Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:900-909

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Giese, N. A.
Right arrow Articles by Hanson, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Giese, N. A.
Right arrow Articles by Hanson, S. R.
Related Collections
Right arrow Animal models of human disease
Right arrow Growth factors/cytokines
Right arrow Smooth muscle proliferation and differentiation
Right arrow Catheter-based coronary and valvular interventions: other
Right arrow Carotid endarterectomy
Right arrow Autonomic, reflex, and neurohumoral control of circulation
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:900-909.)
© 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

Neill A. Giese; Monique M. H. Marijianowski; Oscar McCook; Andrea Hancock; Vanitha Ramakrishnan; Larry J. Fretto; Changyi Chen; Andrew B. Kelly; James A. Koziol; Josiah N. Wilcox; Stephen R. Hanson

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


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 1ADown and 1DDown). 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 1BDown); 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 1EDown). At 30 days after injury, a concentric neointima was readily observed, the luminal region of which stained strongly for beta PDGFR (Figure 1CDown). 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 1EDown and 1FDown).



View larger version (175K):
[in this window]
[in a new window]
  		Figure 1. Effect of endarterectomy on immunohistochemical staining for beta PDGFR and cell proliferation in baboon carotid artery. Staining for beta PDGFR (A through C) or proliferating cells (D through F) from uninjured carotid artery (A and D) was compared with vessels at 7 days after injury (B and E) or 30 days after injury (C and F). Proliferating cell nuclei were stained for BrdU (D and E) or for PCNA (F). Double staining for PCNA and beta PDGFR (F) was performed on endarterectomy sections taken 30 days after injury; arrows indicate proliferating cells.

Femoral artery injury by balloon angioplasty also caused the induction of beta PDGFR expression in association with cell proliferation (Figure 2Down). 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 2ADown and 2DDown). 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 2BDown and 2EDown). 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 2CDown and 2FDown). 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.



View larger version (187K):
[in this window]
[in a new window]
  		Figure 2. Immunohistochemical staining for beta PDGFR and cell proliferation in native or balloon-injured baboon femoral artery. Staining for beta PDGFR (A through C) or BrdU-positive nuclei (D through F) from uninjured femoral artery (A and D) was compared with vessels at 7 days after injury (B and E) or 30 days after injury (D and E). Arrows in panel F indicate BrdU-positive nuclei 30 days after injury.

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 3Down, 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 3Down).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. Comparison of mAb 2H7C5 inhibition of human versus baboon alpha PDGFR autophosphorylation. Human MG63 sarcoma cells (A) and primary baboon SMC (B) that express endogenous alpha PDGFR were stimulated with 50 ng/mL of PDGF AA in the absence or presence of mAb 2H7C5 at 0.4 to 90 nmol/L as indicated. After ligand stimulation, cell lysates were prepared, separated by SDS-polyacrylamide gel electrophoresis, and subjected to Western blot analysis with anti-phosphotyrosine antibodies (see Methods).

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 4Down, 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 4Down). 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 4Down). 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.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 4. Comparison of mAb 2A1E2 inhibition of human versus baboon beta PDGFR autophosphorylation. HR5 cells (A) expressing recombinant human beta PDGFR and baboon SMC (B) expressing endogenous beta PDGFR were stimulated with 50 ng/mL of PDGF AA or PDGF BB in the absence or presence of mAb 2A1E2 at 0.1 to 24 nmol/L as indicated. After ligand stimulation, cell lysates were prepared, separated by SDS-polyacrylamide gel electrophoresis, and subjected to Western blot analysis with anti-phosphotyrosine antibodies (see Methods).

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 5Down). 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 3Up and 4Up).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. mAb plasma concentrations after administration to baboons. Blood samples were drawn just before or 10 minutes after each IV dose and mAb plasma concentrations were determined using a standard ELISA (see Methods). Plotted are the average peak ({blacksquare}) and trough ({square}) 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 1Down. 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 1Down). 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 1Down).


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of Anti-ß-PDGFR mAb in Carotid Artery Endarterectomy

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 1Up). 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 1Up).

