© 1999 American Heart Association, Inc.
Original Contributions |
The Role of Alpha and Beta Platelet-Derived Growth Factor Receptor in the Vascular Response to Injury in Nonhuman Primates
From COR Therapeutics Inc (N.A.G., O.M., A.H., V.R., L.J.F.), South San Francisco; Department of Molecular and Experimental Medicine (J.A.K.), Scripps Research Institute, LaJolla, Calif; and Division of Hematology-Oncology, Department of Medicine (M.M.H.M., A.B.K., J.N.W., S.R.H.) and Department of Surgery (C.C.), Yerkes Regional Primate Research Center, Emory University, Atlanta, Ga.
Correspondence to Stephen R. Hanson, PhD, 1639 Pierce Dr, Room 1129 Woodruff Memorial Bldg, Emory University, Atlanta, GA, 30322. E-mail shanson{at}emory.edu
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Abstract—Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.
Key Words: angioplasty • endarterectomy • tyrosine kinase • smooth muscle cell • monoclonal antibody
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Restenosis is the major complication limiting the usefulness of interventional procedures for improving blood flow through obstructed arteries and small-caliber arterial grafts. Although the mechanisms underlying restenosis are poorly understood, it is well-recognized that procedures such as angioplasty, endarterectomy, stenting, and grafting cause disruption of endothelial cells, exposure of subendothelium, platelet thrombus formation, and eventual medial smooth muscle cell (SMC) proliferation. The most potent SMC mitogen released at the site of injury by platelets, endothelial cells, and SMCs themselves is platelet-derived growth factor (PDGF).1 2 3 4 5 6 7 PDGF is a disulfide-linked dimer of 2 related polypeptide chains, designated A and B, which are assembled as heterodimers (PDGF AB) or homodimers (PDGF AA and PDGF BB).8 9 10 PDGF exerts its biological activity by binding to alpha or beta PDGF receptors (PDGFRs), causing receptor dimerization and induction of intrinsic tyrosine kinase activity.11 The receptor-binding specificity for the PDGF isoforms is such that PDGF AA induces only alpha/alpha receptor dimers, PDGF AB induces alpha/alpha and alpha/beta receptor dimers, and PDGF BB induces all 3 receptor dimer combinations.12 13 14 15
The involvement of PDGF in the processes of arterial injury repair and atherogenesis is suggested by observations of increased expression of this growth factor and its receptor in human atherosclerotic plaques and coronary vessels after angioplasty and in the vessel wall after vascular injury in animal models.1 2 4 7 16 Antibodies that neutralize PDGF have been shown to inhibit neointima formation after balloon dilatation of the rat carotid artery, demonstrating a direct role for PDGF in the vascular response to injury in rodents.17 18 Although the rat carotid artery injury model has been extensively characterized, its usefulness for predicting clinical outcomes in humans has been questioned.19 To study the role of PDGF in more relevant arterial injury models, baboons were chosen because they exhibit a vascular anatomy and homeostatic mechanisms similar to those found in humans.20 21 Experimental arterial injury was produced by 2 methods that simulate therapeutic procedures used in humans, namely, surgical carotid artery endarterectomy and balloon catheter dilatation of the femoral artery. Using these models, the effects of alpha and beta PDGFR antagonism on vascular lesion formation was evaluated.
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Monoclonal Antibody Inhibition of Baboon PDGFR Phosphorylation In Vitro
To compare the relative potency of monoclonal antibody (mAb) 2H7C5 against human and baboon alpha PDGFR, human MG63 cells (American Type Tissue Collection, CRL 1427) and lung primary baboon fibroblasts were used. A similar characterization of anti-beta mAb 2A1E2 was done using HR5 cells that overexpress recombinant human beta PDGFR and primary baboon SMCs, which express endogenous beta PDGFR.22 Confluent monolayers of cells that had been serum-starved for 18 hours were stimulated with 50 ng/mL PDGF AA or PDGF BB for 10 minutes at 37°C in the absence or presence of increasing concentrations of mAb. The cells were solubilized in lysis buffer (20 mmol/L Tris HCl, pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 10 mmol/L tetrasodium pyrophosphate, 50 mmol/L NaF, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF), and lysates were separated by SDS-polyacrylamide gel electrophoresis and subjected to Western blot analysis with PY20 mouse anti-phosphotyrosine mAb (Transduction Labs) followed by detection with [125I]-protein A (Dupont NEN Research).
Carotid Endarterectomy and Femoral Balloon
Angioplasty
Carotid artery endarterectomy procedures
were performed as previously described by Hanson et al.23
Briefly, after a midline neck incision, one common carotid artery was
dissected free of surrounding tissue from the aortic arch proximally to
the carotid bifurcation distally. Standard heparin (100 U/kg) was given
intravenously, and the common carotid artery was occluded
with atraumatic microsurgical vascular clamps placed at the end of the
exposed vessel. The artery was then divided at a point 1 cm proximal to
the distal clamp, and the proximal segment was everted over curved
forceps to obtain exposure of the vessel intimal surface. The everted
surface was held in place using 7-0 polypropylene stay sutures, and the
endarterectomy was begun 1 cm from the divided end
of the artery and continued for 1 cm. The endothelium,
internal elastic lamina, and approximately half of the thickness of the
medial tissue layers were removed using microvascular forceps and a
surgical operating microscope under 32x magnification. After the
endarterectomy, the vessel was restored to its
normal configuration, and an end-to-end anastomosis was accomplished
using continuous 7-0 polypropylene sutures.
