© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Endogenous Neutralizing Antibodies Against Platelet-Derived Growth Factor-AA Inhibit Atherogenesis in the Cholesterol-Fed Rabbit
From the Centre for Clinical Science and Measurement (D.J.L., T.Y.A., G.A.A.F.), School of Biological Sciences, University of Surrey, and the Department of Clinical Biochemistry (G.A.A.F.), Royal Surrey County Hospital, Egerton Road, Guildford, UK. T.Y.A. is currently at the Royal Sussex County Hospital, Eastern Road, Brighton, UK.
Correspondence to Professor G.A.A. Ferns, MD, Centre for Clinical Science and Measurement, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH. E-mail g.ferns{at}surrey.ac.uk
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Abstract—Previous studies have shown that the B chain of platelet-derived growth factor (PDGF) has an important role in atherogenesis. In this study we have investigated the contribution of PDGF-A chain in cholesterol-induced atherogenesis in the New Zealand White rabbit. High titers of antibodies to PDGF-AA or to platelet cytosolic protein (PCP) were induced in these animals by immunization against recombinant human PDGF-AA or human PCP. Rabbits were then fed a 0.25% to 1% cholesterol–containing diet for 10 weeks to induce atherosclerotic lesions; the rabbits were then humanely killed and perfusion-fixed and their aortas were removed. The extent of atherosclerosis in the thoracic aortas was determined by quantitative morphometry after staining with oil red O. The intimal and medial areas in histological sections taken at the level of the first intercostal branch were quantified by image analysis. Immunization against PDGF-AA and PCP, but not against adjuvant alone, resulted in rising titers of antibodies within 2 weeks, the levels of which reached a plateau by 8 weeks. The antibodies to PDGF-AA were isoform-specific, recognized both human and rabbit PDGF-AA, and neutralized the biological activity of PDGF-AA in vitro. Integrated plasma cholesterol levels were similar in both groups. Compared with nonimmune rabbits (n=10), animals immunized against PDGF-AA (n=10) or PCP (n=10) had significantly smaller areas of the aorta covered by atherosclerotic lesions (24.6±5.1% and 18.7±4.2%, respectively, vs 34.4±4.3%; P<0.05). This was associated with a reduced aortic intimal-medial area ratio in PDGF-AA–immunized (0.009±0.006) and PCP-immunized (0.025±0.017) rabbits than in nonimmune animals (0.159±0.066; P<0.05). These data suggest that PDGF-AA is actively involved in cholesterol-induced atherosclerosis in the rabbit.
Key Words: platelet-derived growth factor-AA • endogenous antibody • atherosclerosis • cholesterol-fed rabbit
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Introduction |
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The fatty streak is the earliest macroscopically evident lesion of atherosclerosis. This lesion is predominantly composed of macrophage-derived foam cells, which accumulate beneath the endothelial layer. The formation of the more clinically relevant fibrofatty plaque appears to depend on smooth muscle cell migration into the intima and subsequent proliferation (reviewed in Ross1 ). The factors responsible for these processes are still unknown. Previous studies, however, suggest that smooth muscle cell migration and proliferation may be a consequence of synergistic interplay between growth factors and cytokines released by several arterial or lesional cell types (reviewed in Ross1 ). Of particular importance to atherogenesis may be platelet-derived growth factor (PDGF).2
PDGF is a cationic protein with a molecular weight of 
30
kDa whose active molecule consists of 2 homologous polypeptide chains.
The dimeric molecule can contain a combination of PDGF-A and -B chains;
hence, the 3 isomeric forms found are PDGF-AA, PDGF-AB, and PDGF-BB
(reviewed in Ross et al3 ).
The biological effects of PDGF are mediated by plasma membrane
receptors, which are also composed of dimers of 2 subunits, the
-subunit and ß-subunit. The ß-subunit has a high affinity for
the PDGF-B chain, whereas the -subunit binds the PDGF-A and -B
chains with equal affinity.4
The biological responses of smooth muscle cells to the 3 isoforms of
PDGF are dependent on the relative levels of expression of the PDGF
receptor subunits on their
surfaces.5
Initially, atherogenic growth factors such as PDGF-AA and
PDGF-BB may be derived from dysfunctional
endothelium.6 7
The subsequent accumulation of activated macrophages
within the lesion provides a rich source of potentially atherogenic
growth factors, including transforming growth factor-ß (TGF-ß),
tumor necrosis factor-, insulin-like growth factor-1 (IGF-1), and
interleukin-1ß (IL-1ß) (reviewed in
Ross1 ). Resting monocytes
constitutively express both the PDGF-A and -B chains but express
predominantly the PDGF-B chain as they mature into tissue
macrophages.8
As smooth muscle accumulates and the lesion becomes fibrous,
smooth muscle cells may also secrete mitogenic factors such
as PDGF-AA, IGF-1, and TGF-ß, which may act in a paracrine or
autocrine manner. As the lesion becomes severe, the
endothelium can be compromised, and thrombogenic
elements of the extracellular matrix beneath become exposed. This event
may be associated with platelet deposition and degranulation.
