the cytokine-induced expression of VCAM-1 in en- dothelial cells is altered when the apoAI in HDL is
replaced by the acute phase reactant, serum amyloid-A SAA.
SAA is synthesised by the liver during times of inflammation. Once released into the circulation, SAA
rapidly associates with HDL [5 – 7] in which it may account for up to 87 of HDL protein [8,9]. There
have been suggestions that SAA may be pro-athero- genic. For example, HDL enriched with SAA have a
reduced reactivity with lecithin:cholesterol acyltrans- ferase LCAT which may compromise their efficiency
as promoters of reverse cholesterol transport [10]. SAA- enriched HDL have also been reported to have a
decreased ability to protect LDL against oxidation [11]. Furthermore, SAA which is unassociated with HDL
has been shown to induce the migration, adhesion and tissue infiltration of monocytes, polymorphonuclear
leukocytes and T lymphocytes, and to increase the expression of cellular adhesion molecules [12]. It is not
known whether the expression of endothelial cell adhe- sion molecules is also promoted by the SAA, which is
associated with HDL. Nor is it known whether the presence of SAA in HDL interferes with the docu-
mented ability of HDL to inhibit the cytokine-induced expression of adhesion molecules in endothelial cells.
This latter issue is addressed in the studies described in this report.
2. Methods
2
.
1
. Isolation of high density lipoproteins Blood was collected from subjects by standard
venipuncture in tubes containing disodium EDTA final concentration 1 mgml after a 14 h fast and was placed
immediately on ice. In the subjects undergoing coro- nary artery bypass graft CABG surgery, blood was
collected on the day prior to surgery and again from the same subjects on the third post-operative day.
Blood was collected from healthy control subjects on two occasions, 4 days apart. Plasma was separated by
centrifugation at 4°C. HDL
3
were isolated in the den- sity interval 1.13 – 1.21 gml by sequential ultracentrifu-
gation at 4°C for 17 h at 100 000 rpm in a Beckman TLA 100.4 rotor using a Beckman TL-100 Ultracen-
trifuge; density adjustments were achieved by the addi- tion of solid KBr [13]. Purification was achieved by two
further centrifugations, one at the lower and the other at the higher density. Supernatants and infranatants in
each spin were recovered by tube slicing. The resulting preparations of HDL
3
were dialysed against either 3 × 1 L of endotoxin-free phosphate buffered saline pH
7.4 PBS or Tris-buffered saline TBS pH 7.4. Approval for collection of plasma samples from pa-
tients and normal controls was obtained from the Hu- man Ethics Committee of the Royal Adelaide Hospital.
2
.
2
. Characterisation of HDL HDL particle size distribution was assessed by elec-
trophoresis on 3 – 40 nondenaturing polyacrylamide gradient gels Gradipore, Sydney, Australia as de-
scribed previously [14]. Concentrations of total protein, apoAI, apoAII, triglyceride, total and unesterified
cholesterol and phospholipids were assayed as de- scribed elsewhere [15]. The SAA content of HDL
3
was estimated by two methods. i HDL
3
samples were subjected to SDS polyacrylamide gel electrophoresis.
The gels were then stained and scanned [8] and the percentages of SAA, apoAI and apoAII were calculated
from the integrated curves on the assumption that dye uptake is similar for each peptide and proportional to
apolipoprotein mass. ii HDL
3
were assayed directly for total protein, apoAI and apoAII, with SAA being
estimated by difference. All samples were assayed for SAA by both methods, with the two values never
varying by more than 8. The values presented repre- sent the means of the two methods.
2
.
3
. Isolation of SAA SAA was purified from the plasma collected from
patients 3 days after they had undergone surgery for coronary artery bypass grafting CABG. HDL
3
was isolated as above and delipidated [16]. The apoAI,
apoAII and SAA were separated by a modification of the method of Weisweiler [17]. The modification in-
volved using a salt gradient of 0.5 M NaCl from 0 – 50 over 3 h using a Superose 6 Pharmacia column contain-
ing Q-sepharose. A total of 100 mg protein apoAI, apoAII and SAA was loaded onto the column. The
apoAI, apoAII and SAA each appeared as a single band after electrophoresis on a 20 SDS-polyacryla-
minde gradient gel stained with Coomassie R 350. The purified SAA was dialysed against 3 × 5 L of 20 mM
NH
4
HCO
3
, lyophilised and stored at − 20°C until used. Prior to use, the SAA was reconstituted in 0.01 M
Tris – HCl, 3.0 M guanidine HCl, 0.01 wv EDTA- Na
2
pH 8.2 for 1 – 2 h and then exhaustively dialysed against TBS 5 × 1 l.
2
.
4
. Preparation of SAA-enriched HDL
3
in 6itro HDL
3
was isolated from healthy subjects and incu- bated in vitro with purified SAA at a mass ratio of
SAA:HDL
3
total protein of 2:1. As a control another aliquot of the HDL
3
was incubated in the absence of SAA. Both samples were maintained at room tempera-
ture for 30 min and then reisolated ultracentifugally as
the supernatant following ultracentrifugation for 17 h at 1.25 gml at 100 000 rpm in a Beckman TLA 100.4
rotor in a Beckman TL-100 Ultracentrifuge. The re- covered HDL
3
samples were dialysed against 3 × 1 l of endotoxin- free PBS before use.
2
.
5
. Preparation of discoidal reconstituted HDL Discoidal reconstituted HDL rHDL containing g-
palmityl-b-linoleoyl-phosphatidylcholine PLPC and either apoAI or SAA were prepared by the cholate
dialysis method described by [18].
2
.
