extracellular matrix during the formation of atheroma- tous plaques and, therefore, it is likely that larger
amounts of oxLDL than those circulating in plasma accumulate in these regions [14]. Furthermore the LDL
is likely more heavily oxidized since the entrapment of LDL by the extracellular matrix together with the
presence of microenvironment conditions excluding plasma soluble anti-oxidants in the subendothelial
space creates ideal conditions for oxidation of LDL. Studies showing that LDL eluted from atherosclerotic
lesions has characteristics similar to those of copper-ox- idized LDL support this hypothesis [14].
In this study it was proposed to determine whether or not the expression of adhesion molecules by endothelial
cells is influenced by the degree of LDL oxidation. It was also determined whether or not the expression of
adhesion molecules by endothelial cells can be elicited by oxLDL located in the subendothelial space by im-
mobilizing oxLDL to type I collagen, one of the main components of the subendothelial matrix.
2. Material and methods
2
.
1
. Endothelial cells Human umbilical vein endothelial cells HUVEC
were purchased from Clonetics San Diego, CA and grown on 0.1 gelatin-coated flasks in Medium 199
M199, Life Technologies, Gaithersburg, MD supple- mented with 20 fetal bovine serum Sigma, St. Louis,
MO, endothelial cell growth supplement 30 mgml, Sigma, heparin 1 Uml, antibiotics and antimycotics
Penicillin G 100 Uml, Streptomycin 100 mgml, Am- photericin B 0.25 mgml, Sigma. HUVEC were used in
their fifth or sixth passage in all experiments.
2
.
2
. Isolation of nati6e LDL and lipoprotein deficient serum
Human native LDL nLDL, 1.019 B d B 1.063 gml was isolated from a plasma pool of healthy volunteers
by sequential ultracentrifugation at 60 000 rpm for 24 h at 10°C on a Beckman L-80 ultracentrifuge using a type
60-Ti rotor Beckman, Palo Alto, CA as previously described [15]. The isolated LDL was washed by ultra-
centrifugation, dialyzed against a 0.16 molL NaCl solution containing 300 mmoll EDTA, pH 7.4, passed
through a 0.45 mm filter Gelman Sciences, Ann Arbor, MI in order to sterilize and remove aggregates and
stored under nitrogen in the dark at 4°C.
Lipoprotein deficient serum LPDS was prepared from pooled plasma of healthy volunteers by ultracen-
trifugation at 100 000 × g, 10°C for 36 h on a Beckman L-80 ultracentrifuge, using a type 60-Ti rotor, after
adjusting density to 1.25 gml with KBr [15]. After ultracentrifugation the lipoprotein deficient plasma was
washed
by ultracentrifugation
and clotted
with thrombin 20 Uml and CaCl
2
2.5 mgml. Afterwards the serum was dialyzed against 0.16 M NaCl solution
containing 300 mmolL EDTA, pH 7.4 and sterilized by passage through a 0.45 mm filter.
2
.
3
. Oxidation of LDL Native LDL 1.5 mgml was passed through a PD-
10 column containing Sephadex G-25 M Pharmacia Biotech, Piscataway, NJ to remove EDTA and then
incubated at 37°C in the presence of 40 mM CuCl
2
. Oxidation of LDL was continuously monitored by
measuring the formation of conjugated dienes [16] and fluorescent compounds [17]. The oxidation was stopped
Fig. 1. Graphic representation of a 24 h continuous monitoring of conjugated dienes formation optic density 234 nm, solid line and of fluorescent compounds formation excitation 360 nm, emission 430 nm, broken line during low density lipoprotein LDL oxidation 1.5 mgml in the
presence of CuCl
2
40 mmoll. The arrows indicate the time points when the oxidation reaction was stopped and oxidized LDL oxLDL aliquots were collected. OxLDL collected at the time points depicted in the figure was used to stimulate the expression of adhesion molecules by human
umbilical vein endothelial cells HUVEC.
Table 1 Relative electrophoretic mobility of oxidatively modified low density
lipoprotein LDL
a
Oxidation time OxLDL
Relative electrophoretic h
preparations mobilityfold of mobility for
native LDL OxLDL-1
1 1.25 9 0.11
OxLDL-2 2
1.53 9 0.19 2.59 9 0.12
7 OxLDL-3
OxLDL-4 2.82 9 0.10
24
a
LDL was oxidized with 40 mM of Cu
2+
at 37°C for different times as indicated in Fig. 1. Levels of the relative electrophoretic
mobility are expressed as percent of the mobility of native LDL nLDL. The values represent mean 9 S.D. of the measurements of
three oxidized LDL oxLDL pools.
incubation time 24 h was determined, in pilot experi- ments, to allow maximal stimulation of ICAM-1
expression. The second experimental system was designed to
study the effects of sub-endothelial oxLDL on the expression of adhesion molecules. To perform these
studies, 96-well plates were incubated with rat tail type I collagen 1 mgml Becton Dickinson Labware, Bed-
ford, MA and 0.02 N NaOH at 37°C for 30 min to allow collagen polymerization to occur. After polymer-
ization of collagen, the plates were washed with PBS. After washing, culture medium containing either 10
mgml of LPDS or 10 mgml of LPDS to which 200 mgml of native or oxLDL were added was added to the
wells, according to protocol. Incubation was then car- ried out at 37°C for 24 h, which has been shown to be
the optimal time to achieve maximal diffusion of oxLDL into collagen [20]. After the incubation, the
medium was removed and 3 × 10
4
cells were seeded in each well and grown at 37°C for 24 h. Since the cells
were seeded at high density 93 750cm
2
, a confluent monolayer was obtained within 24 h.