To perform balloon catheter denudation, an incision was made over the medial aspect of the distal thigh. A side branch on the distal superficial femoral artery (near the takeoff of the popliteal artery) was isolated without exposing the proximal superficial femoral artery and controlled using vessel loops. A 3F Fogarty balloon catheter was passed through the branch to the femoral bifurcation proximally, inflated to a diameter of approximately 4 mm by filling with sterile saline, and withdrawn throughout the length of the superficial femoral artery (to the insertion site) using a gentle twisting motion. A moderate but not strong resistance to the passage of the balloon was achieved in all cases. To ensure complete deendothelialization, this procedure was repeated 3 times with no instance of vessel rupture. After ballooning, the catheter was withdrawn, the side branch access vessel was ligated, and the incision site was closed.
Treatment and Control Groups
A total of 38 normal male juvenile baboons (Papio
cynocephalus) weighing 8 to 36 kg were used in these studies; 15
were used as controls. Control animals were untreated but underwent the
same procedures for vessel injury as the animals receiving anti-PDGFR
antibodies. In the treated groups, 5 baboons were given anti-alpha
PDGFR mAb 2H7C5 and 18 were treated with anti-beta PDGFR mAb 2A1E2. The
animals were quarantined and observed to be disease-free for at least
90 days before initiating the studies. All procedures were approved by
the Institutional Animal Care and Use Committee in compliance with
National Institutes of Health guidelines (Guide for the Care and Use of
Laboratory Animals, 1985). In control studies, 11 animals underwent
bilateral carotid artery endarterectomy. Single
vessels from 5 of these baboons were harvested after 7 days for
bromodeoxyuridine (BrdU) staining; the remaining 17 vessels were
harvested after 30 days for morphometric analysis. Two of the
vessels harvested at 30 days were found to be occluded and in 1
additional vessel the injury procedure was deemed technically
incomplete because histological evaluation showed only
partial removal circumferentially of the internal elastic lamina and
subjacent media; these vessels were not included in the subsequent
morphometric analysis. Thus, in the control studies a total of
14 vessels from 10 different animals were patent at 30 days and
available for morphometric analysis.
In addition to the endarterectomy procedures, 9 of the 15 control animals also under went bilateral femoral artery balloon angioplasty, performed concurrent with the endarterectomy procedures. Within this group of 18 injured arteries, 5 single vessels from individual animals were taken at 7 days for BrdU analysis. The remaining 13 vessels and an additional 4 vessels from animals who had balloon angioplasty of only 1 femoral artery were take at 30 days for morphometric analysis.
Eighteen animals were treated with anti-beta PDGFR mAb 2A1E2. Of this number, 10 animals underwent bilateral carotid endarterectomy. In 5 of these 10 animals receiving bilateral endarterectomy, single vessels were harvested for BrdU analysis at day 7. Of the remaining 15 vessels, 4 endarterectomy procedures were considered technically inadequate. Thus, 11 carotid arteries from treated animals were patent and available for analysis at the 30-day point. Concurrently, 9 treated baboons also underwent bilateral balloon angioplasty of the femoral arteries. Five of these vessels were taken for BrdU analysis at 7 days. The remaining 13 vessels, and 7 additional vessels from animals who had undergone balloon angioplasty of only 1 femoral artery, were taken at 30 days for morphometric analysis.
Five additional animals who received anti-alpha PDGFR mAb 2H7C5 were also studied. In these baboons, balloon angioplasty was performed on 1 femoral artery only. All animals in this group were sacrificed at 30 days, at which time all vessels were patent and taken for subsequent morphometric analysis.
