Atherosclerotic plaques develop from complex multi- cellular processes in which recruitment of circulating
monocytes to focal areas of the arterial sub-endothe- lium is an early event. Localized upregulation of adhe-
sive endothelial cell adhesion molecules CAMs, a prerequisite for monocyte adherence and migration
[12], is a dynamic process which is sensitive to inflam- matory cytokines, shear stress and oxidative insults.
Levels of certain CAMs are elevated in human atherosclerotic
tissue with
vascular cell
adhesion molecule-1 VCAM-1, intercellular adhesion molecule-
1 ICAM-1, E-selectin and P-selectin, in particular, being indicators of inflammation and early atheroscle-
rosis [13 – 15]. VCAM-1, a member of the immunoglob- ulin
superfamily of
CAMs, aids
the selective
accumulation of monocytes and T-lymphocytes and is a hallmark of early atherogenesis [16 – 18].
It has been reported that levels of cytokine-induced VCAM-1, ICAM-1 and E-selectin were reduced by
HDL in human umbilical vein endothelial cells HU- VECs [19], a popular and relatively accessible in vitro
model for CAM research [20]. Here, we have investi- gated whether HDL can downregulate CAM expression
in primary human coronary artery endothelial cells HCAECs, a model directly relevant to blood vessels
susceptible to atherosclerosis. Arterial endothelial cells were preincubated with increasing amounts of total
HDL, or different subfractions, and then activated with the inflammatory cytokine, tumor necrosis factor-alpha
TNF-a. However, flow cytometric analysis failed to detect any downregulation of VCAM-1 or E-selectin
expression by HDL, implying that suppression of CAM induction in arterial endothelium is unlikely to con-
tribute to HDL anti-atherogenic effects in vivo.
2. Materials and methods
2
.
1
. Materials O-Phenylenediamine OPD substrate tablets, IgG
1
negative control antibodies, von Willebrand Factor vWF antibodies clone F886, StreptABComplex
HRP Duet kit, fluorescein isothiocyanate FITC-con- jugated antibodies and R-phycoerythrin RPE-conjug-
ated antibodies were from DAKO High Wycombe, UK.
Monoclonal antibodies
against VCAM-1
CD106; clone BBIG-V1, ICAM-1 CD54; clone BBIG-I1, E-selectin CD62E; clone BBIG-E4 and
platelet endothelial CAM-1 or PECAM-1 CD31; clone 9G11 were from RD Systems Abingdon, UK.
Other antibodies used for endothelial cell characteriza- tion were thrombomodulin clone QBEnd40 and en-
dothelial cell CD34 clone QBEnd10 [21], both from Quantum Biosystems Waterbeach, Cambridge, UK.
M199 media, trypsin-EDTA, glutamine and penicillin streptomycin were purchased from Life Technologies
Paisley, UK. S-Nitroso-
L
-glutathione GSNO was supplied by Alexis Nottingham, UK. Recombinant
human TNF-a and other chemicals or tissue culture reagents were from Sigma Poole, UK.
2
.
2
. Cell culture HCAECs were isolated from the vessels of four pa-
tients with dilated cardiomyopathy at the time of ortho- topic heart transplantation. Informed consent was
obtained from each patient and the study was approved by the Ethical Committee of Harefield Hospital. The
methods used and culture conditions were as described previously [22,23]. In brief, the endothelium was
scraped off the vessel lumen, collected in M199 media and cultured in plates pre-coated with 1 gelatin.
Growth media was M199, supplemented with 2 mmoll glutamine, 100 IUml penicillin, 100 mgml strepto-
mycin, and 15 vv foetal calf serum FCS and 15 vv human AB serum. Cells were used between pas-
sages 3 and 10 and the purity of the population was routinely confirmed by positive staining for PECAM-1,
negative staining for smooth muscle a-actin and by uptake
of DiI-acetylated
LDL Biogenesis,
Bournemouth, UK. HUVECs were isolated and cul- tured as described previously [24]. Cultures were exten-
sively characterized on the basis of ‘cobblestone’ morphology and by expression of endothelial antigens:
vWF, CD34, thrombomodulin, PECAM-1 and ICAM- 1 [24]. Experiments were carried out in M199 media
containing 3.6 mmoll glutamine, 100 IUml penicillin, 100 mgml streptomycin and 20 vv FCS. Cells were
used between passages 2 and 4.
