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
3
.
1
. Inhibition of HUVEC VCAM-
1
expression by HDL
:
effect of the in 6i6o enrichment of HDL
3
with SAA
Preparations of HDL
3
were isolated on two separate occasions from the plasma of each of 19 subjects: the
first sample was collected 1 day before and the second 3 days after undergoing coronary artery bypass graft
surgery. The total protein content of the HDL
3
was unaffected by the surgery Table 1. The composition of
the protein, however, was considerably changed Table 2. Whereas the preoperative HDL
3
sample contained no SAA, in the postoperative sample SAA had replaced
a proportion of both the apoAI and apoAII and ac- counted for an average of 42 of the HDL
3
protein Table 2. The postoperative HDL
3
were also larger, were enriched in unesterified cholesterol, triglyceride
and phospholipids and were depleted in cholesteryl esters when compared with the preoperative samples
Table 1. As a control, samples of HDL
3
were also isolated on two separate occasions 4 days apart from
each of six healthy volunteers who did not undergo surgery. In contrast to the surgical patients, the compo-
sition and size of the HDL
3
isolated from the normal subjects did not change over the 4 days Tables 1 and
2. The preoperative HDL
3
and postoperative SAA-en- riched HDL
3
were compared in terms of their ability to inhibit the TNF-a-induced expression of VCAM-1 in
HUVECs. Both samples inhibited VCAM-1 expression in a concentration dependent manner Fig. 1. Further-
more, when added at equal cholesterol concentrations, the extent of inhibition of VCAM-1 expression was
identical for the two samples. The samples of HDL
3
obtained from control subjects also inhibited VCAM-1 expression in a concentration dependent manner, with
no significant difference in the degree of inhibition between the first and second samples of HDL
3
Fig. 2.
3
.
2
. Inhibition of HUVEC VCAM-
1
expression by HDL
:
effect of enriching HDL
3
with SAA in 6itro To assess the effect of having an even greater propor-
tion of the HDL
3
protein replaced by SAA, samples of HDL
3
were incubated in vitro with purified SAA. This resulted in SAA replacing almost all of the apoAI and
about 40 of the apoAII; SAA accounted for an average of 81.5 of the protein in these in vitro
modified HDL
3
Table 3. The in vitro incorporation of SAA into HDL
3
was associated with an increase in the particle size from 8.8 to 10.2 nm Table 3, comparable
Table 2 Protein composition of the different preparations of native HDL
3
Percentage by mass ApoAI
ApoAII SAA
CABG patients n =
19
Preoperative
a
74.8 9 1.3 25.2 9 1.3
44.9 9 1.7 12.3 9 1.0
42.2 9 1.4 Postoperative
b
Normal controls n =
6
c
28.5 9 3.3 71.5 9 3.3
Sample 1 74.5 9 2.9
25.5 9 2.8 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 postopera- tively 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 ignore any
contribution made by other minor HDL apolipoproteins. Values represent means 9 SEM.
PB0.05 for the difference between the two samples.
Fig. 1. The Effect of preoperative HDL
3
and post-op SAA-HDL
3
on VCAM-1 expression in HUVECs. HUVECs were preincubated for 1
h with preoperative HDL
3
and postoperative SAA-enriched HDL
3
before being activated with TNF-a 100 Uml and incubated for a further 5 h. The expression of VCAM-1 was quantitated by flow
cytometry. Values are presented as a percentage of that in a sample without HDL. The preoperative HDL
3
and postoperative SAA- enriched HDL
3
2 were added to the HUVECs according to total cholesterol concentration. The results are expressed as the mean and
standard error of the mean, n = 19. Table 3
Protein composition of in vitro modified native HDL
3 a
Percentage by mass ApoAI
ApoAII SAA
Unmodified 72.9 9 1.6
27.1 9 1.6 HDL
3
81.5 9 1.7 17.2 9 1.3
1.4 9 0.4 SAA-enriched
HDL
3 a
Samples of HDL
3
were isolated from each of three normal subjects and incubated in vitro with lipid-free SAA which displaced
both apoAI and apoAII from the particles. Values represent the means 9 SEM.
manner Fig. 3. As in the studies using HDL
3
enriched with SAA in vivo, the in vitro SAA-enriched HDL
3
were identical to the unmodified HDL
3
in terms of the magnitude of the inhibition.