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 6Down). 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 6Down), 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).



View larger version (11K):
[in this window]
[in a new window]
  		Figure 6. Effect of anti-beta PDGFR mAb treatment on neointima formation at 30 days after endarterectomy. The ratio of area of neointima to the area of uninjured media in the vessel segment adjacent to the endarterectomy site is shown for 11 vessels from animals treated with anti-beta PDGFR mAb 2A1E2. The treated animals had a mean body weight of 28.1 kg (Table 1Up). These data are compared with results from 9 vessels from large baboons ({bullet}; mean weight, 26.8 kg) and 5 vessels from smaller baboons ({blacktriangleup}; 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 2Down 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 2Down). 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).


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of Anti-ß-PDGFR mAb in Femoral Artery Balloon Angioplasty

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 2Up. 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 7Down. 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.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 7. Effect of anti-beta PDGFR mAb (2A1E2) and anti-alpha PDGFR mAb (2H7C5) on baboon femoral artery neointima formation at 30 days after balloon angioplasty. The ratio of the area of neointima to the area of media at sites of femoral balloon angioplasty is shown. A, Results for baboons having body weights averaging 9 to 12 kg (Table 2Up). B, Results for animals having body weights averaging 29 to 30 kg. A consistent benefit of therapy is seen with anti-beta PDGFR mAb, but not anti-alpha PDGFR mAb.