Platelets contain several mitogenic and potentially
proatherogenic growth factors, including PDGF-AA, PDGF-BB, epidermal
growth factor, IGF-1, and TGF-ß.
Recent studies have placed particular emphasis on the role of PDGF-BB in atherosclerosis. The PDGF-B chain has been found to be expressed in both atherosclerotic lesions induced in primates2 and in advanced lesions found in humans.2 9 The uptake of oxidized LDL by macrophages and the subsequent cholesterol accumulation, thought to be an initially important process in atherogenesis, are also accelerated by PDGF-BB.10 We have previously reported that endogenous neutralizing antibodies against PDGF-BB elicited in the cholesterol-fed rabbit inhibits atherosclerotic lesion formation in the aortas of these animals.11 These antibodies were found to inhibit atherosclerosis to a similar extent as did antibodies raised against proteins from rabbit platelets.
The role of PDGF-AA in atherosclerosis is
less clear, although PDGF-AA is expressed in human atherosclerotic
lesions12 and is upregulated
in areas of neointimal
thickening.13 In addition,
smooth muscle cells isolated from spontaneously hypertensive rats
express greater numbers of PDGF- receptors than do cells from
control animals.14 Both
lisinopril (an angiotensin-converting enzyme
inhibitor) and verapamil (a calcium channel
blocker), which inhibit arterial neointimal
thickening in vivo, downregulate the expression of the PDGF-A chain
mRNA in endothelial
cells.15 This may be a
subsidiary mechanism by which these pharmacological agents reduce the
incidence of coronary heart disease.
In this study, we have investigated the effect of eliciting endogenous neutralizing antibodies against PDGF-AA and human platelet cytosolic protein (PCP) in the cholesterol-fed rabbit on the formation of atherosclerotic lesions in their aortas. This may help to clarify the importance of PDGF-A chain in atherogenesis in this model.
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Materials
Cholesterol-containing rabbit diets were supplied by Special Diet Services. All reagents were of analytical grade unless stated otherwise.
Rabbit Colonies
Juvenile New Zealand White rabbits (10 weeks old)
weighing 2.0 kg were housed in the Experimental Biology Unit at the
University of Surrey, Guildford, in accordance with Home Office
regulations. Food and water were allowed ad
libitum.
Immunization Groups
Each experimental group consisted of 10 rabbits.
Human recombinant PDGF-AA was purchased from NBS Biologicals. Human and
rabbit PCPs were prepared from heparinized whole blood.
Platelet-rich plasma was obtained by centrifugation
at 200g for 20 minutes at
4°C. Prostacyclin (Sigma) was added to a final concentration of 0.33
µg/mL, and the platelet-rich plasma was centrifuged at
800g for 15 minutes at 4°C.
The platelet pellet was resuspended in phosphate-buffered saline
(PBS, pH 7.4), and the platelets were lysed by 5 cycles of
freeze-thawing. Platelet debris was removed by
centrifugation at
10 000g for 1 hour at 4°C,
and the protein concentration of the supernatant was determined by a
modification of the Lowry protein
assay.16 Immunogens were
administered subcutaneously at 4 sites in a volume of 0.25 mL per site
in complete nonulcerative Freund’s adjuvant (NUFA). In brief, primary
immunizations of 75 µg PDGF-AA, 75 µg PCP, or saline were prepared
in 0.9 mL saline and mixed with 0.1 mL BCG vaccine (John Bell &
Croydon) in a 5-mL syringe. Two volumes of incomplete NUFA (a kind gift
from Mr B.A. Morris, School of Biomedical and Life Sciences, University
of Surrey, UK) were taken in a second syringe, and a stable
emulsion was formed by mixing the contents of both syringes by several
passes through a twin Luer-hubbed syringe connector with a fine channel
(Guildhay). Four weeks after the primary immunization, booster
injections containing 50 µg PDGF-AA, 50 µg PCP, or saline were
prepared without BCG vaccine in incomplete NUFA and administered as
described above. Antibody titers were measured
biweekly.