In order to determine whether there would be an effect of SAA if there was a longer preicubation of the
HUVECs with the HDL
3
, the duration of the preincu- bation HUVECs was increased from 1 to 17 h. The
longer preincubation was associated with a greater de- gree of VCAM-1 inhibition for a given cholesterol
concentration of each preparation Fig. 4. However, as with the shorter preincubations, there was no difference
between the two preparations in terms of the degree of the inhibition.
to the increase in HDL
3
size associated with SAA enrichment in vivo.
The unmodified HDL
3
and the in vitro SAA-enriched HDL
3
both inhibited the TNF-a-induced expression of VCAM-1 in HUVECs in a concentration dependent
Fig. 3. The effect of enriching HDL
3
with SAA during in vitro incubation on cytokine-induced VCAM-1 expression in HUVECs.
SAA-enriched HDL
3
was prepared in vitro by displacing apoAI with lipid-free SAA as descibed in Methods. HUVECs were preincubated
for 1 h with either the unmodified HDL
3
or the SAA-enriched HDL
3
before being activated with TNF-a 100 Uml and incubated for a further 5 h. The expression of VCAM-1 was quantitated by flow
cytometry. Values are presented as a percentage of that in a sample without HDL. The unmodified HDL
3
and in vitro SAA-HDL
3
were added to the HUVECs according to total cholesterol concentration. The results are presented as the mean and standard
error of the mean from three experiments, where each was performed in duplicate.
Fig. 2. The effect of samples of HDL
3
collected 4 days apart from normal subjects on VCAM-1 expression in HUVECs. HUVECs were
preincubated for 1 h with pre HDL
3
and post HDL
3
before being activated with TNF-a 100 Uml and incubated for a further 5 h.
The expression of VCAM-1 was quantitated by flow cytometry. Values are presented as a percentage of that in a sample without
HDL. The first sample of HDL
3
and the second sample of HDL
3
2 were added to the HUVECs according to total cholesterol concentration. The results are presented as the mean and standard
error of the mean, n = 6.
Fig. 4. The effect of longer preincubation with unmodified and SAA-enrched HDL
3
on the inhibition of VCAM-1 expression in HUVECs. SAA-enriched HDL
3
was produced in vitro as described in the legend to Fig. 3. HUVECs were preincubated for 17 h with either
unmodified HDL
3
or with the SAA-enriched HDL
3
before being activated with TNF-a 100 Uml and incubated for a further 5 h.
The expression of VCAM-1 was quantitated by flow cytometry. Values are presented as a percentage of that in a sample without
HDL. The unmodified HDL
3
and SAA-enriched HDL
3
were added to the HUVECs according to total cholesterol concentration.
The results are presented as the mean values obtained in a single experiment, which was performed in duplicate.
3
.
3
. Inhibition of HUVEC VCAM-
1
expression by HDL
:
effects of discoidal rHDL containing apoAI or SAA
As a final investigation of whether SAA influenced the inhibitory activity of HDL, studies were conducted
with discoidal rHDL which contained PLPC as the sole lipid component and SAA or apoAI as the sole protein
component. The AI-rHDL were 26 protein and 74 phospholipid and, as reported previously [13], com-
prised a homogeneous population of particles with of diameter 9.4 nm as determined by non-denaturing gra-
dient gel electrophoresis results not shown. The SAA- rHDL were 28 protein and 72 phospholipid but,
unlike the AI-rHDL, were heterogeneous in size, con- sisting of three distinct subpopulations of particles with
diameters of 19.7, 9.3 and 7.6 nm results not shown.
The preparations of both AI-rHDL and SAA-rHDL inhibited the TNF-a-induced expression of VCAM-1 in
HUVECs in a concentration-dependant fashion. When equated for phospholipid composition, if anything the
inhibitory activity of the SAA-rHDL was superior to that of the AI-rHDL Fig. 5. However, we have
demonstrated previously that the inhibition is a func- tion of the HDL particle molarity. Our inability to
determine the number of SAA molecules per particle of SAA-rHDL made it impossible to equate them with the
AI-rHDL in terms of particle molarity. Thus, all that can be concluded is that both preparations were effec-
tive inhibitors but that it cannot be determined whether either is superior to the other.
We also investigated the effects of lipid-free SAA on VCAM-1 expression. As we have observed previously
with lipid-free apoAI, the SAA had no effect: it neither stimulated nor inhibited the TNF-a-induced expression
of VCAM-1 in HUVECs result not shown.
