junction communication, induction of carcinogen-me- tabolizing enzymes, and photoprotection, in addition to
their antioxidant properties [6]. More than 600 distinct carotenoids have been isolated from natural sources
and among these approxim. 50 are components of the US diet, with identification of 21 carotenoids in human
plasma [7]. There are five well-characterized carotenoids in most blood samples comprising approximately 90
of the total plasma pool of carotenoids. These are a-carotene, b-carotene, b-cryptoxanthin, lutein and ly-
copene [8].
Regulated expression of numerous cell surface adhe- sion molecules is a critical event in the binding of
normally non-thrombogenic circulating leukocytes such as the monocyte to the aortic endothelial surface and is
one of the earliest detectable events in human and experimental atherosclerosis [9,10]. The subsequent
transendothelial migration of these adherent leukocytes, their accumulation in the aortic intima, transformation
of monocytes into lipid-engorged foam cells, and secre- tion of cytokines and growth factors are important
events in the initiation and progression of atheroscle- rotic plaques [11]. Cell surface adhesion molecules are
important regulators of direct cell – cell interactions; inflammatory responses in atherogenesis are directed by
regulation and expression of these molecules [12]. Ad- hesion molecule expression in low-density lipoproteins
LDL receptor-knockout mice fed atherogenic diets showed a significantly reduced incidence of fatty streaks
supporting a role for adhesion molecules in atherogene- sis [13]. Constituents of each of the main families of
adhesion molecules are involved in the interactions of endothelial cells EC and immune cells. Intercellular
adhesion molecule ICAM-1 and -2 CD54 and CD102 and vascular cell adhesion molecule VCAM-
1, CD106 are members of the immunoglobulin super- family expressed on EC. Of these, ICAM-1 and
VCAM-1 increase in response to various inflammatory cytokines. E-Selectin and P-selectin CD62E and
CD62P on EC also play an early role in adhesion between these two cell types. ICAM-1 and -2 bind to
the LFA-1 counterligand; VCAM-1 binds to VLA-4; the selectins recognize certain carbohydrate structures
on opposing cells [14,15].
Oxidative stress
and expression
of adhesion
molecules on vascular EC are considered to be impor- tant features in the pathogenesis of atherosclerosis and
other inflammatory diseases [16,17]. Studies suggest a molecular linkage between an antioxidant-sensitive
transcriptional regulatory mechanism and expression of adhesion molecule genes that expands on the notion of
oxidative stress as an important regulatory signal in the pathogenesis of atherosclerosis [18]. In the inflamma-
tory response of the arterial wall, leukocyte recruitment to the endothelium is mediated by the interaction of
adhesion molecule receptors expressed on the surface of EC and immune cells.
While the pathogenic oxidation of LDL is currently one
postulated mechanism
for the
etiology of
atherosclerosis, the role of dietary antioxidants in sup- pressing the deleterious oxidation of LDL has produced
conflicting results, suggesting that other factors and possibly alternate mechanisms are involved. The spe-
cific contribution of carotenoids in the atherosclerosis process is inconclusive since carotenoids have been
shown to protect LDL in some studies but not in others. In fact, the extent of in vitro LDL oxidation
and protection by an antioxidant may not indicate in vivo atherosclerotic status as well as was recently em-
phasized [19]. However, in one study b-carotene supple- mentation in rabbits retarded aortic lesion formation
[20]. Also, in a more recent study b-carotene supple- mentation in combination with vitamins E and C re-
duced
aortic valve
lesion formation
in LDL
receptor-knockout mice [21], which may indicate a po- tential effect by carotenoids in the final pathological
outcome. Our contention is that carotenoids may act via alternate mechanisms within the endothelial cell to
modulate adhesion molecule expression and thus reduce subsequent leukocyte binding. As a result, the focus of
this research was to examine in vitro the effect of the five most prevalent plasma carotenoids on expression of
key adhesion molecules involved in the atherosclerosis process, and determine the subsequent binding of U937
monocytic cells when carotenoids are incorporated into human aortic endothelial cells HAEC. Our results
indicate that among the test carotenoids, lycopene ap- pears to be the most effective in reducing immune and
endothelial cell interaction.
2. Materials and methods
2
.
