esterbauer H, Dieber-Rotheneder M, Waeg G, Striegl G, Jürgens G. Biochemical, structural, and functional properties of oxidized low-density lipoprotein. Chem Res Toxicol. 1990;3:77-92. [Medline] [Order article via Infotrieve]
39. Esterbauer H, Gebicki J, Puhl H, Jürgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med. 1992;13:341-390. [Medline] [Order article via Infotrieve]
40. Ito Y, Breslow JL, Chait BT. Apolipoprotein C-IIIo lacks carbohydrate residues: use of mass spectrometry to study apolipoprotein structure. J Lipid Res. 1989;30:1781-1787. [Abstract]
41. Mahley RW, Innerarity TL, Rall SC Jr, Weisgraber KH. Plasma lipoproteins: apolipoprotein structure and function. J Lipid Res. 1984;25:1277-1294. [Abstract]
42.
Remaley AT, Wong AW, Schumacher UK, Meng MS, Brewer HB
Jr, Hoeg JM. O-linked glycosylation modifies the association of
apolipoprotein A-II to high density lipoproteins. J
Biol Chem. 1993;268:6785-6790.
43.
Agnani G, Bard JM, Candelier L, Delattre S, Fruchart
JC, Clavey V. Interaction of LpB, LpB:E, LpB:C-III, and
LpB:C-III:E lipoproteins with the low density lipoprotein receptor of
HeLa cells. Arterioscler Thromb. 1991;11:1021-1029.
44. Clavey V, Agnani G, Bard JM, Lestavel-Delattre S, Fruchart JC. Interaction entre le LDL-récepteur et les lipoprotéines contenant de l'apo B. Ann Endocrinol (Paris). 1991;52:459-463. [Medline] [Order article via Infotrieve]
45. Chapman MJ, Cadman H, Laplaud PM. Heterogeneity in apo-B,E receptor binding of human LDL subspecies. Circulation. 1984;70(suppl II):II-137. Abstract.
46. Gabelli C, Gregg RE, Zech LA, Manzato E, Brewer HB. Abnormal low density lipoprotein metabolism in apolipoprotein E deficiency. J Lipid Res. 1986;27:326-333. [Abstract]
47.
Flavahan NA. Atherosclerosis or
lipoprotein-induced endothelial dysfunction:
potential mechanisms underlying reduction in EDRF/nitric oxide
activity. Circulation. 1992;85:1927-1938.
48. Dicorleto PE, Soyombo AA. The role of endothelium in atherogenesis. Curr Opin Lipidol. 1993;4:364-372.
49. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]
50. Bruckdorfer KR. Nonenzymatic oxidation of lipids and lipoproteins: the role of metals and nitric oxide. Curr Opin Lipidol. 1993;4:238-243.
51. Pech MA, Myara I, Moatti N. Influence of culture medium characteristics on the ability to oxidize low-density lipoproteins. Bioelectrochem Bioenerg. 1993;29:277-288.
52. Sparrow CP, Olszewski J. Cellular oxidation of low density lipoproteins is caused by thiol production in media containing transition metal ions. J Lipid Res. 1993;34:1219-1228. [Abstract]
53. Morel DW, Hessler JR, Chisolm GM. Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid. J Lipid Res. 1983;24:1070-1076. [Abstract]
54.
Pettersen KS, Boberg KM, Stabursvik A, Prydz H.
Toxicity of oxygenated cholesterol derivatives
toward cultured human umbilical vein endothelial
cells. Arterioscler Thromb. 1991;11:423-428.
55. Dousset N, Dousset JC, Taus M, Ferretti G, Curatola G, Soléra ML, Valdiguié P. Effect of desialylation on low density lipoproteins: comparative study before and after oxidative stress. Biochem Mol Biol Int. 1994;32:555-563. [Medline] [Order article via Infotrieve]
56. Myara I, Haberland ME, Demuth K, Chappey B, Moatti N. Susceptibility to copper oxidation of neuraminidase-treated LDL. Clin Chim Acta. 1995;240:221-223. [Medline] [Order article via Infotrieve]
57. Holvoet P, Perez G, Zhao Z, Brouwers E, Bernar H, Collen D. Malondialdehyde-modified low density lipoproteins in patients with atherosclerotic disease. J Clin Invest. 1995;95:2611-2619.