2
.
3
. Preparation of HDL Blood was freshly drawn into EDTA-containing
tubes from normolipaemic volunteers in the morning, a few hours after their previous meal although some
subjects were fasted overnight. Sequential, isopycnic ultracentrifugation was used to isolate total plasma
HDL d, 1.063 – 1.21 gml or HDL
2
d, 1.063 – 1.125 gml and HDL
3
d, 1.125 – 1.21 gml [25]. All fractions were washed by recentrifugation at the limiting density
and then extensively dialysed against Dulbecco’s phos- phate buffered saline PBS. In some cases, preparation
time was reduced by precipitating apo B-containing lipoproteins prior to isolating total HDL at d = 1.21
gml [26] and by rapid desalting with a PD-10 column Amersham Pharmacia Biotech, St Albans, UK. All
HDL was used on the day of preparation or, if stored at 4°C, within 2 weeks; immediately before addition to
cells, samples were further dialysed against supple- mented M199 media excluding FCS, sterile filtered
and assayed for total protein concentration Bradford
reagent; Bio-Rad Laboratories, Hemel Hempstead, UK.
2
.
4
. Flow cytometry analysis of cell-surface CAMs FACS analysis for cell-surface VCAM-1 and E-se-
lectin was carried out on confluent HCAEC and HU- VEC monolayers as described previously [22,24]. Cells
were preincubated for either 1 or 16 h with HDL 0.25 – 2 mg HDL proteinml in serum-containing me-
dia, washed with warm HDL-free media and then stimulated for 4 h with TNF-a 10 ngml for HCAECs
and 5 ngml or 100 Uml for HUVECs. For positive controls showing VCAM-1 downregulation, cells were
preincubated for 40 h with 10 mmoll 17b-estradiol prior to TNF-a addition [24]. Primary antibody binding
and negative control antibody binding at equivalent IgG
1
protein concentrations was detected using either goat anti-mouse FITC- or RPE-conjugated antibodies,
and 5 × 10
3
cells were analysed per well by FACS Coulter Epics Elite or Epics XL-MCL; Coulter,
Hialeah, FL.
2
.
5
. Cell-associated VCAM-
1
ELISA in HUVECs The ELISA for measuring total VCAM-1, that is
both cell-surface and intracellular VCAM-1, by fixation prior to antibody incubations, was carried out as de-
scribed previously [24]. HUVECs were grown in 96-well plates pre-coated with 1 gelatin wv until confluent,
preincubated for 16 h with HDL 1.5 mgml and then, after washing with warm media to remove HDL,
treated with 100 Uml TNF-a or suboptimal concen- trations as indicated for a further 6 h. Variations to
this protocol included the use of serum-free media supplemented with bovine serum albumin BSA 1
wv and the replacement of TNF-a with interleukin-1b IL-1b 1 ngml or 100 Uml or lipopolysaccharide
LPS 5 mgml or 50 Uml. For positive controls of VCAM-1 downregulation, parallel wells were preincu-
bated with 17b-estradiol 10 mmoll prior to TNF-a addition [24] or were coincubated with S-nitroso-
L
-glu- tathione GSNO 200 mmoll. Any binding of mouse
anti-human monoclonal VCAM-1 antibodies or iso- type-matched irrelevant antibodies at equivalent IgG
1
protein concentrations was detected by the sensitive StreptABComplexHRP Duet kit and OPD chro-
mogenic substrate. Absorbances were read at 492 nm in a Titertek Multiskan MCC340 plate reader against
blank substrate. The cell protein per well was measured using the Bradford reagent.
2
.
6
. Statistical analysis The statistical analysis of independent experiments
was performed using Student’s t-test; results are shown as mean 9 S.E. and P B 0.05 was considered significant.
3. Results