Administration of mAbs
mAb 2H7C5 or 2A1E2 was given by intravenous bolus at
2.0 mg/kg beginning 2 hours before surgery and once every 24 hours
thereafter at 1.0 mg/kg for a total of 6 doses. Plasma samples were
obtained just before and 10 minutes after each dose of antibody. Plasma
concentrations of mAb were determined by standard ELISA using purified
alpha or beta PDGFR extracellular domain immobilized in
microtiter plates.24 25
Immunohistochemistry and Morphometric Analysis
Carotid and femoral arteries were harvested fresh at 7 days or
30 days after injury. The contralateral femoral artery or a region
adjacent to the endarterectomy site of the carotid
artery was used to provide uninjured control sections. All tissues were
fixed for 2 hours in 4% paraformaldehyde, transferred
to 15% sucrose buffer overnight, embedded in ornithine
carbamyl-transferase (Miles Inc) and stored at -80°C before
sectioning. Sections 10 µm thick were placed on slides, thawed
at 25°C, dried 1 to 3 hours at 60°C, and rehydrated in PBS for 10
minutes at 25°C before antibody staining. Rehydrated vessel sections
were blocked in 5% donkey sera for 5 to 10 minutes at 25°C and then
incubated with 10 µg/mL of rabbit anti-alpha or anti-beta PDGFR
IgG24 in 1% donkey sera, 0.01% Brij 35, and 0.01%
NaN3 in PBS at pH 7.4 overnight at 4°C. After
rinsing the slides with PBS, biotin-conjugated donkey anti-rabbit IgG
(Jackson Immuno Research Labs) at 2 µg/mL in 1% donkey sera/PBS was
added and incubated for 40 minutes at 25°C. The avidin horseradish
peroxidase conjugate, Vectastain Elite ABC (Vector Laboratories), was
used according to the manufacture's protocol with
3,3'-diaminobenzidine (DAB) substrate. Slides were counterstained with
Gills hematoxylin for 1.5 minutes at 25°C.
To determine the effects of vascular injury on cell proliferation, BrdU immunostaining was done on baboon vessels obtained 7 and 30 days after injury. BrdU (Sigma Chemical Co) was administered intravenously in 3 doses of 50 mg/kg at 24, 16, and 8 hours before tissue harvest. BrdU staining was performed on rehydrated tissue sections by first denaturing with 5N HCl at 50°C to 60°C for 10 minutes followed by digestion with 1% trypsin for 10 minutes at 37°C. Slides were washed in PBS and incubated with 3 µg/mL mouse anti-BrdU mAb (Boehringer Mannheim) in 1% donkey sera/PBS overnight at 4°C. For detection of bound anti-BrdU mAb, slides were washed and incubated with 2 µg/mL biotin-conjugated donkey anti-mouse IgG for 40 minutes at 25°C followed by the addition of Vectastain Elite ABC with DAB/nickel chloride as substrate.
To stain for proliferating cell nuclear antigen (PCNA), rehydrated slides were blocked with 5% donkey sera, 0.01% Brij 35 in PBS for 10 minutes at 25°C, washed, and incubated with 5 µg/mL mouse anti-PCNA mAb (Boehringer Mannheim), 0.01% Brij 35 in PBS for 6 to 8 hours at 4°C. Bound anti-PCNA mAb was detected as described above for anti-BrdU mAb.
Quantitative examination of cell proliferation after BrdU administration was also performed, as described previously.26 Briefly, cells containing BrdU were identified using a specific mAb (Dako, 1/20 dilution) after predigestion of the tissue with proteinase K (1 mg/mL) and 4N HCl. Color digital images of the immunostained sections were analyzed using the IP Laboratory Spectrum software package (Signal Analytics Corp) to count BrdU and hematoxylin stained cells. Cells were counted in 4 to 5 randomly selected fields from 3 to 4 cross-sections 4 to 6 mm apart from each vessel.
Morphometric analysis was performed on carotid and femoral arteries harvested 30 days after injury. Vessel cross-sections were treated with Verhoeff-van Gieson elastic stain to facilitate the identification of neointima. Morphometric measurements were performed on femoral and carotid arteries using a Nikon Optiphot-2 microscope (Nikon Inc) coupled with a Hitachi HV-C 20 U color video camera (Hitachi Inc). Two sections from each site of carotid endarterectomy (1 cm in length) and 2 sections from the adjacent uninjured portion of each carotid artery were analyzed to determine medial and neointimal areas (millimeter squared) using Image-Pro Plus software (Media Cybermetics, Inc). Injured femoral arteries (approximately 5 cm in length) were divided into multiple segments longitudinally. An average of 8 sections were analyzed from each vessel to determine the vessel-averaged medial and intimal areas (millimeter squared).
Statistics
The lesion sizes for vessels taken at each location were
analyzed with a statistical analysis of
covariance procedure that afforded an efficient comparison of
lesion size in the control and treated groups, as well as
between-animal and between-vessel components of
variability.27 In addition, because animal weights in the
different study groups varied approximately 3-fold, analysis of
covariance was used to assess whether weight was significantly
related to the neointima/media ratio while
simultaneously accounting for potential treatment effects
and animal-to-animal variability. Computations were performed in
Systat28 on an IBM-compatible PC. All values are reported
as the mean±SD.
