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
3
.
1
. Characterization of the oxLDL preparations Four oxLDL preparations oxLDL-1 to oxLDL-4
were obtained by stopping the oxidative reaction cata- lyzed by copper chloride at different steps of the reac-
tion, as shown in Fig. 1. Approximately 2 h after the addition of copper chloride, the formation of conju-
gated dienes, used as a surrogate for lipid oxidation, reached its peak Fig. 1. Two LDL preparations were
collected during the period of conjugated dienes forma- tion, one at the middle of conjugated dienes formation
oxLDL-1 and another when the formation of conju- gated dienes reached its peak oxLDL-2. The forma-
tion of fluorescent compounds reached its peak approximately 7 h after addition of copper chloride. A
LDL preparation was collected at this stage oxLDL-3. Another preparation was obtained 24 h after starting
the oxidation reaction oxLDL-4. For all the experi- ments performed LDL obtained from three different
plasma pools was used. The pattern of oxidation of the three LDL preparations was similar although the maxi-
mum values of O.D. and fluorescence at peak levels were slightly different for each pool. Representative
results are shown in Fig. 1. Electrophoretic mobility of all oxLDL preparations used was also determined and
the data is summarized in Table 1.
3
.
2
. Concentration-dependent effect of oxLDL-
3
on ICAM-
1
expression by HUVEC To determine the concentration of oxLDL needed to
elicit maximal stimulation of ICAM-1 expression by HUVEC the cells were incubated with medium contain-
ing different concentrations of oxLDL-3. As depicted in Fig. 2, oxLDL-3 stimulated ICAM-1 expression in a
concentration-dependent manner. ICAM-1 expression was significantly increased P B 0.01 in the cells stimu-
lated with 50-200 mgml of oxLDL. The minimum concentration of oxLDL-3 that stimulated ICAM-1
expression was 5 mgml. Native LDL added at the same concentrations to the medium did not show a concen-
tration-dependent stimulation of ICAM-1 expression. Time course experiments from 2 to 48 h revealed that
oxLDL preparations led to maximal stimulation of ICAM-1 at 24 h. Thus all experiments thereafter were
performed with oxLDL concentrations above 50 mgml and an incubation time of 24 h.
3
.
3
. Influence of the degree of LDL oxidation on ICAM-
1
expression by HUVEC As depicted in Fig. 3, ICAM-1 expression was
slightly but significantly increased in cells incubated with medium containing any of the four oxLDL prepa-
rations. The levels observed, expressed as percentages of those obtained in cells incubated with medium alone,
were 113.8 9 7 for oxLDL-1, 132.3 9 4 for oxLDL- 2, 147.7 9 5 for oxLDL-3 and 123.0 9 7 for
oxLDL-4. Of the four oxLDL preparations, oxLDL-3 was the most effective in stimulating the expression of
ICAM-1.
Fig. 2. Concentration-dependent effect of oxidized low density lipo- protein LDL-3 on intercellular adhesion molecule-1 ICAM-1 ex-
pression by human umbilical vein endothelial cells HUVEC. HUVEC were incubated at 37°C for 24 h with medium alone or
medium containing different concentrations of native LDL or oxLDL-3 1 – 400 mgml. After the incubation the cells were washed
with Dulbecco’s PBS and fixed with glutaraldehyde. ICAM-1 expres- sion was then determined by cell ELISA using a monoclonal anti-
ICAM-1 antibody. The results presented are the mean 9 S.E.M. of three different experiments run in triplicate.
Fig. 3. Effects of oxidized low density lipoprotein oxLDL, at different stages of oxidation, on intercellular adhesion molecule-1
ICAM-1 expression by human umbilical vein endothelial cells HU- VEC. HUVEC were incubated at 37°C for 24 h with medium alone
or medium containing 100 mgml of native LDL or oxLDL-1, -2, -3 or -4 see Fig. 1. After the incubation the cells were washed with
Dulbecco’s PBS and fixed with glutaraldehyde. ICAM-1 expression was then determined by cell ELISA using a monoclonal anti-ICAM-1
antibody. The results presented are the mean 9 S.E.M. of three different experiments run in triplicate.
molecules other than ICAM-1 was increased when HU- VEC were incubated in the presence of oxLDL, HU-
VEC were incubated with medium containing 100 mgml of oxLDL-3 for 24 h and the expression of
VCAM-1 and E-selectin was measured. E-selectin ex- pression by HUVEC was significantly increased by
exposure to oxLDL-3 188.6 9 55.1 of control, P B 0.05. Expression of VCAM-1 was not stimulated by
oxLDL-3. Native LDL at the same concentrations and using the same experimental conditions did not stimu-
late the expression of E-selectin or VCAM-1.
