(Circulation. 2001;103:108.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
From the British Heart Foundation Cardiovascular Research Unit, National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital Campus, London, UK (G.W.C., T.Y.H., A.W., C.S., D.O.H.); the ZLB Central Laboratory, Blood Transfusion Service SRC, Bern, Switzerland (P.G.L.); and the Department of Cardiovascular Biochemistry, St Bartholomews and the Royal School of Medicine and Dentistry, Queen Marys and Westfield College, London, UK (N.E.M.).
Correspondence to G.W. Cockerill, Department of Cardiovascular Biochemistry, St Bartholomews and the Royal London SMD, Queen Marys and Westfield College, Charterhouse Square, London EC1 M 6BQ, UK. E-mail g.w.cockerill{at}mds.qmw.ac.uk
| Abstract |
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Methods and ResultsWe
used a porcine model of inflammation previously established in our
laboratory, in which the level of vascular endothelial cell expression
of E-selectin in interleukin (IL)-1
induced skin lesions was
measured by the uptake of a radiolabeled antiE-selectin antibody
(1.2B6). Porcine plasma HDL levels were elevated by use of a bolus
injection of reconstituted discoidal HDL (recHDL). These particles
resemble nascent HDL particles in shape and contain apolipoprotein A-I
as the sole protein and soybean phosphatidylcholine as the sole
phospholipid. We found that recHDLs inhibited the expression of
IL-1
induced E-selectin by porcine aortic endothelial cells in
vitro, confirming that the inhibitory effect is conserved with
synthetic HDLs and demonstrating that the phenomenon is not restricted
to human endothelial cells. In vivo, elevating the circulating level of
HDLs
2-fold led to significant inhibition of basal and
IL-1
induced E-selectin expression by porcine microvascular
endothelial cells.
ConclusionsThese observations demonstrate the potential anti-inflammatory action of HDLs and provide support for the further investigation of the mechanisms underlying the inhibitory effects of HDLs on endothelial cell activation.
Key Words: inflammation atherosclerosis proteins endothelium apolipoproteins
| Introduction |
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There is now considerable support for the importance of
leukocyte recruitment into the arterial intima, both for the
development of atheroma and for the maintenance of the mature plaque.
Indeed, the earliest observable cellular event in the genesis of
atheroma is the binding of leukocytes to the
endothelium.19 20
Also, several studies have now demonstrated the presence of
cytokine-induced adhesion molecules for leukocytes (eg, E-selectin,
intercellular adhesion molecule [ICAM]-1, and vascular cell adhesion
molecule [VCAM]-1), both in animal models of
atherosclerosis21 22
and in human atherosclerotic
tissue.23 24 25
In previous studies, we have explored the possibility that the
protective effect of HDLs may be related to an ability to inhibit
cytokine-induced endothelial cell adhesion molecule expression. HDLs
were found to inhibit the upregulation of E-selectin, VCAM-1, and
ICAM-1 by interleukin (IL)-1ß or tumor necrosis factor-
in
cultured human umbilical vein endothelial cells at the level of both
steady-state mRNA and surface protein
expression.26 27
In the present study, we used a radiolabeled monoclonal antibody
targeting technique in the pig, developed in our
laboratory,28 29 30
to show that inhibition of E-selectin expression by HDLs also occurs in
vivo.
| Methods |
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(2x107 U/mg) was a kind gift from Glaxo
Wellcome (Geneva, Switzerland). Reconstituted HDLs (recHDLs) were
prepared from plasma-derived human apoA-I and soybean
phosphatidylcholine by cholate dialysis in the ZLB Central
Laboratory,31 32
resulting in the generation of discoidal particles with an average
protein:phospholipid molar ratio of 1:150. The physical properties of
these proteoliposomes have been described previously in
detail.32
Isolation and Growth of Porcine Aortic
Endothelial Cells
Porcine aortic endothelial cells (PAECs) were
isolated and grown according to the modified method of Jaffe et
al33 34 from
aortas obtained from the local abattoir and transported in Hanks
balanced salt solution (GIBCO) containing 50 U/mL penicillin, 50
µg/mL streptomycin, 100 µg/mL gentamicin, 1 mmol/L sodium pyruvate,
and 0.1 µg/mL amphotericin (all from GIBCO). After removal of loose
connective tissue and ligation of branching vessels, the aortas were
filled with collagenase A (0.25 mg/mL, Boehringer Mannheim) for 15
minutes at 37°C. Endothelial cells were harvested and seeded into
25-cm2 flasks (Costar) precoated with 1%
gelatin (Sigma) in RPMI 1640 (GIBCO) supplemented with 20%
heat-inactivated FCS (GIBCO), antibiotics (as above), 15 U/mL sodium
heparin, 10 mmol/L
l-glutamine, and 10 µg/mL
endothelial cell growth supplement (Sigma). Cells were used for
experiments before the fourth passage.