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.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 1Up and 2Up, 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 3Up and 4Up). 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 6Up and 7Up; Tables 1Up and 2Up). 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 5Up and 7Up). 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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. 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]
  2. Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest. 1988;82:1134–1143.
  3. Mattsson E, Clowes AW. Current concepts in restenosis following balloon angioplasty. Trends Cardiovasc Med. 1995;5:200–204.
  4. Tanizawa S, Ueda M, van der Loos CM, van der Wal AC, Becker AE. Expression of platelet-derived growth factor B chain and beta receptor in human coronary arteries after percutaneous transluminal coronary angioplasty: an immunohistochemical study. Heart. 1996;75:549–556.[Abstract/Free Full Text]
  5. Hart CE, Bailey M, Curtis DA, Osborn S, Raines E, Ross R, Forstrom JW. Purification of PDGF-AB and PDGF-BB from human platelet extracts and identification of all three PDGF dimers in human platelets. Biochemistry. 1990;29:166–172.[Medline] [Order article via Infotrieve]
  6. Heldin CH, Westermark B. Platelet-derived growth factor: mechanism of action and possible in vivo function. Cell Reg. 1990;1:555–566.[Medline] [Order article via Infotrieve]
  7. Linder V, Reidy MA. Platelet-derived growth factor ligand and receptor expression by large vessel endothelium in vivo. Am J Pathol. 1995;146:1488–1497.[Abstract]
  8. Hammacher A, Hellman U, Johnsson A, Gunnarsson K, Ostman A, Wasterson A, Heldin C-H. A major part of PDGF purified from human platelets is a heterodimer of one A chain and one B chain. J Biol Chem. 1988;263:16493–16498.[Abstract/Free Full Text]
  9. Strooband P, Waterfield MD. Purification and properties of porcine platelet-derived growth factor. EMBO J. 1984;3:2963–2967.[Medline] [Order article via Infotrieve]
  10. Heldin C-H, Johnsson A, Wennergren S, Wernstedt C, Betsholtz C, Wasteson A. A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A chains. Nature. 1986;319:511–515.[Medline] [Order article via Infotrieve]
  11. Williams LT. Signal transduction by the platelet-derived growth factor receptor. Science. 1989;243:1564–1570.[Abstract/Free Full Text]
  12. Seifert RA, Hart EC, Phillips PE, Forstrom JW, Ross R, Murry M, Bowen-Pope DF. Two different subunits associate to create isoform-specific platelet-derived growth factor receptors. J Biol Chem. 1989;264:8771–8778.[Abstract/Free Full Text]
  13. Bishayee S, Majumdar S, Khire J, Das M. Ligand-induced dimerization of the platelet-derived growth factor receptor: monomer-dimer interconversion occurs independent of receptor phosphorylation. J Biol Chem. 1989;264:11699–11705.[Abstract/Free Full Text]
  14. Heldin C-H, Ernlund A, Rorsman C, Ronnstrand L. Dimerization of B type PDGF receptors occurs after ligand binding and is closely associated with receptor kinase activation. J Biol Chem. 1989;264:8905–8912.[Abstract/Free Full Text]
  15. Kanakaraj P, Raj S, Khan SA, Bishayee S. Ligand-induced interaction between {alpha}- and ß-type platelet-derived growth factor (PDGF) receptor: role of receptor heterodimers in kinase activation. Biochemistry. 1991;30:1761–1767.[Medline] [Order article via Infotrieve]
  16. Masjesky 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]
  17. Ferns GAA, Raines AW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle cell accumulation after angioplasty by an antibody to PDGF. Science. 1991;253:1129–1132.[Abstract/Free Full Text]
  18. Rutherford C, Martin W, Salame M, Carrier M, Anggard E, Ferns G. Substantial inhibition of neo-intimal response to balloon injury in the rat carotid artery using a combination of antibodies to platelet-derived growth factor-BB and basic fibroblast growth factor. Atherosclerosis. 1997;130:45–51.[Medline] [Order article via Infotrieve]
  19. Muller DWM, Ellis SG, Topol EJ. Experimental models of coronary artery restenosis. J Am Coll Cardiol. 1992;19:418–432.[Abstract]
  20. Hampton JW, Matthews C. Similarities between baboon and human clotting. J Appl Physiol. 1966;21:1713–1716.[Free Full Text]