58. Sevanian A, Hodis HN, Hwang J, McLeod LL, Peterson H. Characterization of endothelial cell injury by cholesterol oxidation products found in oxidized LDL. J Lipid Res. 1995;36:1971-1986. [Abstract]
59.
Ji ZS, Brecht WJ, Miranda RD, Hussain MM, Innerarity
TL, Mahley RW. Role of heparan sulfate proteoglycans in the
binding and uptake of apolipoprotein E–enriched remnant lipoproteins
by cultured cells. J Biol Chem. 1993;268:10160-10167.
60. Parks JS, Gebre AK, Edwards IJ, Wagner WD. Role of LDL subfraction heterogeneity in the reduced binding of low density lipoproteins to arterial proteoglycans in cynomolgus monkeys fed a fish oil diet. J Lipid Res. 1991;32:2001-2008.[Abstract]
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Conxita De Castellarnau, J. L. Sanchez-Quesada, S. Benitez, R. Rosa, L. Caveda, L. Vila, and J. Ordonez-Llanos Electronegative LDL From Normolipemic Subjects Induces IL-8 and Monocyte Chemotactic Protein Secretion by Human Endothelial Cells Arterioscler Thromb Vasc Biol, October 1, 2000; 20(10): 2281 - 2287. [Abstract] [Full Text] [PDF]
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 O. Ziouzenkova, L. Asatryan, M. Akmal, C. Tetta, M. L. Wratten, G. Loseto-Wich, G. Jurgens, J. Heinecke, and A. Sevanian Oxidative Cross-linking of ApoB100 and Hemoglobin Results in Low Density Lipoprotein Modification in Blood. RELEVANCE TO ATHEROGENESIS CAUSED BY HEMODIALYSIS J. Biol. Chem., July 2, 1999; 274(27): 18916 - 18924. [Abstract] [Full Text] [PDF]
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G. Gardner, C. L. Banka, K. A. Roberts, A. E. Mullick, and J. C. Rutledge Modified LDL–Mediated Increases in Endothelial Layer Permeability Are Attenuated With 17ß-Estradiol Arterioscler Thromb Vasc Biol, April 1, 1999; 19(4): 854 - 861. [Abstract] [Full Text] [PDF]
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B. Vedie, X. Jeunemaitre, J. L. Megnien, I. Myara, H. Trebeden, A. Simon, and N. Moatti Charge Heterogeneity of LDL in Asymptomatic Hypercholesterolemic Men Is Related to Lipid Parameters and Variations in the ApoB and CIII Genes Arterioscler Thromb Vasc Biol, November 1, 1998; 18(11): 1780 - 1789. [Abstract] [Full Text] [PDF]
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B. Chappey, B. Beyssen, E. Foos, F. Ledru, J. L. Guermonprez, J. C. Gaux, and I. Myara Sialic Acid Content of LDL in Coronary Artery Disease: No Evidence of Desialylation in Subjects With Coronary Stenosis and Increased Levels in Subjects With Extensive Atherosclerosis and Acute Myocardial Infarction : Relation Between Desialylation and In Vitro Peroxidation Arterioscler Thromb Vasc Biol, June 1, 1998; 18(6): 876 - 883. [Abstract] [Full Text] [PDF]
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D. M. Smalley, N. Hogg, B. Kalyanaraman, and K. A. Pritchard Jr Endothelial Cells Prevent Accumulation of Lipid Hydroperoxides in Low-Density Lipoprotein Arterioscler Thromb Vasc Biol, December 1, 1997; 17(12): 3469 - 3474. [Abstract] [Full Text]
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I. Maor, T. Hayek, R. Coleman, and M. Aviram Plasma LDL Oxidation Leads to Its Aggregation in the Atherosclerotic Apolipoprotein E-Deficient Mice Arterioscler Thromb Vasc Biol, November 1, 1997; 17(11): 2995 - 3005. [Abstract] [Full Text]
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