Animals
The animals used in this study were healthy Large
White pigs weighing 15 to 20 kg derived from lines of stock lacking the
halothane-sensitivity gene. Animals were obtained from a commercial
supplier and housed under standard husbandry conditions. Animals were
studied according to a protocol approved under the UK Animals
(Scientific Procedures) Act, 1986.
Monoclonal Antibodies
1.2B6 is a mouse IgG1 monoclonal antibody (mAb) that
recognizes human
E-selectin35 and
P-selectin.36 This antibody
also recognizes porcine E-selectin but not porcine P-selectin, as shown
by reactivity with COS-7 cells transfected with porcine E-selectin or
P-selectin cDNA.37 MOPC21 is
a nonbinding mouse IgG1 myeloma protein and was kindly supplied by Dr
M. Robinson (Celltech Ltd, Slough, Berkshire, UK). Antibodies were
radiolabeled with 111In and
99mTc as previously
described.38
Apo A-I Assay
ApoA-I was determined immunoturbidimetrically on a
Cobas-Fara centrifugal analyzer (Roche Diagnostic). The apoA-I antibody
and standards were obtained from Boehringer Mannheim. The human apoA-I
antibody did not cross-react with porcine apoA-I.
Model of Cutaneous Inflammation
Animals were anesthetized for the bolus injections of
recHDL and intradermal injections of IL-1
. Anesthesia was induced
initially with halothane by inhalation, resulting in rapid induction of
sedation with minimal stress. Anesthesia was maintained by repeated
intravenous boluses of propofol (1 mg/kg; Diprivan, Zeneca
Pharmaceuticals) given every 15 to 20 minutes. The animals did not
require intubation or other external support. Cutaneous inflammatory
sites were induced by intradermal injections of IL-1
or saline
control, as previously
described.28 29
For the dose-response experiments, the animals were lightly sedated for
the entire time (3 hours), after which
111In-labeled 1.2B6 and
99mTc-labeled MOPC21 were injected as
a bolus via an ear vein and allowed to circulate for 5 minutes. Animals
were then exsanguinated under deep anesthesia, after which standard
skin disks
20 mm in diameter were excised and weighed. Radioactivity
of the excised tissue was measured in a gamma-counter (Minaxi 5530,
Canberra-Packard). Radioactivity in cpm/g for each sample was adjusted
for decay and spillover from the 111In
channel into the 99mTc channel. Specific
antiE-selectin mAb uptake in skin disks, which is a measure of
luminal endothelial E-selectin expression, was calculated by
subtracting the percentage of injected 99mTc
per gram disk tissue from the percentage of injected
111In per gram disk tissue. During the
time-course experiments, animals were allowed to return to their
holding pens after the IL-1
skin spots had been administered under
mild sedation.
Statistical Analysis
Data comparing the dose response to HDLs in vitro
(Figure 1
) were analyzed by 1-factor ANOVA followed by
Dunnetts test for multiple comparisons. The in vivo dose-response
data
(Figure 2
) and time-course data
(Figure 3
) were compared by 2-factor
ANOVA.
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| Results |
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(10 ng/mL), the level
of expression of E-selectin, measured by flow cytometry, was found to
be reduced in a dose-dependent fashion
(Figure 1
In Vivo Clearance of Reconstituted HDL
To establish the kinetics of clearance of intravenously
injected recHDL in the pig, we measured changes in the plasma
concentration of human apoA-I. We found that the concentration of
apoA-I had decreased by
30% of the initial level after 6 hours
(Table
).
|
Effect of recHDL on Basal and
IL-1
Dependent E-Selectin Expression
For our study of the effect of recHDL on expression of
E-selectin in vivo, 1 pig received a bolus injection of recHDL (50
mg/kg), and a control animal received a similar volume of PBS. Fifteen
minutes later, the animals were injected in multiple sites
intradermally with various amounts of IL-1
in 50 µL PBS per site.
It is known that unstimulated microvascular endothelium in pig skin
expresses readily detectable
E-selectin,28 29 30
and this was significantly reduced in pigs treated with recHDL, as
measured by the specific uptake of intravenously injected
antiE-selectin mAb 1.2B6 after 3 hours
(Figure 2
). Furthermore, recHDL abrogated the upregulation of
E-selectin in response to concentrations of IL-1
up to 100
ng/site.