1
. Human aortic endothelial cell and U
937
cell cultures
HAEC were purchased from Clonetics Laboratories San Diego, CA and cultured in M-199 medium
Gibco, Grand Island, NY. Culture medium contained 10 fetal bovine serum FBS Sigma, St. Louis, MO,
5 mgml EC-derived growth factor, 100 Uml heparin, 100 Uml penicillin, 100 Uml streptomycin, and 1.25
mgml amphotericin B Sigma. HAEC were cultured on 1 gelatin-coated flasks in Corning 24-well and 96-well
plates, and T-25 and T-75 flasks Corning, NY. The medium was replaced every second day until the cells
attained confluence. Fourth to eighth passage cells were employed, and all experiments conducted within 72 h
post-confluence using triplicate and quadruplicate wells per experimental treatment. Under inverted microscopy
Zeiss, West Germany, confluent HAEC monolayers displayed a cobblestone phenotype typical of quiescent
EC and were characterized by the presence of von
Willebrand factor antigen using immunofluorescent mi- croscopy. Cells were grown to confluence in a humi-
dified atmosphere of 95 air and 5 CO
2
. Following treatment of HAEC with 0.05 trypsin for 1 – 2 min or
until 80 of the cells were detached, cell viability was determined by trypan blue exclusion and expressed as
the percent of cells that excluded the dye. The mono- cytic U937 cell line was originally derived from a
human histiocytic lymphoma and was procured from American Tissue Type Collection Rockville, MD.
U937 cells were seeded at 5 × 10
5
cellsml, grown in suspension culture in RPMI-1640 medium containing
10 FBS, 100 Uml penicillin, and 100 Uml strepto- mycin, and routinely subcultured at a 1:5 ratio two
times per week.
Confluent HAEC grown in 24-well plates were sup- plemented with carotenoids by incubating them in
medium alone or containing 1 mmoll a-carotene, b-carotene, b-cryptoxanthin, lutein or lycopene for 24
h. Based on our pilot dose response studies and the absence of any apparent pro-oxidant effects, we se-
lected 1 umoll as a biologically relevant test concentra- tion. Following supplementation, supernatant was
removed, HAEC were washed with phosphate buffered saline PBS, and medium alone or containing 5 ngml
human recombinant interleukin IL-1b Pharmingen, San Diego, CA was added to wells for 6 h to activate
cells. Expression of adhesion molecules and adhesion of U937 monocytes were determined see below.
2
.
2
. Carotenoid supplementation Stock solutions of carotenoids containing 5 mmoll
were prepared in absolute ethanol and stored under a nitrogen blanket in amber tubes at -70°C. To supple-
ment the culture media with carotenoids, the required amount of carotenoid was transferred from stock solu-
tion and dried under nitrogen. Carotenoids were redis- solved in tetrahydrofuran THF to achieve a final
concentration of 0.7 vv in the culture medium. Carotenoids were then mixed with aliquots of FBS and
incubated at 37°C for 15 min with gentle mixing by inversion every 5 min. The HAEC were incubated for
24 h in medium containing vehicle or the indicated carotenoid incorporated into the FBS.
2
.
3
. Carotenoid determination Cellular association of test carotenoids by HAEC
and concentrations in media were measured by high performance liquid chromatography HPLC. Briefly, 2
mg echinenone gift from Hoffmann-La Roche, Nutley, NJ was added to homogenates of HAEC cells or
media as an internal standard. Aliquots 0.5 ml of HAEC homogenates were saponified in the presence of
2 pyrogallol Sigma at 70
o
C for 30 min after addition of 30 KOH 0.1 ml. After extraction of cells or
medium with hexane containing 0.02 butylated hy- droxytoluene BHT, the samples were dried under
nitrogen and reconstituted in absolute ethanol 0.04 ml. Carotenoid peaks were separated according to the
method of Yeum et al. [22] using a Pecosphere C18 reverse phase column 0.46 × 8.3 cm × 3 mm protected
by a Perkin-Elmer Norwalk, CT guard column 0.46 × 3.3 cm × 3 mm. The mobile phase was acetoni-
trile-tetrahydrofuran-water 50:20:30: vvv, solvent A, and 50:44:6, vvv, solvent B, with 1 ammonium
acetate in water. The gradient procedure at a flow rate of 1 mlmin was as follows: 60 solvent A and 40
solvent B were used for 1 min followed by a 9 min linear gradient to 83 solvent B, a 4 min hold at 83
solvent B, then a 2 min linear gradient to 100 solvent B, a 2 min hold at solvent B, and finally a 2 min
gradient back to 60 solvent A and 40 solvent B. The HPLC system was equipped with a Waters 490E pro-
grammable multiwavelength detector Waters Corpora- tion, Milford, MA with the wavelength set at 450 nm;
peaks were integrated using the Waters Millenium chro- matography system.