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Effect of Vascular Injury on Expression of Beta PDGFR and Cell Proliferation
Histologic cross-sections of carotid and femoral arteries were stained with rabbit polyclonal IgG directed against the alpha or beta PDGFR extracellular domain.24 Proliferating cells were identified in adjacent sections by staining with antibodies to PCNA or BrdU. Uninjured segments of carotid artery did not stain positive for beta PDGFR and contained very few BrdU-positive nuclei (Figure 1A
and 1D). In contrast, at 7 days after
injury, sections of the carotid artery that had undergone
endarterectomy stained strongly for beta PDGFR at
the luminal boundary of the media and in the surrounding medial area
(Figure 1B); neointima was not easily identified at
this time. Coincident with beta PDGFR expression was a significant
increase in the number of BrdU-positive nuclei, which were most
frequently observed in regions of receptor staining but were also
present in the adventitia (Figure 1E). At 30 days after
injury, a concentric neointima was readily observed, the
luminal region of which stained strongly for beta PDGFR (Figure 1C). Nuclei that stained positively for PCNA were found
primarily in the region of beta PDGFR staining although the number of
proliferating cells was decreased compared with the findings at 7 days
after injury (Figure 1E and 1F).
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Femoral artery injury by balloon angioplasty also caused the induction
of beta PDGFR expression in association with cell proliferation (Figure 2). Immunohistochemistry of uninjured
femoral artery cross-sections did not show beta PDGFR staining, and the
level of cell proliferation, as measured by BrdU staining, was very
low, as expected (Figure 2A and 2D). However, sections obtained
at 7 days after injury stained strongly for beta PDGFR in the luminal
region of the media, and proliferating cells were detected in the same
region by BrdU staining (Figure 2B and 2E). At 30 days after
injury, most of the femoral neointima stained strongly for
beta PDGFR and contained most of the BrdU-positive nuclei, which were
now infrequent (Figure 2C and 2F). Interestingly, no alpha PDGFR
was detected in control vessels, and injury of either the carotid or
femoral arteries failed to induce its expression as determined by
staining with anti-alpha receptor IgG (data not shown). In
control experiments, IgG obtained from rabbits before immunization with
beta PDGFR failed to stain vessel sections, whereas anti-beta PDGFR
mAbs gave the same pattern of staining as the immune polyclonal IgG
(data not shown). These results indicate that alpha and beta PDGFR
expression is low in normal baboon vessels, but that injury produced by
endarterectomy or balloon dilatation causes a
selective upregulation of beta PDGFR in regions of the vessel
undergoing cellular proliferation.
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Anti-PDGFR mAbs Block Baboon PDGFR Autophosphorylation
mAbs 2H7C5 and 2A1E2 are potent selective antagonists
of the alpha and beta PDGFR, respectively.24 Both mAbs
block PDGF binding, receptor autophosphorylation, and
mitogenic signaling.24 The relative potency of
mAb 2H7C5 against human and baboon alpha PDGFR was compared using the
human sarcoma cell line MG63 and baboon primary lung fibroblasts. As
shown in Figure 3, treatment of each cell
line with 50 ng/mL PDGF AA resulted in the anti-phosphotyrosine
antibody detection of the 180-kDa alpha PDGFR band, which was not
detected in unstimulated cells. Preincubation of each cell line with
increasing concentrations of mAb 2H7C5 caused a reduction in alpha
PDGFR phosphorylation with an
IC50 of 1 to 3 nmol/L and >90% inhibition at 10
nmol/L (Figure 3).
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Similarly, the ability of mAb 2A1E2 to inhibit human and baboon beta
PDGFR phosphorylation was compared using HR5, a CHO
cell line that overexpresses recombinant human beta PDGFR, and baboon
SMC, which express endogenous beta PDGFR.22 As
shown in Figure 4, stimulation of each
cell line with 50 ng/mL PDGF BB generated only a 180-kDa PDGFR band
detected by anti-phosphotyrosine antibody, whereas no such band was
detected in unstimulated cells. Incubation of HR5 or baboon SMC with
mAb 2A1E2 at 2.7 nmol/L caused a marked reduction in PDGF BB-induced
beta PDGFR phosphorylation that was almost completely
blocked at 8.1 nmol/L (Figure 4). PDGF AA at 50 ng/mL induced
only a faint band at 180 kDa in baboon SMC and no detectable
phosphotyrosine signal in HR5, demonstrating that the level of alpha
PDGFR phosphorylation in these cell lines was very low
relative to that of beta PDGFR (Figure 4). These results
demonstrate that mAbs 2H7C5 and 2A1E2 are equally potent for inhibiting
human and baboon PDGFR activation at concentrations that should be
readily achievable in vivo.
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Effect of mAb 2H7C5 or 2A1E2 Treatment on Neointima
Formation in Baboons
To examine the effect of alpha or beta PDGFR inhibition on
neointima formation, mAb 2H7C5 or 2A1E2 was administered by
IV bolus at 2 mg/kg 2 hours before injury and at 1 mg/kg every 24 hours
thereafter for a total of 6 doses. Treated baboons had average daily
trough and peak mAb 2H7C5 plasma concentrations of 107 and 179 nmol/L
whereas these values for 2A1E2 were 122 and 207 nmol/L (Figure 5). These plasma levels, sustained during
the first 7 days after injury, were 10 to 20 times those required to
completely inhibit alpha or beta PDGFR activation in vitro (Figures 3
and 4).
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) and trough
(
) concentrations ±SD of mAb 2H7C5 (A) and 2A1E2 (B) as determined
for 5 and 17 treated animals, respectively.