Blood Sampling
Blood was drawn before the start of the experimental
diet and biweekly thereafter from the ear vein into heparinized
containers, and plasma was obtained by centrifugation
at 4°C. Plasma was stored at -70°C before
analysis.
Dietary Manipulation
Rabbits were maintained on a chow diet for the first
8 weeks of the experiment. Animals were first fed a chow diet
supplemented with 1% cholesterol for 10 weeks after
the first immunization. The plasma cholesterol levels were
measured biweekly. This schedule enabled modification of the
cholesterol content of the
cholesterol-containing diets to maintain the plasma
cholesterol level at 20 mmol/L. This was achieved
by mixing the 1% cholesterol–containing diets with a
standard chow diet to produce individually tailored diets with
different cholesterol contents.
Cholesterol Measurement
Plasma cholesterol levels were measured
on a Boehringer Accutrend meter with Accutrend test strips
(Boehringer Mannheim) after the plasma samples were diluted in
PBS if necessary.
Plasma Antibody Titers
Plasma antibody titers to PDGF-AA, PCP, and
mycobacterial tuberculin (John Bell & Croyden) were measured with a
sandwich ELISA. Microtiter plates (Nunc Maxisorp, Merck Ltd) were
coated with 10 ng recombinant PDGF-AA or PCP in 0.1 mol/L carbonate
buffer, pH 9.6, per well for 18 hours at 4°C under humidified
conditions. The wells were washed 3 times in wash buffer (PBS
containing 0.05% Tween-20). Nonspecific binding was reduced by
blocking each well with 20% (wt/wt) milk in PBT (PBS containing 1%
bovine serum albumin and 0.1% Tween-20) for 1 hour at 37°C.
Wells were washed 3 times with wash buffer. Plasma was diluted 1:100
with PBT, and 100 µL per well was incubated for 30 minutes at 37°C.
After being washed, the wells were incubated with 100 µL per well of
biotinylated anti-rabbit IgG antibody (Sigma) diluted 1:20 000 in PBT
for 30 minutes at 37°C. After being washed, the wells were incubated
with 100 µL premixed avidin D and biotinylated horseradish peroxidase
(both from Vector Laboratories) diluted 1:500 in PBT for 30 minutes at
37°C.
o-Phenylenediamine
(0.04%, Sigma) was dissolved in 0.05 mol/L citrate/0.1 mol/L phosphate
buffer, pH 5, containing 10 µL
H2O2 per 25 mL. Substrate
(100 µL per well) was incubated at room temperature for 5 minutes,
and the reaction was terminated by adding 50 µL of 3 mol/L HCl.
Optical density at 492 nm was measured with a Labsystems iEMS reader MF
microtiter plate reader with Genesis 2 software (Life
Sciences).
Antibody Partial Purification
Plasma was isolated from whole, heparinized blood by
centrifugation at
1000g for 10 minutes at 4°C.
Saturated ammonium sulfate (Fisher) was added dropwise until 25%
saturation was achieved. The large proteins and lipoproteins were
allowed to precipitate at 4°C overnight. The supernatant containing
the smaller-molecular-weight species was retained after
centrifugation at
15 000g at 4°C for 1 hour.
The low-molecular-weight proteins were precipitated by adding an equal
volume of saturated ammonium sulfate to the supernatant and incubating
the resulting mixture at 4°C overnight. The precipitate was pelleted
by centrifugation at
15 000g at 4°C for 1 hour
and resuspended in 0.1 mol/L PBS. The fractions in the 50 000- to
300 000-Da molecular-weight range were obtained by filtration through
a 300 000-Da molecular-weight-cutoff Mini-Ultrasette filtration system
(Filtron Technology Corp) according to the manufacturer’s
instructions. The filtrate was then filtered through a 50 000-Da
molecular-weight-cutoff Mini-Ultrasette filtration system (Filtron) and
washed with several volumes of 0.1 mol/L PBS. The retentate containing
the IgG fraction was sterilized by membrane filtration and assayed for
protein by a modified Lowry
assay.16
Rabbit Smooth Muscle Cell Lysate
Preparation
Rabbit smooth muscle cells were grown from medial
explants from rabbit aortas and cultured in Dulbecco’s modified
Eagles’ medium containing 10% fetal bovine serum, 10 IU/mL
penicillin, and 10 µg/mL streptomycin at 37°C under 5%
CO2. Cells were washed with ice-cold PBS and
lysed in 10 mmol/L Tris, 158 mmol/L NaCl, 0.1 mmol/L
EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mmol/L
PMSF, 1 mmol/L DTT, and 1:100 Sigma protease inhibitor
cocktail, pH 7.4, for 30 minutes at 4°C. Lysates were
centrifuged at 15 000g
for 30 minutes at 4°C, and the supernatants were stored at -80°C
until required.