3
.
4
. Binding of unmodified and SAA-enriched HDL
3
to endothelial cells
Having found that SAA did not influence the ability of HDL
3
to inhibit the cytokine-induced expression of VCAM-1 in HUVECs, further studies were conducted
to determine whether other aspects of the interaction of HDL with endothelial cells may be influenced by the
presence of SAA in the HDL. Specifically, the effect of SAA on the binding of native HDL
3
to BAECs was assessed.
In competition displacement experiments, both unla- belled HDL
3
and SAA-enriched HDL
3
prepared by incubating native HDL
3
in vitro with purified SAA as described above competed almost identically for bind-
ing and internalisation of
125
I-labelled HDL
3
, with 43 – 50 of the label being displaced by the addition of 100
mg of the competitor Fig. 6A. Similarly, in cross-com- petition experiments, unlabelled HDL
3
and SAA-en- riched HDL
3
competed almost equally for binding of
Fig. 5. Effect of discoidal AI rHDL and SAA rHDL on the cytokine- induced expression of VCAM-1 in HUVECs. The discoidal rHDL
preparations were prepared as described in Section 2. HUVECs were preincubated for 17 h with either AI rHDL or SAA rHDL before
being activated with TNF-a 100 Uml and incubated for a further 5 h. The expression of VCAM-1 was quantitated by flow cytometry.
Values are presented as a percentage of that in a sample without HDL. The AI rHDL 2 and the SAA rHDL were added to the
HUVECs according to phospholipid concentration. The results are presented as the mean and standard error of the mean obtained from
three experiments, each of which was performed in duplicate.
125
I-labelled SAA-enriched HDL
3
to cultured BAECS. SAA-enriched HDL
3
were less effective in reducing internalisation of
125
I-SAA-enriched HDL
3
, suggesting that factors other than specific binding sites may con-
tribute to uptake of HDL
3
. Concentration dependence studies were also carried
out to investigate further possible differences in cell interaction between the two lipoprotein preparations.
Specific binding and internalisation is shown in Fig. 7A and B. For both lipoprotein preparations, saturation
Fig. 7. Concentration dependent binding and internalisation of
125
I- HDL
3
or
125
I-SAA-HDL
3
to BAEC. Data shown is specific binding which was determined by subtracting non-specific binding from total
binding as described in the Methods section. Panel A shows specific binding and internalisation
of
125
I-HDL
3
determined in the presence of unlabeled HDL
3
. Panel B shows specific binding and internalisation
of
125
I-SAA-HDL
3
, with non-specific binding having been determined in the presence of unlabeled HDL
3
; corre- sponding parameters {binding , internalisation } were de-
termioned in incubations performed in the presence of excess SAA-HDL
3
.
Fig. 6. Competitive displacement of
125
I-HDL
3
A or
125
I-SAA- HDL
3
B by unlabeled lipoprotein. BAEC cells were incubated with 3 ng protein of
125
I-labeled HDL
3
and increasing concentrations of either unlabeled HDL
3
or SAA-HDL
3
. Surface bound or internalised labeled lipoprotein was determined as trypsin releasable or trypsin
resistant radioactivity, respectively, as described in the Methods section. Panel A shows the surface bound
125
I-HDL
3
displace by either excess HDL
3
or SAA-HDL
3
and the amount of
125
I-HDL
3
internalised after incubating with increasing concentra- tions of HDL
3
or SAA-HDL
3
. Panel B shows the corre- sponding
displacement of
125
I-SAA-HDL
3
bound ,
or internalised ,
in the presence of HDL
3
,
or SAA-HDL
3
, , respectively.
was approached at 100 mg protein. Binding parameters were calculated from Scatchard plots. Kds for specific
binding of SAA-enriched HDL
3
were 1.18 × 10
− 9
and 0.3 × 10
− 9
M for native HDL
3
. Capacity of binding B
max
was correspondingly higher for SAA-enriched HDL
3
, being 648 ngmg protein compared with 198 ngmg protein for native HDL
3
. Although the cells appeared to show a higher capacity of binding for
SAA-enriched HDL
3
than for native HDL
3
, the num- bers of binding sites for SAA-enriched HDL
3
could not be calculated since the precise molarity of the SAA-en-
riched HDL
3
could not be determined.
4. Discussion