Results After Carotid Endarterectomy
The results obtained from vessels after carotid
endarterectomy are given in Table 1. The untreated control baboons were
stratified into 2 groups that were homogeneous on the basis
of body weight: a group with low body weight (average, 10.8 kg) and one
with high body weight (average, 26.8 kg). To preclude animal weight as
an independent variable in these measurements, animals were
subsequently selected for treatment with anti-PDGFR mAb on the basis of
size so that the treated group (average, 28.1 kg) was closely
weight-matched with the group of larger control animals (Table 1). However, it is noteworthy that for control animals of all
sizes the ratio of areas of injured media to normal media were similar,
averaging 0.51 to 0.55, indicating that the
endarterectomy procedure consistently
removed approximately half of the medial thickness of the vessel wall
(Table 1).
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After 30 days, animals that had been treated with anti-beta PDGFR
antibody 2A1E2 had a significant reduction in the area of
neointima formed (Table 1). When 11 vessels from
treated animals were compared with 9 arteries obtained from
weight-matched control animals, there was a 37% reduction in
neointimal area in the treated versus control group
(1.85±0.86 mm2 versus 2.92±1.28
mm2; P=0.05). Between these treated
and control groups there was no difference in the area of media in
normal vessel segments taken adjacent to the injury site
(1.97±0.51 mm2 versus 2.37±0.54
mm2, respectively; P=0.15), indicating
that vessel size was comparable in the 2 groups. Similarly, the ratios
of areas of the injured media to adjacent normal media were not
different between the treated and control groups (0.47±0.24
mm2 versus 0.51±0.17
mm2, respectively; P=0.56), indicating
that the extent of vessel injury was equivalent between the groups
(Table 1).
Although the endarterectomy procedure removed about
half of the vessel media in each study group, in individual vessel
segments the fraction of media removed varied from approximately 20%
to 80%. Because the variable removal of media by this method
invalidates normalization of neointimal areas by the area
of underlying (ie, endarterectomized) media, the benefit of anti-beta
PDGFR mAb 2A1E2 for reducing neointima formation was
further documented by comparing the ratio of neointimal
area to the medial area in normal vessel segments taken immediately
adjacent to the endarterectomy site (Figure 6). This measurement was similar for
control animals in both the low body weight and high body weight groups
(1.42±0.13 versus 1.23±0.39, respectively; P=0.36). This
ratio therefore provides an appropriate normalized injury index for
endarterectomized vessels of different size. When all control animals
were pooled using this comparison (Figure 6), the
neointima/media ratio was reduced significantly for the
treated versus control group (0.93±0.36 versus 1.29±0.33,
respectively; P=0.019). Analysis of
covariance indicated that the benefit of treatment was
independent of animal size (P=0.885).
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; mean weight, 26.8 kg) and 5 vessels from smaller
baboons (
; mean weight, 10.8 kg). The overall
neointima/media ratio was reduced significantly by antibody
therapy (P=0.019).Subgroup analysis was also performed to assess the possible influence of other interventional procedures on neointima formation in carotid arteries harvested at 30 days. In these studies, animals underwent either (1) bilateral carotid endarterectomy and bilateral femoral angioplasty with 1 carotid artery and 1 femoral artery harvested at 7 days, and the remaining vessel pair harvested at 30 days (5 control animals and 5 treated animals), or (2) bilateral carotid endarterectomy and bilateral femoral angioplasty with all vessels harvested at 30 days (6 control animals and 5 treated animals). When results obtained with vessels taken at 30 days from animals studied under these 2 procedural protocols were compared, the neointima/media ratios were equivalent (ie, were unaffected by differences in the interventional protocols) for both the control and treated animal groups (P>0.45 in both cases).
To determine whether the decrease in neointima formation after treatment with 2A1E2 was associated with a reduction in SMC proliferation, BrdU staining was done 7 days after injury. Quantitative evaluation of 5 vessels harvested from treated animals showed no difference compared with results in 5 control arteries, with respect to the percentage of BrdU-positive medial SMC (7.9±1.2% versus 5.5±2.0%, respectively; P>0.05).
Results After Femoral Artery Angioplasty
In Table 2 the results with 10
vessels from baboons weighing an average of 11.5 kg were compared with
5 vessels from weight-matched animals treated with anti-alpha PDGFR mAb
2H7C5 and with 7 vessels from weight-matched animals treated with
anti-beta PDGFR mAb 2A1E2. Medial areas were equivalent in the 3 study
groups (P>0.4). In the animals treated with 2H7C5, neither
the area of neointima nor the neointima/media
ratio was decreased versus the control results (P>0.5 in
both cases, Table 2). Conversely, in animals treated with 2A1E2,
the area of neointima was reduced by 48%
(P=0.005 versus controls), and the
neointima/media ratio was reduced by 43%
(P=0.006 versus controls). Thus, a striking benefit for
reducing femoral artery intimal thickening was shown after blockade of
the beta PDGFR, but not the alpha PDGFR. In the 5 femoral arteries
harvested after 7 days for quantitative evaluation of cell
proliferation by BrdU staining, there was no difference in the
2A1E2-treated animals versus results in 5 control arteries in the
percentage of BrdU-positive medial SMC (4.6±2.1% versus 5.5±3.8%,
respectively; P>0.5).