Antibody Specificity Assessment
The specificity of the raised antibodies was assessed
by Western blotting analysis under denaturing, nonreducing
conditions by using the Hoefer Mighty Small system (Amersham Pharmacia
Biotech). A 15% gel (15% concentration of the acrylamide
monomer; 2.7% concentration of the cross-linker relative to the
concentration of the acrylamide monomer) of 1.5-mm thickness was cast
according to the manufacturer’s instructions. Samples were boiled for
5 minutes in sample buffer (0.19 mol/L Tris, 2% SDS, 10% glycerol,
and 0.005% bromophenol blue, pH 8.8), and 30 µL (100 ng protein) was
then applied to each well. The samples were separated by
electrophoresis at 80 mA for 2 hours in a 12.5 mmol/L Tris, 0.2
mol/L glycine, and 0.1% SDS, pH 8.3, tank buffer. Samples were
transferred to nitrocellulose membranes at 200 mA for 3 hours in
25 mmol/L Tris, 0.1 mol/L glycine, 10% methanol, and 0.05% SDS,
pH 8.3, transfer buffer. Nonspecific protein binding was reduced by
incubation with Superblock (Pierce & Warriner) at room temperature for
1 hour. The partially purified anti–PDGF-AA or anti-human PCP
antibodies were diluted to 100 µg/mL in 0.1 mol/L Tris, 0.1%
Tween-20, and 0.1 mol/L NaCl, pH 7.5, and incubated with the membrane
for 30 minutes at room temperature. After being washed, the blot was
developed by using Vector Elite ABC anti-rabbit kit and the Vector DAB
substrate kit (both from Vector Laboratories) according to the
manufacturer’s instructions.
Antibody Neutralization Capability
Murine (BALB/c) 3T3 cells were cultured in
Dulbecco’s modified Eagles’ medium containing 10% fetal bovine
serum, 1x glutamax, 50 IU penicillin per mL, 50 µg streptomycin per
mL, and 2.5 µg amphotericin B per mL (all from Life Technologies) and
plated into 24-well tissue culture plates (Costar, Fisher Scientific).
Cells were made quiescent by incubation in Dulbecco’s modified
Eagles’ medium containing 0.4% fetal bovine serum, 1x glutamax, 50
IU penicillin per mL, 50 µg streptomycin per mL, and 2.5 µg
amphotericin B per mL for 48 hours. The cells were then restimulated
with 30 ng/mL PDGF-AA in the presence or absence of increasing
concentrations of anti–PDGF-AA antibody. After 18 hours, 1 µCi of
[3H]methyl thymidine (1 mCi/mL, Amersham
Pharmacia Biotech) was added to each well and incubated for a further 4
hours. The medium was removed, the wells were washed with 5% ice-cold
trichloroacetic acid at 4°C, and the resulting material was dissolved
in 1 mol/L NaOH at room temperature.
[3H]methyl thymidine uptake was measured
in a Wallac 1410 Wizard scintillation counter (EG&G Wallac), and cell
protein was measured by a modification of the Lowry
assay.16
Animal Sacrifice
Eighteen weeks after the primary immunization and 10
weeks after initiating a cholesterol-containing diet, the
animals were anesthetized with xylazine (3.5 mg Rompun per kg
IM; Bayer Plc) and ketamine (18 mg Ketaset per kg IM; Willows
Francis Veterinary) and subsequently heparinized (300 IU/kg IV; Sigma),
and the abdominal aorta was cannulated. Rabbits were then killed with
an overdose of pentobarbitone (Rhone Merieux), and the jugular veins
were transected for perfusion runoff. Rabbits were perfused with
isotonic saline at a rate of 100 mL/min and a pressure of 120
mm Hg. When the runoff was clear, the saline was replaced with 4%
paraformaldehyde in isotonic saline as described
previously.11 After
perfusion, the entire thoracic aorta was isolated, cleaned of fascia,
and placed in fresh paraformaldehyde for 48 hours. The
aortas were split longitudinally into 2 halves: 1 half was placed in
fresh paraformaldehyde before being stained with oil
red O and the other half was divided into segments and dehydrated in
increasing ethanol concentrations before being embedded in paraffin wax
for histology.