3
.
5
. ICAM-
1
expression by HUVEC induced by oxLDL immobilized by type I collagen
In order to determine whether oxLDL located in the subendothelial space was able to stimulate the expres-
sion of ICAM-1, a novel experimental model was de- signed in which oxLDL 50 ml of 200 mgml oxLDL
preparation was added to type I collagen-coated wells prior to the seeding of HUVEC cells. Only 150 ng from
the 10 mg of oxLDL added to each well bound to collagen 1.5 of total oxLDL added. As shown in
Fig. 4, the expression of ICAM-1 by HUVEC exposed to type I collagen-bound oxLDL-3 or -4 was signifi-
cantly increased 147.2, P B 0.01, 127.0, P B 0.05, respectively. The stimulatory effect of oxLDL-4 was
less than that of oxLDL-3 P B 0.05. OxLDL-1 and -2, which stimulated ICAM-1 expression when added to
medium, did not significantly increase ICAM-1 expres- sion in these experiments. Also, none of the different
preparations of oxLDL oxLDL-1, -2, -3, and -4 when added to collagen stimulated the expression of VCAM-
1 and E selectin by HUVECs data not shown. As expected, native LDL did not stimulate the expression
of any of the adhesion molecules studied.
3
.
6
. Concentration-dependent effect of collagen-bound oxLDL-
3
on ICAM-
1
expression by HUVEC Type I collagen-bound oxLDL-3 stimulated ICAM-1
expression on HUVEC in a concentration dependent manner as depicted in Fig. 5. Maximal ICAM-1 levels
were observed when the HUVEC were seeded and grown on type I collagen gel pre-incubated in presence
of 200 mgml of oxLDL-3 for 24 h 140 of levels observed in cells incubated in presence of medium
alone. Type I collagen-bound native LDL did not significantly change ICAM-1 expression by HUVEC
Fig. 5. On average, as previously stated, 1.5 of oxLDL and native LDL remained attached to type I
collagen, as determined by incubation of radiolabeled LDL and oxLDL with type I collagen in the same
experimental conditions. Thus the amount of oxLDL, once immobilized to collagen, needed to stimulate
Fig. 4. Effect of oxidized low density lipoprotein oxLDL immobi- lized subendothelially on the expression of intercellular adhesion
molecule-1 ICAM-1. Two hundred mgml of native or oxLDL were incubated with type I collagen gel adhered to 96-well plates at 37°C
for 24 h. Human umbilical vein endothelial cells HUVEC were then seeded and incubated at 37°C for 24 h. After the incubation the cells
were washed with Dulbecco’s PBS and fixed with glutaraldehyde. ICAM-1 expression was then determined by cell ELISA using a
monoclonal antibody against ICAM-1. The data presented are the mean 9 SEM of three different experiments run in triplicate.
3
.
4
. Effects of oxLDL-
3
on VCAM-
1
and E-selectin expression by HUVEC
To determine whether the expression of adhesion
ICAM-1 expression by HUVEC is in the nanogram range.
3
.
7
. The stimulation of ICAM-
1
expression by oxLDL is not dependent on cytokine release
Since it has been shown that cytokines such as TNFa and IL-1b are potent stimulators for adhesion molecule
expression in endothelial cells [4], it is possible that oxLDL induces release of cytokines that stimulate
ICAM-1 expression in HUVECs. Thus, the effect of oxLDL on secretion of TNFa and IL-1b from HU-
VECs was examined. HUVECs were incubated with or without oxLDL for 24 h and secreted TNFa and IL-1b
in culture medium was measured by ELISA. The data showed that both TNFa and IL-1b in control medium
were undetectable by ELISA, and oxLDL did not stimulate TNFa and IL-1b secretion. This study indi-
cates that oxLDL-stimulated ICAM-1 expression is not cytokine-dependent.
4. Discussion