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Also studied were animals of substantially higher body weight, both as
controls (6 animals) and after treatment with 2A1E2 (9 animals). These
findings are given in Table 2. When compared with the results
obtained in control animals, treatment with the anti-beta PDGFR mAb
reduced the area of neointima by 46% (P=0.015)
and the neointima/media ratio by 52%
(P=0.0003), results that were consistent with the
observed benefit of 2A1E2 in animals of smaller size, as noted above.
The overall comparison of individual data (neointima/media
ratio) from all treated and control animals is provided in Figure 7. When all of the data from animals
having different body weights were grouped, analysis of
covariance documented that the benefit of treatment was
independent of animal size (P=0.969), thereby reinforcing
the generality of these results.
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The possible influence on neointima formation of performing several interventional procedures in individual animals was also assessed. In these studies, femoral arteries were analyzed from animals that underwent (1) bilateral carotid endarterectomy and bilateral femoral angioplasty with 1 carotid artery and 1 femoral artery harvested at 7 days, and the remaining vessel pair harvested at 30 days (5 control animals, 5 treated with 2A1E2); (2) bilateral carotid endarterectomy and bilateral femoral angioplasty with all vessels harvested at 30 days (4 control animals, 5 animals treated with 2A1E2); or (3) angioplasty of 1 femoral artery with all vessels harvested at 30 days (4 control animals, 7 animals treated with 2A1E2). When vessels from animals studied under the 3 interventional protocols were compared, control values for the neointima/media ratio at 30 days were equivalent for animals of comparable body weight (P>0.5 in each case). Within each procedural group, the benefit of therapy was also equivalent (range, 44% to 48% reduction in the neointima/media ratio), indicating that surgical procedural variables (ie, concurrent endarterectomy, bilateral angioplasty, or the harvesting of some vessels at 7 days) did not affect femoral artery neointima formation when measured 30 days after balloon angioplasty.
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This study demonstrates that in nonhuman primates the PDGF response to vascular injury is mediated by beta PDGFR and that its antagonism can effectively inhibit neointima formation. The biological activity of PDGF is mediated by 2 PDGFRs, alpha and beta, which interact differentially with PDGF isoforms.8 9 10 12 13 14 15 PDGF BB and, to a much lesser extent, PDGF AB activate beta PDGFR, whereas all PDGF isoforms efficiently activate alpha PDGFR. Therefore, the responsiveness of vascular SMC to PDGF will be determined by the PDGFR subtype that is expressed and by the ligand isoforms that are present. Immunostaining failed to detect alpha or beta PDGFR in our uninjured baboon vessels, but after balloon injury or endarterectomy there was a sharp increase in beta, but not alpha, PDGFR (Figures 1
and 2, and data not
shown). This finding is consistent with previous observations
showing beta PDGFR expression in injured rat carotid arteries, in
injured pig coronary arteries, in the neointima of
vascular grafts in baboons, in human atherosclerotic plaques, and in
human coronary arteries after
angioplasty.2 4 16 26 29 30 31 32 33 34 35 Although alpha PDGFR was not
detected in baboon arteries in the present study, low levels of
this receptor have been detected in the media of the rat carotid artery
and in the neointima of healing vascular grafts in
baboons.16 29 32 33 35 Alpha PDGFR expression by medial
SMC could be functionally important because PDGF A chain, which only
interacts with the alpha receptor, has been routinely observed in
atherosclerotic plaques and in medial cells after
injury.1 2 16 26 29
To address the relative importance of alpha and beta PDGFR in the
vascular response to injury, we used the isotype-matched neutralizing
mAbs 2H7C5 and 2A1E2 directed against alpha and beta PDGFRs,
respectively.24 These mAbs have very similar properties in
that they recognize epitopes within the PDGF binding domain, and they
block PDGF binding, receptor autophosphorylation, and
mitogenic signaling.24 More importantly, they
show almost complete inhibition of baboon PDGFR
phosphorylation at concentrations >10 nmol/L (Figures 3 and 4). Treatment of baboons with mAb 2A1E2 for 6 days
after injury resulted in a significant 37% to 48% reduction in
neointima formation 30 days after femoral artery balloon
injury or carotid artery endarterectomy, whereas
such treatment with mAb 2H7C5 had no measurable effect (Figures 6 and 7; Tables 1 and 2). This result
implies that alpha receptor signaling is not required for the PDGF
response to vascular injury in baboons. Beta PDGFR may also play a
predominant role in the rat carotid artery because its level of
expression and autophosphorylation were increased after
injury, whereas alpha PDGFR was unaffected.33 Also,
periadventitial treatment of the rat carotid artery with antisense
oligonucleotide that blocked beta PDGFR expression
caused a 60% to 80% reduction in neointima
formation.35 Taken together, these data strongly support
the conclusion that beta PDGFR is important in the response to vascular
injury, and that the alpha PDGFR is unlikely to play a major role.