Quantification of Lesional Area
Aortas were rinsed in 80% propan-2-ol and
stained for 90 seconds in 80% propan-2-ol containing 2% (wt/vol) oil
red O (Sigma). Aortas were rinsed in 80% propan-2-ol, restained for a
further 90 seconds in 80% propan-2-ol containing 2% (wt/vol) oil red
O, and destained in 80% propan-2-ol and PBS. The sections were pinned
out on a cork board and photographed en face. The area stained
positively with oil red O was quantified by using an MCID image
analysis system (Imaging Research) and expressed as a
percentage of the total area analyzed.
Tissue Processing and Staining
Five-micron-thick sections of thoracic aorta were
taken at the level of the first intercostal branch and stained with
Verhoeff–van Gieson’s elastin stain. The extent of intimal expansion
was measured by morphometry by measuring the intimal-medial ratio with
the use of a Zeiss Axioskop microscope (Carl Zeiss Ltd) equipped with a
10x Achroplan objective, video camera, and MCID image analysis
system (Imaging Research). The intimal and medial cross-sectional areas
were measured, and the mean intima-media area ratio was calculated for
each animal as previously
described.11
Statistical Analysis
The integrated plasma cholesterol levels
were calculated as the area under the variable versus time curve
and expressed as (mmol/L) · weeks. The comparison between aortic
intimal-medial ratios was performed with unpaired
t tests. The comparison of oil
red O positivity was performed by using the nonparametric
Mann-Whitney U test after the
data were found to be not normally distributed. The relationship
between antibody titers and aortic area positively staining for oil red
O was calculated by linear regression analysis. Statistical
significance was assumed for a
P value <0.05. All
analyses were performed with
SPSS
software.
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Immunization With PDGF-AA and Human PCP Elicited Endogenous Specific Antibodies of High Titer
Immunization with recombinant PDGF-AA elicited a rapid increase in plasma levels of antibodies that had a high reactivity with PDGF-AA and a lower reactivity with human PCP as measured by ELISA (Figure 1A
). The anti–PDGF-AA antibodies isolated from these
animals recognized recombinant human PDGF-AA, but not PDGF-BB or other
growth factors, in Western blotting experiments. They also exhibited
weaker reactivity with recombinant PDGF-AB and fractions of human PCP
but did not bind to human serum albumin
(Figure 1B). These data suggest that the antibodies raised
against PDGF-AA were PDGF-A chain–specific. These antibodies
cross-reacted with rabbit PDGF-AA contained within rabbit smooth muscle
cells and platelets
(Figure 1C).
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The antibodies raised to human PCP also attained high
titers. They exhibited no detectable cross-reactivity with recombinant
PDGF-AA as measured by ELISA
(Figure 1D
). The anti–human PCP antibodies recognized
components of human PCP in Western blotting experiments, exhibited weak
binding to human albumin, and additionally showed no reactivity
with either PDGF-AB or -BB isoforms
(Figure 1E). All immunization were performed in NUFA, which
elicited a rapid increase in plasma levels of antibodies that had a
high reactivity to mycobacterial tuberculin
(Figure 1F). The plasma level of these antibodies was not
significantly different in any of the immunization
groups.
Endogenous Antibodies to PDGF-AA
Were Neutralizing In Vitro
In vitro, increasing concentrations of the partially
purified rabbit anti–PDGF-AA antibody inhibited the
mitogenic effects of PDGF-AA on 3T3 fibroblasts in a
dose-dependent manner
(Figure 2). However, at high concentrations of the antibody,
the mitogenic response to PDGF-AA was enhanced. This may
have been due to the presence of small quantities of contaminating
mitogenic factors in the antibody preparations. This may
also explain why the partially purified antisera did not completely
inhibit the mitogenic effects of
PDGF-AA.
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Cholesterol Feeding Significantly
Raises Plasma Cholesterol
The cholesterol content of the diet was
manipulated to range between 0.25% and 1% so that plasma
cholesterol levels were allowed to peak at 20 to 25
mmol/L in all animals. There were no significant differences in plasma
cholesterol levels between the experimental groups, either
during the experimental period (expressed as integrated
cholesterol levels) or at sacrifice
(the
Table). There was also no significant difference in
mean body weights of the animals from the 3 experimental groups
(the
Table).