To determine the amount of oxLDL and native LDL immobilized to collagen radiolabeled oxLDL and na-
tive LDL were used. It was found that the amount of immobilized oxLDL or immobilized native LDL, at the
concentrations tested 1 – 400 mgml, had a linear corre- lation with the concentrations of oxLDLnative LDL
used. An average of 1.5 of the total oxLDL or native LDL added to the wells coated with type I collagen was
immobilized. Therefore, 0.15 mg oxLDLnative LDL were immobilized after 50 ml of a 200 mgml of oxLDL
or native LDL were incubated with collagen at 37°C for 30 min. There was no difference, in the experimen-
tal conditions, between the percentage of binding of oxLDL and native LDL to collagen.
2
.
5
. Cell ELISA The expression of adhesion molecules on HUVEC
was determined by cell ELISA. After stimulation with oxLDL, HUVEC were washed with Dulbecco’s phos-
phate-buffered saline PBS twice and fixed with 0.025 glutaraldehyde for 10 min. After fixation, the cells were
washed once with PBS containing 0.05 Tween 20 PBSTween. Afterwards the cells were incubated at
room temperature for 1 h with 1 mgml of monoclonal antibody against ICAM-1, VCAM-1 or E-selectin
Pharmingen, San Diego, CA or with 1 mgml of a non-specific mouse IgG
1
Sigma in PBS containing 1 bovine serum albumin PBSBSA. The later was cho-
sen as a control since the monoclonal antibodies against ICAM-1, VCAM-1 and E-selectin are IgG
1.
After incubation, the cells were washed three times with PBSTween and incubated at room temperature for 1 h
with horseradish peroxidase-conjugated rabbit anti- at the times indicated in Fig. 1 by addition of 100 mM
EDTA and 200 mM butylhydroxytoluene. Two oxLDL preparations were collected during the period of conju-
gated dienes formation Fig. 1, one at the middle of conjugated dienes formation oxLDL-1 and another
when the formation of conjugated dienes reached its peak oxLDL-2. Two more LDL preparations were
collected during the formation of fluorescent com- pounds Fig. 1, one immediately after the formation of
the fluorescent compounds reached its peak oxLDL-3, and another 24 h after the oxidation was started or 16
h after the peak in fluorescence was reached oxLDL- 4. Each oxLDL preparation was dialyzed against a
solution containing 0.16 M NaCl and 300 mM EDTA and sterilized by passage through a 0.45 mm filter. The
electrophoretic mobility of all oxLDL preparations, as summarized in Table 1, was determined by agarose gel
electrophoresis [18] in barbital buffer, pH 8.6. The protein content of native LDL and oxLDL prepara-
tions was measured by the Lowry assay [19]. Endotoxin levels in the native and oxidized LDL preparations
were measured using an endotoxin assay kit Sigma. The endotoxin levels of the LDL preparations used in
the studies were found to be below the lower limit of detection 0.015 Uml.
2
.
4
. Stimulation of HUVEC by nati6e and oxidized LDL
To investigate the effects of native or oxidized LDL on the expression of adhesion molecules by HUVEC,
two in vitro experimental systems were employed. In the first experimental system, HUVEC 2 × 10
4
cells well were seeded in a 96-well plate coated with 0.1
gelatin and grown for 48 h to reach confluence. After reaching confluence the cells were washed with Dulbec-
co’s phosphate buffered saline PBS and incubated, at 37°C for 24 h, with culture medium M199 to which 10
mgml of LPDS and several concentrations 5 – 400 mgml of native or oxidized LDL were added. The
mouse IgG Organon Teknika Corporation, Durham, NC diluted at 1:5000 in PBSBSA. The cells were
washed again with PBSTween three times, and 2,2- azin-bis-3-ethylbenzthiazoline-6-sulfonic acid ABTS
with 0.003 H
2
O
2
was added to each well and incu- bated for 30 min. The absorbance was measured at 414
nm in a microplate reader. The optical densities of the wells incubated with non-specific mouse IgG
1
were subtracted from those of the wells incubated with mon-
oclonal antibody. Each experiment was performed in triplicate and repeated at least three times.
2
.
6
. Statistic analysis Data was presented as mean 9 S.E.M. Comparison
between treatments was performed using the one-way analysis of variance ANOVA. The Tukey – Kramer
multiple comparison test was used to compare the different treatments with each other. A value of P B
0.05 was considered significant.
3. Results