  21. Todd ME, NcDevitt E, Goldsmith EI. Blood clotting mechanisms of nonhuman primates: choice of the baboon model to simulate man. J Med Primatol. 1972;1:132–141.[Medline] [Order article via Infotrieve]
  22. Escobedo JA, Navankasatussas S, Cousens LS, Coughlin SR, Bell GI, Williams LT. A common PDGF receptor is activated by the homodimeric A and B forms of PDGF. Science. 1988;240:1532–1534.[Abstract/Free Full Text]
  23. Hanson SR, Powell JS, Dodson T, Lumsden A, Kelly AB, Anderson JS, Clowes AW, Harker LA. Effects of angiotensin converting enzyme inhibition with cilazapril on intimal hyperplasia in injured arteries and vascular grafts in the baboon. Hypertension. 1991;18:II-70–II-76.
  24. Lokker NA, O'Hare JP, Barsoumian A, Tomlinson JE, Ramakrishnan V, Fretto LJ, Giese NA. Functional importance of platelet-derived growth factor (PDGF) receptor extracellular immunoglobulin-like domains: identification of PDGF binding site and neutralizing monoclonal antibodies. J Biol Chem. 1997;272:33037–33044.[Abstract/Free Full Text]
  25. Fretto LJ, Snape AJ, Tomlinson JE, Seroogy JJ, Wolf DL, LaRochelle WJ, Giese NA. Mechanism of platelet-derived growth factor (PDGF) AA, AB, and BB binding to {alpha} and ß PDGF receptor. J Biol Chem. 1993;268:3625–3631.[Abstract/Free Full Text]
  26. Scott NA, Cipolla GD, Ross CE, Dunn B, Martin FH, Simonet L, Wilcox JN. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation. 1996;93:2178–2187.[Abstract/Free Full Text]
  27. Dunn OJ, Clark VA. Applied Statistics: Analysis of Variance and Regression. New York: John Wiley; 1987.
  28. Wilkinson L. The System for Statistics. Evanston, IL: Systat, Inc; 1990.
  29. Kraiss LW, Raines EW, Wilcox JN, Seifert RA, Barrett TB, Kirkman TR, Hart CE, Bowen-Pope DF, Clowes AW. Regional expression of the platelet-derived growth factor and its receptors in a primate graft model of vessel wall assembly. J Clin Invest. 1993;92:338–348.
  30. Bohm M, Roessner A. Immunochemical localization of PDGF B, PDGF beta receptors and IGF I receptors during atherogenesis. Zentralbl Pathol. 1994;140:357–362.[Medline] [Order article via Infotrieve]
  31. Rubin K, Tingstrom A, Hansson GK, Larsson E, Ronnstrand L, Klareskog L, Claesson-Welsh L, Heldin C-H, Fellstrom B, Terracio L. Induction of B-type receptors for platelet-derived growth factor in vascular inflammation: possible implications for developments of vascular proliferative lesions. Lancet. 1988;1:1353–1356.[Medline] [Order article via Infotrieve]
  32. Abe J-I, Deguchi J-O, Matsumoto T, Takuwa N, Noda M, Ohno M, Makuuchi M, Kurokawa K, Takuwa Y. Stimulated activation of platelet-derived growth factor receptor in vivo in balloon-injured arteries. Circulation. 1997;96:1906–1913.[Abstract/Free Full Text]
  33. Panek RL, Dahring TK, Olszewski BJ, Keiser JA. PDGF receptor protein tyrosine kinase expression in the balloon-injured rat carotid artery. Atherioscler Thromb Vasc Biol. 1997;17:1283–1288.[Abstract/Free Full Text]
  34. Sirois MG, Simons M, Kuter DJ, Rosenberg RD, Edelman ER. Rat arterial wall retains myointimal hyperplastic potential long after arterial injury. Circulation. 1997;96:1291–1298.[Abstract/Free Full Text]
  35. Sirois MG, Simons M, Edelman ER. Antisense oligonucleotide inhibition of PDGF-beta receptor subunit expression directs suppression of intimal thickening. Circulation. 1997;95:669–676.[Abstract/Free Full Text]
  36. Bowen-Pope DF, Hart CE, Seifert RA. Sera and conditioned media contain different isoforms of platelet-derived growth factor (PDGF) which binds to different classes of PDGF receptor. J Biol Chem. 1989;264:2502–2508.[Abstract/Free Full Text]
  37. Friedman RJ, Stemerman MB, Wenz B, Moore S, Gauldie J, Gent M, Tiell ML, Spaet H. The effect of thrombocytopenia on experimental atherosclerotic lesion formation in rabbits: smooth muscle cell proliferation and re-endothelialization. J Clin Invest. 1977;60:1191–1201.
  38. Fingerle J, Johnson R, Clowes AW, Majesky MW, Reidy MA. Role of platelets in smooth muscle cell proliferation and migration after vascular injury in the rat carotid artery. Proc Natl Acad Sci U S A. 1989;86:8412–8416.[Abstract/Free Full Text]
  39. Ross R, Masuda J, Raines EW, Gown AM, Katsuda S, Sashara M, Malden LT, Masuko H, Sato H. Localization of PDGF-B protein in macrophage in all phases of atherogenesis. Science. 1990;248:1009–1012.[Abstract/Free Full Text]