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Endogenously Elicited Antibodies to
PDGF-AA and PCP Inhibit Aortic Lesion Development
Fatty streaks developed in the thoracic aortas of all
animals and were visualized by positive staining to oil red O
(Figure 3A). Lesions were most severe in the aortic arch and
became less prominent in the distal thoracic aorta, where lesions were
mostly confined to intercostal artery branch points. Immunization of
cholesterol-fed rabbits with PDGF-AA significantly
(P<0.05) reduced the area of
the aortas staining positively with oil red O compared with those from
control animals
(Figure 3B). The oil red O–stained area was also
significantly (P<0.01) reduced
in animals immunized with human PCP compared with control animals.
There was a highly significant positive relation between integrated
plasma cholesterol levels and the area of aorta staining
positively to oil red O in the control animals
(R2=0.792,
P<0.001). However, there was a
weak correlation
(R2=0.465,
P<0.05) in animals immunized
with PDGF-AA and a nonsignificant
(R2=0.125,
P>0.05) relation in animals
from the human PCP group (data not shown). The weaker associations
between integrated cholesterol levels and lesion severity
in the latter 2 groups may be related to the variability in antibody
responses of the individual animals within these groups.
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A similar relation between lesion development and antigen
administered was found when the extent of
atherosclerosis was measured by the ratio of thickness
of the intima and media at the level of the first intercostal branch
(Figure 4). Animals immunized with either PDGF-AA or human
PCP exhibited significantly
(P<0.05) smaller aortic
intimal-medial area ratios compared with animals within the control
group.
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The Role of PDGF-AA and Other Platelet Proteins in Atherosclerosis
Platelets contain several mitogenic and potentially proatherogenic growth factors, including PDGF-AA, -AB, and -BB; epidermal growth factor; IGF-1; and TGF-ß. PDGF-AA has been shown to mediate the mitogenic effect of exogenous TGF-ß17 and IL-ß18 on smooth muscle cells via autocrine pathways. The presence of neutralizing antibodies to PDGF-AA may therefore be expected to modulate the potential atherogenic effects of PDGF-AA, IL-1ß, and TGF-ß. Immunization of cholesterol-fed rabbits with PDGF-AA was associated with a significant reduction in the extent of atherosclerotic lesions in the thoracic aorta. This reduction was of similar magnitude to that observed in rabbits immunized with cytosolic protein from human platelets. The antibodies isolated from rabbits immunized with PDGF-AA showed cross-reactivity with human recombinant PDGF-AA, rabbit platelet protein, and rabbit smooth muscle cell lysates, but not with human platelet protein. The antibodies from rabbits immunized with PCP showed no cross-reactivity with PDGF-AA. The presence of high titers of plasma anti–PDGF-AA immunoglobulin may inhibit atherogenesis by neutralizing the effects of PDGF-AA. However, it is uncertain whether PDGF-AA is the principle growth factor involved or whether its importance is due to its mediation of the effects of other growth factors and cytokines, including IL-1, TGF-ß, and PDGF-BB.
The antiatherogenic effect of the antibodies raised against PCP, however, appear to be mediated through mechanisms independent of PDGF-AA activity. Nevertheless, it is likely that the platelet antibody exerted its antiatherogenic effects by inhibiting other platelet-associated factors. The strong association between plasma cholesterol and lesional area in the control animals was attenuated in the animals immunized against PGDF-AA. This finding probably reflects the action of the anti–PDGF-AA antibodies inhibiting lesion formation. The association between integrated plasma cholesterol and lesional area was even weaker in the animals immunized against PCP, indicating that human platelets contain other factors that contribute to atherogenesis in this model. Hence, although PDGF-AA appears to play an important role in atherogenesis in the cholesterol-fed rabbit, other agents such as growth factors, chemokines, or other vasoactive molecules associated with platelets are also likely to be important. Although the atherogenic immunogen(s) were platelet derived, it does not exclude the possibility that these factors may be secreted by other atherogenic cells in this model.
The Importance of Immunomodulation in
Atherosclerosis
The inflammatory nature of
atherosclerosis was first described by Virchow in
1856.19 It has since been
proposed that modulating the immune response during lesion formation in
the arterial wall may influence both the size and
morphology of these
lesions.20 It is therefore
possible that eliciting antibodies against either PDGF-AA or
platelet proteins may influence atherogenesis independently of
their ability to neutralize the effects of growth factors. The adjuvant
administered to both experimental and control groups, however,
contained Mycobacterium bovis,
which is well known to produce both strong humoral and cellular immune
responses.21 22 23 24 25
Indeed, we found equivalent levels of
anti-Mycobacterium antibodies
in the plasma of animals from all groups. We consider it unlikely that
the immune responses to PDGF-AA, platelet proteins, and saline
(other than the specific activity of the antibodies) differentially
effected atherogenesis in the 3 groups against the background of the
immune response to Mycobacterium
bovis.