2
.
4
. Antibodies Purified mouse anti-human monoclonal antibodies
Pharmingen were used to quantify the expression of VCAM-1. Mab 51-10C9 VCAM-1 CD 106; mouse
IgG1, k reacts with the 110 kD glycoprotein of VCAM-1 CD106, also known as INCAM-110, that is
expressed at high levels on the surface of cytokine-stim- ulated endothelium. Mab 68-5H11 reacts with the 97 –
115 kD cell surface glycoprotein E-selectin CD62E also known as endothelial-leukocyte adhesion molecule-
1 ELAM-1 that is expressed on cytokine-stimulated endothelium and is thought to be involved in the early
interaction of leukocytes with inflamed endothelium. Mab HA58 reacts with the 85 – 110 kD integral mem-
brane glycoprotein ICAM-1 CD54 that is expressed on EC and both resting and activated lymphocytes and
monocytes.
2
.
5
. Enzyme-linked immunosorbent assay ELISA
for adhesion molecule expression
Endothelial cells were cultured in 96-well plates 3.2 × 10
3
cells200 mlwell until confluent, as described above. After 24 h pre-incubation with carotenoids,
cultures were washed PBS with 0.5 sodium dodecyl sulfate, SDS, fixed for 30 min with 1 paraformalde-
hyde in PBS containing 2 FBS and incubated with blocking buffer PBS containing 10 FBS for an
additional 60 min. After washing, monoclonal primary antibodies were added by incubating with blocking
buffer containing either VCAM-1 2.5 mgml, E-se-
lectin 2.5 mgml or ICAM-1 1 mgml for 2 h at 25°C. After thorough washing, horseradish peroxidase-conju-
gated goat anti-mouse antibody Bio-Rad, Hercules, CA was diluted and added to the fixed monolayers.
After 1 h, cultures were washed and peroxide substrate Bio-Rad, Hercules, CA added for 30 min with subse-
quent addition of stop solution 48 N,N-dimethylfor- mamide
containing 20
SDS. After
color development, absorbances were determined at 405 nm
to estimate relative adhesion molecule expression.
2
.
6
. Assay for U
937
monocytic adhesion to HAEC Adhesion of U937 cells to HAEC cultures was deter-
mined as previously described [23,24]. Briefly, HAEC were plated into 24-well plates and grown to confluence
before experimental use. At 100 confluence, M199 alone or containing the indicated concentration of
carotenoid was added, and the monolayers were incu- bated for 24 h. After washing to remove unincorpo-
rated carotenoid, M199 alone or containing IL-1b 5 ngml was added for 6 h at 37°C. U937 cells 10
7
were labeled for 60 min at 37°C with 100 mCi
51
Cr as sodium chromate New England Nuclear, Boston, MA in 1 ml
RPMI-1640 containing 10 FBS. The labeled cells were washed by centrifugation, resuspended in RPMI-
1640, and 10
5
viable cells were added per well after washing the HAEC monolayers to remove cytokine.
After 10 min at 37°C to allow binding, non-adherent
51
Cr-U937 cells were removed by repeated washes with PBS, and the cells HAEC and bound U937 lysed by
addition of 0.5 ml harvest solution PBS containing 0.1 SDS and 1 mM EDTA. After mechanical disrup-
tion, each solution was transferred to a 5 ml polypropy- lene tube for quantitation of total radiolabel associated
with each well by g-ray spectroscopy Canberra, Syd- ney, Australia. The number of U937 cells bound to
endothelial cells was determined from the specific activ- ity cpmcell of the initial cell suspensions and is
expressed as the percentage of added cells that are bound to HAEC. Spontaneous release of
51
Cr by
51
Cr- U937 was less than 5 during the binding phase of the
assay. Experiments were repeated 2 – 3 times with each performed in triplicate or quadruplicate.
2
.
7
. Statistical analysis Significant differences in the measured parameters
between treatment groups were calculated using the unpaired, two-tailed Student’s t-test. Values are pre-
sented as means 9 SEM and P values B 0.05 were considered significant.
3. Results