  40. Golden MA, Au YPT, Kirkman TR, Wilcox JN, Raines EW, Ross R, Clowes AW. Platelet-derived growth factor activity and mRNA expression in healing vascular grafts in baboons. J Clin Invest. 1991;87:406–414.
  41. Myllarniemi M, Calderon L, Lemstrom K, Buchdunger E, Hayry P. Inhibition of platelet-derived growth factor receptor tyrosine kinase inhibits vascular smooth muscle cell migration and proliferation. FASEB J. 1997;11:1119–1126.[Abstract]
  42. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury: I. Smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327–333.[Medline] [Order article via Infotrieve]
  43. Fishman JA, Ryan GB, Karnovsky MJ. Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening. Lab Invest. 1975;32:339–351.[Medline] [Order article via Infotrieve]
  44. Baumgartner HR. Eine neue Methode Zur Erzeugung der Thromben durch gezielte uberdehnung der Gefasswand. A Ges Exp Med. 1963;137:227–249.
  45. Haudenschild CC, Schwartz SM. Endothelial regeneration: II. Restitution of endothelial continuity. Lab Invest. 1978;41:407–418.[Medline] [Order article via Infotrieve]
  46. Clowes AW, Clowes MM, Reidy MA. Kinetics of cell proliferation after arterial injury: III. Endothelial and smooth muscle cell growth in chronically denuded vessels. Lab Invest. 1986;54:295–303.[Medline] [Order article via Infotrieve]
  47. Linder V, Reidy MA. Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1991;88:3739–3743.[Abstract/Free Full Text]
  48. Jackson CL, Raines EW, Ross R, Reidy MA. Role of endogenous platelet-derived growth factor in arterial smooth muscle migration after balloon catheter injury. Arterioscler Thromb. 1993;13:1218–1226.[Abstract/Free Full Text]
  49. Jawien A, Bowen-Pope DF, Linder V, Schwartz SM, Clowes AW. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest. 1992;89:507–511.
  50. Bendeck MP, Irvin C, Reidy MA. Inhibition of matrix metalloproteinase activity inhibits smooth muscle cell migration but not neointimal thickening after arterial injury. Circ Res. 1996;78:38–43.[Abstract/Free Full Text]
  51. Jenkins GM, Crow M, Bilato C, Ryu LZ, Froehlich J, Lakatta E, Cheng L. The role of MMP-2 in neointimal formation following balloon injury in the rat. FASEB J. 1994;8:A51. Abstract.
  52. Zempo N, Koyama N, Kenagy RD, Lea HJ, Clowes AW. Regulation of vascular smooth muscle cell migration and proliferation in vitro and in injured rat arteries by a synthetic matrix metalloproteinase inhibitor. Arterioscler Thromb Vasc Biol. 1996;16:28–33.[Abstract/Free Full Text]
  53. Carmeliet P, Moons L, Ploplis V, Plow E, Collen D. Impaired arterial neointima formation in mice with disruption of the plasminogen gene. J Clin Invest. 1997;99:200–208.[Medline] [Order article via Infotrieve]
  54. Carmeliet P, Moons L, Herbert JM, Crawley J, Lupu F, Lijnen R, Collen D. Urokinase but not tissue plasminogen activator mediates arterial neointima formation in mice. Circ Res. 1997;81:829–839.[Abstract/Free Full Text]
  55. Sibinga NES, Foster LC, Hsieh C-M, Perrella MA, Lee W-S, Endege WO, Sage EH, Haber E. Collagen VIII is expressed by vascular smooth muscle cells in response to vascular injury. Circ Res. 1997;80:532–541.[Abstract/Free Full Text]
  56. LaFleur DW, Fagin JA, Forrester JS, Rubin SA, Sharifi BG. Cloning and characterization of alternatively spliced isoforms of rat tenascin: platelet-derived growth factor-BB markedly stimulates expression of spliced variants of tenascin mRNA in arterial smooth muscle cells. J Biol Chem. 1994;269:20757–20763.[Abstract/Free Full Text]
  57. Bendeck MP, Regenass S, Tom WD, Giachelli CM, Schwartz SM, Hart C, Reidy MA. Differential expression of alpha 1 type VIII collagen in injured platelet-derived growth factor-BB-stimulated rat carotid arteries. Circ Res. 1996;79:524–531.[Abstract/Free Full Text]
  58. Wang X, Louden C, Ohlstein EH, Stadel JM, Gu J-L, Yue T-L. Osteopontin expression in platelet-derived growth factor-stimulated vascular smooth muscle cells and carotid artery after balloon angioplasty. Atheroscl Thromb Vasc Biol. 1996;16:1365–1372.[Abstract/Free Full Text]
  59. Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a component of human atherosclerotic plaques. J Clin Invest. 1993;92:1686–1696.
  60. Bochaton-Piallar M-L, Gabbiani G, Redard M, Desmouliere A, Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol. 1995;146:1–6.[Medline] [Order article via Infotrieve]