Conversely, exogenous administration of immunoglobulins has been reported to inhibit atherosclerosis in the apoE-knockout mouse.26 This effect may be due to the endogenous superoxide dismutase activity found in isolated immunoglobulins.27 However, we found that animals immunized with saline alone did not exhibit significantly different levels of atherosclerosis from animals immunized with adjuvant alone (D.J. Lamb and G.A.A. Ferns, unpublished data, 1998).
Possible Clinical Implications
Our data indicate that PDGF-AA plays an important role
in the development of aortic atherosclerosis in the
cholesterol-fed rabbit. PDGF-AA may be released from
degranulating platelets, and PDGF-A chain is also expressed by
macrophages. PDGF- receptors are found on the plasma
membrane of endothelial
cells28 and smooth muscle
cells.29 Although the 3 PDGF
isoforms display a marked cross-species homology, there are also
interspecies differences in cellular distribution and expression.
Hence, the extrapolation of these data to humans should be treated with
some caution.
These data suggest that PDGF-AA plays a critical role in the evolution of atherosclerotic plaque formation, either directly or indirectly by mediating the effects of other growth factors such as PDGF-BB and TGF-ß. This effect appeared to be independent of the immunomodulatory effect of immunization. In addition, PDGF-AA does not appear to be the only platelet-associated growth factor that contributes to cholesterol-induced atherogenesis.
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Acknowledgments |
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This work was supported by a grant from the British Heart Foundation. The authors would like to thank Brian Morris for his gift of NUFA and Graham Mooray for his technical assistance.
Received November 1, 2000; accepted January 22, 2001.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
-
Ross R, Matsuda J,
Raines EW, Gown AM, Katsuda S, Sasahara M, Malden LT, Masuko H, Stao H.
Localisation of PDGF-B protein in macrophages in all phases of
atherosclerosis.
Science. 1990;248:1009–1012.
[Abstract/Free Full Text] - Ross R, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell. 1986;46:155–169.[Medline] [Order article via Infotrieve]
-
Seifert RA, Hart
CE, Phillips CE, Forstrom JW, Ross R, Murray MJ, Bowen-Pope DF. Two
differential subunits associate to create isoform-specific
platelet-derived growth factor receptors.
J Biol Chem. 1989;264:8771–8778.
[Abstract/Free Full Text] - Ferns GAA, Sprugel KH, Seifert R, Bowen-Pope D, Kelly JD, Murray M, Rains EW, Ross R. Relative platelet-derived growth factor receptor subunit expression determines cell migration to different dimeric forms of PDGF. Growth Factors. 1990;3:315–324.[Medline] [Order article via Infotrieve]
- Katsuda S, Coltrera MD, Ross R, Gown AM. Human atherosclerosis, IV: immunocytochemical analysis of cell activation and proliferation in lesions of young adults. Am J Pathol. 1993;142:1787–1793.[Abstract]
- Bohm M, Blaisius S, Roessner A. Immunohistochemical localisation of PDGF-B, PDGF ß-receptors and IGF-1 receptors during atherogenesis. Zentralbl Pathol.. 1994;140:357–362.[Medline] [Order article via Infotrieve]
- Nagaoka I, Honma S, Someya A, Iwabuchi K, Yamashita T. Differential expression of platelet derived growth factor-A and -B genes during the maturation of monocytes to macrophages. Comp Biochem Physiol. 1992;103:349–356.
-
Barrett TB, Benditt
EP. Platelet-derived growth factor gene expression in human
atherosclerotic plaques and normal artery wall.
Proc Natl Acad Sci
U S A. 1988;85:2810–2814.
[Abstract/Free Full Text] -
Furhman B, Brook
GJ, Aviram M. Proteins derived from platelet -granules modulate
the uptake of oxidised low density lipoprotein by macrophages.
Biochim Biophys Acta. 1992;117:15–21.