6
. Expression of VCAM-
1
in human umbilical 6ein endothelial cells
Human umbilical vein endothelial cells HUVECs were isolated as described previously [2]. They were
cultured on gelatin coated culture flasks in medium M199 with Earles Salts Trace Biosciences, Australia
supplemented with 20 foetal calf serum FCS Commonwealth
Serum Laboratories,
Melbourne, Australia, 20 mmoll HEPES, 2 mmoll glutamine, 1
mmoll sodium pyruvate, non essential amino acids, penicillin, streptomycin, 20 mgml endothelial growth
supplement Collaborative Research, Australia and 20 mgml heparin Sigma. Confluent preparations of
passage three, four or five HUVECs were pre-incu- bated for 1 h or for 17 h in some experiments in
the presence of the various preparations HDL3. TNF-a 100 Uml RD Systems, USA was then
added to the culture medium and 5 h later the cell surface expression of VCAM-1 was measured by flow
cytometry analysis.
2
.
7
. Flow cytometry analysis The expression of VCAM-1 on the surface of HU-
VECs was measured as described previously [2]. In brief, the HUVECs were first washed with FACS
wash RPMI 1640, containing 10 mmoll HEPES, 0.02 azide and 2.5 foetal calf serum. They were
then incubated with mouse monoclonal antibody to VCAM-1 51-10C9 for 30 min at 4°C, then washed
again with FACS wash. Binding of the primary anti- body was detected by incubating with FITC conju-
gated
secondary antibody
Immunotech FITC
conjugated Fab2 fragment goat mouse IgG for 30 min at 4°C. After washing with PBS, the cells were
harvested by trypsinization. FACS wash was added to neutralise the trypsin. The cells were centrifuged and
the cell pellet resuspended in FACS fixative PBS containing 2 glucose, 0.02 azide and 2.5 formal-
dehyde. The expression of VCAM-1 was measured as fluorescence intensity using a Coulter Epics Profile II
flow cytometer Coulter, USA.. A total of 10 000 cells was counted for each sample. Controls included
absence of primary antibody and substitution of an isotype-matched, nonrelevant antibody.
2
.
8
. Labeling of HDL Unmodified HDL
3
and the HDL
3
that were en- riched with SAA during incubation in vitro were la-
belled with Na
125
I using the iodo-bead iodination method according to the manufacturers’ instructions.
Free
125
I was removed by gel filtration through Sep- hadex 6-25M PD10 column, Pharmacia Biotech;
samples were then further dialysed against 0.05M Tris – HCl, pH 7.4. Specific activities of metabolised
HDL ranged from 70 – 110 cpmng protein.
2
.
9
. Binding studies The binding studies were conducted with bovine
aortic endothelial cells BAECs. The BAECs were prepared from primary culture and characterised as
previously described [19]. Cells were grown in 75cm
2
flasks in RPMI 1640 modified medium ICN supple- mented with 10 foetal calf serum. 4 mM
L
-glu- tamine
and 1
penicillinstreptomycin antibiotic
solution ICN. Cells were incubated at 37°C in a 5 CO2, 95 air, water-jacketed incubator.
Cells were seeded into 12 well micro plates at 1 – 2 × 10
5
cells per well and when 90 confluent, the 3ml of medium RPMI with 10 FBS was replaced
with 1ml serum free RPMI and incubated for 2 – 3 h to remove adherent lipoproteins. After washing the
cells once with serum free RPMI, binding studies were commenced by the addition of 1 ml fresh RPMI
serum free containing varying amounts of
125
I-la- belled HDL preparations or unlabelled HDL see Sec-
tion 3 and incubated for 3 h at 37°C. To determine bound HDL, the medium was removed, cells were
washed four times with 0.1 BSA in PBS and twice with PBS. Surface bound lipoproteins were removed
by adding 1 ml 0.05 wv trypsinEDTA ICN to each well and the cells were quickly detached by gen-
tle scraping and pelleted by centrifugation at 1000 × g for 5 min in a Heraeus Christ Minifuge GL. Surface
bound lipoproteins were collected in the supernatant trypsin releasable and internalised lipoproteins were
released from the cell pellet trypsin resistant HDL using 1 ml 0.1 M NaOH to dissolve cell material.
2
.
10
. Statistical analyses Students’ t test for paired samples was used to de-
termine whether differences between values were sig- nificant. Statistical significance was set at P B 0.05.
Table 1 Composition and particle size of the different preparations of native HDL
3
Percentage by mass Phospholipid
Unesterified cholesterol Cholesteryl
Triglyceride Total protein
Particle diameter esters
nm CABG patients
n =
19
24.0 9 0.5 2.6 9 0.2
16.6 9 0.6 Preoperative
a
3.9 9 0.3 52.9 9 0.5
8.5 9 0.1 26.3 9 0.4
3.1 9 0.2 11.9 9 1.0
6.1 9 0.6 52.7 9 0.3
9.7 9 0.1 Postoperative
b
Normal controls n =
6
c
23.5 9 0.3 2.4 9 0.5
18.6 9 0.8 3.4 9 0.6
Sample 1 8.7 9 0.1
52.2 9 0.4 21.3 9 1.3
2.2 9 0.5 17.3 9 1.2
2.9 9 0.5 8.6 9 0.1
56.4 9 2.7 Sample 2
a
Samples of HDL
3
were isolated from each of 19 patients on the day before they underwent coronary artery bypass graft CABG surgery.
b
Another sample was taken from each subject 3 days postoperatively for isolation of a second preparation of HDL
3
.
c
Samples of HDL
3
were also collected on two separate occasions 4 days apart from normal control subjects. All values represent the means 9 SEM.
pB0.05 for the difference between the two samples.
3. Results