Another experiment was conducted to exclude the possibility
that the apparent inhibition of E-selectin expression by recHDL was due
to interference of the binding of mAb 1.2B6 to E-selectin. Pigs were
pretreated intravenously with either recHDL (50 mg/kg) or a similar
volume of PBS and then were given intradermal injections of IL-1
at
1.5, 3.5, and 5.5 hours before injection of radiolabeled antibodies.
During the skin spot injections, the animals recovered from sedation
and were returned to holding pens. A third pig received intravenous
recHDL (50 mg/kg) 5 minutes before injection of radiolabeled
antibodies. As shown in Figure 3
, no inhibition of E-selectin expression was seen in
animals that received recHDL immediately before the injection of
radiolabeled antibodies, indicating that recHDL did not influence the
measurement of E-selectin expression with this technique. The relative
distribution of both the nonspecific and the E-selectin antibodies in
heart, kidney, liver, spleen, lung, and gut did not differ
significantly between experiments.
| Discussion |
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The only physiological way of quickly elevating the plasma HDL concentration is to give a bolus injection of discoidal apoA-I/lecithin proteoliposomes, which effectively mimics an increase in nascent HDL secretion rate by the liver.41 The use of phospholipid liposomes as a control would have been valid only if this had had no effect on the number and types of HDLs in plasma. However, we know that this would not have been the case. Under the influence of plasma phospholipid transfer protein,42 43 some of the infused phospholipid would have been rapidly incorporated into existing HDLs.44 45 46 Addition of phospholipid to HDLs has been shown to destabilize the particles, leading to fusion of particles and release of apoA-I, which subsequently associates with other liposomes to produce apoA-I/phospholipid disks.47 48 49 Thus, the use of phospholipid liposomes as a control would not merely have increased the plasma phospholipid concentration but would also have had major effects on the number of HDLs, including the production of discoidal particles similar to those used in the test animals. The use of lipid-free apoA-I alone would also have been an unsuitable control, because this is very rapidly cleared in the plasma.50
Because the original in vitro data were obtained in human
endothelial cells of umbilical origin, it was important to establish
that HDLs were capable of inhibiting cytokine-induced adhesion molecule
expression by porcine endothelial cells before embarking on an in vivo
study. We found that PAECs isolated from 50- to 60-kg animals and used
at early passage were able to support a high level of expression of
E-selectin on stimulation with IL-1
, as previously
reported.37 The ability of
HDLs to ablate this induction was established by preincubation of cell
cultures with a physiologically relevant concentration of
recHDLs.
Our results clearly indicate that recHDLs are able to reduce
expression of E-selectin in vivo, both basally and in response to
IL-1
. The possibility that recHDLs hindered the radiolabeled
antiE-selectin antibody from binding to its endothelial antigen was
excluded by an experiment showing that recHDLs administered immediately
before injection of radiolabeled antibodies had no inhibitory effect.
This study therefore provides the first direct evidence that HDLs
inhibit endothelial cell activation and adhesion molecule expression.
Although HDL has been found to inhibit E-selectin gene transcription,
the precise mechanism for this is still unknown. Because HDLs do not
inhibit I
B
degradation or the nuclear translocation of nuclear
factor-
B, the mechanism must involve a process independent of this
ubiquitous family of transactivating
factors.27
In summary, we have shown for the first time that elevation of the circulating level of HDLs, in a genetically normal large mammal, can inhibit cytokine adhesion molecule expression in vivo. Our data support the anti-inflammatory function of HDLs as a potential mechanism for the recent demonstration that recA-IMilano particles reduce macrophage infiltration in lesions in apoE-null mice,51 a mechanism further supported by the recent demonstration that recHDLs were also able to inhibit neointimal thickening and VCAM-1 expression in the same mouse model.52
From consideration of data showing that HDLs are able to mediate differential gene expression and activate signal transduction pathways, leading to Ras activation, we hypothesize that HDLs may act through modulation of cAMP-responsive elements. We have found that HDLs modulate cAMP response element binding protein retardation complexes in endothelial cells (unpublished observation). These observations provide the basis of our future work, which may provide an explanation as to how HDLs mediate their anti-inflammatory actions. Establishing the precise mechanism of action of HDLs will be crucial if raising levels is to be considered therapeutically advantageous.
| Acknowledgments |
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Received May 9, 2000; revision received July 12, 2000; accepted July 18, 2000.
| References |
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