For beta PDGFR signaling to occur in injured vessels, PDGF BB must be
present locally. Recruitment of platelets to the site of injury
provides a primary source of PDGF BB.36 The importance of
platelets in the vascular injury response is evidenced by the fact
that thrombocytopenic rats and rabbits show reduced intimal thickening
after balloon denudation.37 38 In the present study,
an endarterectomy procedure that is a more severe
form of injury than balloon angioplasty, and consequently produces more
platelet thrombus deposition at the site of injury, was
used.23 Therefore, a higher local concentration of PDGF
would be expected at this site, perhaps making PDGFR antagonism more
difficult. This could explain the reduced effectiveness of mAb 2A1E2
that was seen in this setting compared with the balloon angioplasty
model (Figures 5 and 7). After platelet deposition
has subsided, a continuous source of PDGF BB is provided by
macrophages and the regenerating
endothelium.1 2 4 7 29 30 39 40 In the
denuded rat carotid artery, endothelial cells at the
wound edge begin to express PDGF B within 8 hours; by several days
after injury, when the endothelium is undergoing
regrowth and migration, it is present at and near the leading edge
of the regenerating monolayer.7 This localized source of
PDGF could act on the underlying SMC to promote intimal thickening.
Interestingly, even 6 weeks after injury, when replication is no longer
detectable, PDGF B mRNA is still being expressed in the denuded
zone.7 In our baboon model of restenosis,
endothelial regeneration and neointima
formation continued for 30 days or longer,23 and in rats,
arterial wall myointimal hyperplastic potential has been
shown to persist long after the initial injury.34
Therefore, extending the treatment period of PDGFR antagonism beyond
the 6 days used in the present study might inhibit lesion formation
to an even greater extent.
Previous attempts to establish a direct role for PDGF in the vascular response to injury have been conducted primarily using the rat carotid injury model.17 18 35 41 In this model, balloon injury initially causes some cell death followed by 15% to 25% of medial SMC undergoing proliferation by 48 to 72 hours.42 43 44 45 46 This medial cell proliferation was inhibited by 85% with a neutralizing anti–basic fibroblast growth factor antibody administered before injury.47 This antiproliferative effect may have been caused by the neutralization of basic fibroblast growth factor that was released on cell death. In contrast, anti-PDGF antibodies have little effect on this initial phase of cell proliferation.17 However, the subsequent SMC migration across the internal elastic lamina, which leads to neointima formation, is strongly mediated by PDGF.17 48 For example, neutralizing antibodies against PDGF were found to reduce SMC migration across the internal elastic lamina at 4 days after injury by 80% and inhibited subsequent neointima formation by 40%.17 48 Conversely, infusion of PDGF BB caused a 20-fold increase in neointima formation with little change in medial SMC proliferation.49 In the baboon, PDGF may act similarly because beta PDGFR antagonism by mAb 2A1E2 had no detectable effect on the rate of medial cell proliferation in either carotid or femoral arteries at 7 days after injury but inhibited vascular lesion formation by 48% at 30 days after injury. These results support the view that enhanced SMC migration is an important mechanism by which PDGF mediates vascular lesion formation. SMC migration across the internal elastic lamina requires the formation of plasmin and the activation of matrix metalloproteinases, which have been shown to be regulated by PDGF.48 50 51 52 53 54 It is noteworthy that inhibitors of matrix metalloproteinases and tissue-type plasminogen activator effectively block PDGF-induced SMC migration after balloon injury,48 50 52 but not the subsequent neointima formation.52 Therefore, other effects of PDGF, including its ability to induce synthesis of extracellular matrix,55 56 57 58 59 protect SMC against apoptosis,60 61 and possibly enhance intimal SMC proliferation41 may contribute to neointima formation.