- Rutherford C, Martin W, Carrier M, Anggard EE, Ferns GAA. Endogenously elicited antibodies to platelet derived growth factor-BB and platelet cytosolic protein inhibit aortic lesion development in the cholesterol-fed rabbit. Int J Exp Pathol. 1997;78:21–32.[Medline] [Order article via Infotrieve]
-
Rekhter MD,
Gordon D. Does platelet-derived growth factor A chain stimulate
proliferation of arterial mesenchymal cells in human
atherosclerotic plaques? Circ
Res. 1994;75:410–417.
[Abstract/Free Full Text] -
Lemstrom KB,
Koskinen PK. Expression and localisation of platelet-derived growth
factor ligand and receptor protein during acute and chronic rejection
of rat cardiac allografts.
Circulation. 1997;96:1240–1249.
[Abstract/Free Full Text] - Scott-Burden T, Resink TJ, Hahn AA, Buhler FR. Platelet-derived growth factor-A chain homodimer stimulated growth of cultured smooth muscle cells from spontaneously hypertensive rats. Clin Exp Hypertens. 1991;13:763–772.
- Yamaguchi M, Gallati H, Baur W, Cruess DF, Sharefkin JB. Both lisinopril and verapamil reduced platelet-derived growth factor-A chain mRNA levels in human saphenous vein endothelial cells stimulated by thrombin. Surgery. 1994;115:495–502.[Medline] [Order article via Infotrieve]
-
Lowry OH,
Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin
phenol reagent. J Biol
Chem. 1951;193:265–275.
[Free Full Text] - Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross R. TGFß induces bimodal proliferation of connective tissue cells via a complex control of an autocrine PDGF loop. Cell. 1990;63:515–524.[Medline] [Order article via Infotrieve]
- Raines EW, Dower SK, Ross R. Interleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA. Science. 1989;43:393–396.
- Virchow R. Der ateromatose prozess der arterien. Wien Med Wochenschr. 1856;6:825–827.
- Minick CR, Murphy GE. Experimental induction of atheroarteriosclerosis by synergy of allergic injury to arteries and lipid-rich diet, II: effect of repeatedly injecting foreign protein in rabbets fed a lipid-rich, cholesterol-poor diet. Am J Pathol. 1973;73:265–300.[Medline] [Order article via Infotrieve]
-
Banerjee DK,
Sharp AK, Lowrie DB. The effect of
-interferon during
Mycobacterium bovis (BCG)
infection in athymic and euthymic mice.
Microb Pathog. 1986;1:221–224.[Medline]
[Order article via Infotrieve]
-
Andersen P,
Askgaard D, Ljungqvist L, Bentzon MW, Heron L. T-cell proliferative
response to antigens secreted by
Mycobacterium tuberculosis.
Infect Immun. 1991;59:1558–1563.
[Abstract/Free Full Text] - Tokunaga T, Yano O, Kuramoto E, Kimura Y, Yamamoto T, Kataoka T, Yamamoto S. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells. Microbiol Immunol. 1992;36:55–66.[Medline] [Order article via Infotrieve]
-
Ladel CH, Hess J,
Daugelat S, Mombaerts P, Toneegawa S, Kaufmann SHE. Contribution of
/ß and /
T-lymphocytes to immunity against
Mycobacterium bovis Bacillus
Calmette-Guerin: studies with T-cell receptor-deficient mice.
Eur J Immunol. 1995;25:838–846.[Medline]
[Order article via Infotrieve]
- Jackson AM, Lien E, Alexandroff AB, Prescott S, Espevik T, James K, Selby PJ, Sundan A. Soluble urinary CD14 after intravesical bacille Calmette-Guerin immunotherapy for carcinoma in situ. Br J Urol. 1997;80:766–771.[Medline] [Order article via Infotrieve]
- Nicoletti A, Kaveri S, Caligiuri G, Bariety J, Hansson GK. Immunoglobulin treatment reduces atherosclerosis in apo-E knockout mice. J Clin Invest. 1998;102:910–918.[Medline] [Order article via Infotrieve]
- Petyaev IM, Hunt JV. Apparent superoxide dismutase-like activity of immunoglobulin. Redox Report. 1996;2:365–372.
-
Eriksson A,
Siegbahn A, Westermark B, Heldin CH, Claesson-Welsh L. PDGF - and
ß-receptors activate unique and common signal transduction
pathways. EMBO J. 1992;11:543–550.[Medline]
[Order article via Infotrieve]
-
Majesky MW, Reidy
MA, Bowen-Pope DF, Hart CE, Wilcox JN, Schwartz SM. PDGF ligand and
receptor gene expression during repair of arterial injury.
J Cell Biol. 1990;111:2149–2158.
[Abstract/Free Full Text]
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