The rat carotid artery model of restenosis has proven to be of limited value for identifying agents to prevent restenosis after coronary angioplasty in humans.19 Some agents that tested positive and then failed to show benefit in the clinic include heparin, calcium-channel blockers, antiplatelet drugs and angiotensin-converting enzyme (ACE) inhibitors.19 62 63 64 65 66 67 When tested in the baboon model, ACE inhibitors did not decrease intimal thickening after balloon angioplasty or endarterectomy.23 Heparin, the only other of these agents tested in baboons, did not inhibit lesion formation at the usual antithrombotic doses that were effective in the rat.68 Thus, the factors mediating the vascular response to injury in humans and nonhuman primates are different from those in rodents. Interspecies differences that could be of considerable consequence include the greater propensity for thrombus formation at arterial injury sites in primates compared with rodents, which would entail a larger accumulation of platelets in primates and most likely cause more PDGF to be released at the injury site. Also, rat platelets contain only the PDGF BB isoform, whereas human and baboon platelets also contain significant levels of the PDGF AB and PDGF AA isoforms.36 Although the potential biological significance of having multiple PDGF isoforms is unknown, this characteristic is unique to primates.36
A major drawback of all commonly used animal models of restenosis is that they do not duplicate the underlying disease state that exists in human atherosclerotic vessels. The response to acute mechanical injury of normal arteries in animals primarily involves SMC proliferation and migration. In humans, restenosis after angioplasty is a considerably more complex process involving multiple components, including plaque compression, vasoconstriction, thrombosis, recoil, matrix production, inflammatory cell reactions, and vascular remodeling, as well as SMC proliferation and migration.69 The use of coronary stent implantation as a method of revascularization potentially eliminates problems caused by vasoconstriction, recoil, and vascular remodeling, which could account for the reported reduction in restenosis rates compared with those observed after balloon angioplasty without stenting.70 71 Nevertheless, restenosis after vascular stent placement still occurs in 20% to 30% of cases, and the occlusive hyperplastic lesions that develop are highly cellular and arise from SMC migration and intimal proliferation.72 73 74 75 76 Therefore, beta PDGFR blockade may be most effective for prevention of in-stent restenosis, a possibility we are currently exploring. In summary, the studies reported here indicate that pharmacological antagonism of beta PDGFR may provide a therapeutic strategy for limiting clinical restenosis after interventional procedures for coronary ischemic syndromes and after other procedures for peripheral artery revascularization or repair.
|
Acknowledgments |
|---|
This work was supported in part by grants HL-31469, HL-47838, and RR-00165 from the National Institutes of Health. We thank Donna Forestner, Steven Marzec, and Debbie White for their expert technical assistance.
Received July 24, 1998; accepted September 29, 1998.
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 C. Horvath, F. G.P. Welt, M. Nedelman, P. Rao, and C. Rogers Targeting CCR2 or CD18 Inhibits Experimental In-Stent Restenosis in Primates: Inhibitory Potential Depends on Type of Injury and Leukocytes Targeted Circ. Res., March 8, 2002; 90(4): 488 - 494. [Abstract] [Full Text] [PDF]
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Y. Zhan, S. Kim, H. Yasumoto, M. Namba, H. Miyazaki, and H. Iwao Effects of Dominant-Negative c-Jun on Platelet-Derived Growth Factor-Induced Vascular Smooth Muscle Cell Proliferation Arterioscler. Thromb. Vasc. Biol., January 1, 2002; 22(1): 82 - 88. [Abstract] [Full Text] [PDF]
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 J.-C. Yu, N. A. Lokker, S. Hollenbach, M. Apatira, J. Li, A. Betz, D. Sedlock, S. Oda, Y. Nomoto, K. Matsuno, et al. Efficacy of the Novel Selective Platelet-Derived Growth Factor Receptor Antagonist CT52923 on Cellular Proliferation, Migration, and Suppression of Neointima following Vascular Injury J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 1172 - 1178. [Abstract] [Full Text] [PDF]
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 H. Sano, T. Sudo, M. Yokode, T. Murayama, H. Kataoka, N. Takakura, S. Nishikawa, S.-I. Nishikawa, and T. Kita Functional Blockade of Platelet-Derived Growth Factor Receptor-{beta} but Not of Receptor-{alpha} Prevents Vascular Smooth Muscle Cell Accumulation in Fibrous Cap Lesions in Apolipoprotein E-Deficient Mice Circulation, June 19, 2001; 103(24): 2955 - 2960. [Abstract] [Full Text] [PDF]
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O. Leppanen, N. Janjic, M.-A. Carlsson, K. Pietras, M. Levin, C. Vargeese, L. S. Green, D. Bergqvist, A. Ostman, and C.-H. Heldin Intimal Hyperplasia Recurs After Removal of PDGF-AB and -BB Inhibition in the Rat Carotid Artery Injury Model Arterioscler. Thromb. Vasc. Biol., November 1, 2000; 20 (11): e89 - e95. [Abstract] [Full Text] [PDF]
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J. N. Topper Genes, Matrix, and Restenosis Arterioscler. Thromb. Vasc. Biol., October 1, 2000; 20(10): 2173 - 2174. [Full Text] [PDF]
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M. G. Davies, E. L. Owens, D. P. Mason, H. Lea, P. K. Tran, S. Vergel, S. A. Hawkins, C. E. Hart, and A. W. Clowes Effect of Platelet-Derived Growth Factor Receptor-{alpha} and -{beta} Blockade on Flow-Induced Neointimal Formation in Endothelialized Baboon Vascular Grafts Circ. Res., April 14, 2000; 86(7): 779 - 786. [Abstract] [Full Text] [PDF]
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C. Horvath, F. G.P. Welt, M. Nedelman, P. Rao, and C. Rogers Targeting CCR2 or CD18 Inhibits Experimental In-Stent Restenosis in Primates: Inhibitory Potential Depends on Type of Injury and Leukocytes Targeted Circ. Res., March 8, 2002; 90(4): 488 - 494. [Abstract] [Full Text] [PDF]
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