PDGFβ Receptor Blockade Inhibits Intimal Hyperplasia in the Baboon
Background—We have evaluated the use of a mouse/human chimeric anti–platelet-derived growth factor-β receptor antibody in combination with heparin to inhibit intimal hyperplasia in the saphenous artery of the baboon after balloon angioplasty.
Methods and Results—The study evaluated lesion development in sequential injuries made 28 days apart. Each animal received control treatment after the first injury and antibody/heparin therapy after the second injury to the contralateral artery. The antibody was administered by bolus intravenous injections (10 mg/kg) on study days 1, 4, 8, 15, and 22 and heparin coadministered by continuous intravenous infusion at a dose of 0.13 mg/kg per hour. Morphometric analysis of tissue sections showed a 53% decrease in intimal area after antibody/heparin treatment (P=0.005), corresponding to a 40% decrease in the intima-to-media ratio (P=0.005). Smooth muscle cell proliferation in the injured wall, measured at both 4 and 29 days after balloon injury, were similar in the control and antibody/heparin-treated animals.
Conclusions—These data suggest that platelet-derived growth factor plays a key role in the development of intimal lesions at sites of acute vascular injury in the nonhuman primate.
Numerous studies have addressed the potential role of platelet-derived growth factor (PDGF) on the development of intimal hyperplasia at sites of acute vascular injury.1 2 3 4 With the use of an antiplatelet antibody to make rats thrombocytopenic, Fingerle et al2 demonstrated that platelets were required for the development of intimal hyperplasia in the rat carotid artery after balloon injury. Subsequently, Ferns et al3 demonstrated that administration of a goat polyclonal antibody that reacts with all 3 dimeric forms of PDGF (AA, AB, BB) was able to inhibit intimal hyperplasia in the rat carotid artery after balloon injury but had no effect on smooth muscle cell (SMC) proliferation. Jawien et al4 showed that addition of PDGF-BB to a rat after gentle injury to the carotid artery led to an increase in lesion size, probably by stimulating SMC migration. Recently Sirois et al1 used an antisense approach to address the role of the PDGF-β receptor (PDGFR-β) in lesion development in the rat balloon injury model. They observed that blockade of PDGFR-β production through local administration of a PDGFR-β antisense construct directly to the injured carotid artery also led to a marked decrease in the extent of lesion development.
PDGF ligand and PDGF receptors have been detected in developing vascular lesions in humans and baboons,5 6 7 8 but their function has not been defined in part because specific inhibitors have not been available. The development of a specific neutralizing antibody to the PDGF-β receptor now makes these studies possible.9 Both the α and β receptors, when activated, can mediate a mitogenic signal. In contrast, only activation of the β receptor appears to be capable of eliciting a chemotactic response in vascular SMCs. Blockade of the β receptor with a monoclonal antibody, followed by the addition of either PDGF-BB or PDGF-AA, activates the α receptor and leads to inhibition of SMC migration stimulated by fibronectin.9
Unfractionated and low-molecular-weight heparin are potent inhibitors of intimal hyperplasia in the rat balloon injury model.10 When low-molecular-weight heparin was used in a baboon balloon injury model, no inhibitory activity was observed on intimal lesion development, even at a dose of 0.6 mg/kg per hour over a 28-day period.11 However, we have recently shown that heparin, given together with a blocking antibody to the PDGF-β receptor, inhibits SMC migration from fresh aortic explants obtained from the baboon (R. Kenagy, C. Hart, A. Clowes, unpublished observation, 1997).
The observation that heparin treatment alone has no inhibitory activity but in association with anti-PDGF receptor antibodies blocked SMC migration provided the basis for the in vivo experiments described in this report.
Source of Antibodies
A murine monoclonal antibody, 184.108.40.206, was generated against the human PDGF-β receptor.9 12 A mouse/human chimeric version of this antibody, containing the variable domains for the heavy and light chains of the parent murine antibody, and the constant domains of human IgG4 and human κ for the heavy and light chains, respectively, was subsequently generated with the use of previously described methods.13 14 The antibody was formulated at 10 mg/mL in 50 mmol/L sodium acetate, 125 mmol/L NaCl, pH 5.0.
Source of Heparin
Porcine heparin, grade II, 157 U/mg (Sigma Chemical Co), was formulated into a 0.9% sterile saline solution. Two Alzet osmotic pumps (Alza Corp) were placed subcutaneously into each animal, and the heparin (0.13 mg/kg per hour) or vehicle control was delivered through a catheter inserted into the femoral vein.
In Vivo Study Protocols
Study 1 evaluated the ability of the chimeric antibody, in combination with heparin, to inhibit intimal hyperplasia in the saphenous artery of baboons, measured 29 days after balloon pullback injury. The study was divided into a control phase and an antibody treatment phase. For each study phase, a 10-cm section of 1 saphenous artery was injured with a 2F Fogarty embolectomy catheter as previously described.11 In the first phase, the animals received antibody vehicle control injections on study days 1, 4, 8, 15, and 22 after balloon injury and continuous intravenous infusion into the femoral vein of saline control. Study day 1 was the day of the surgery. On day 28 after the injury, the animals received 3 injections of bromodeoxyuridine (BrdU) (300 mg/dose IM) at 17 hours, 9 hours, and 1 hour before tissue collection. At study day 29, the injured artery was surgically removed and processed for tissue analysis. After removal of the first artery, the contralateral saphenous artery was immediately balloon-injured. Anti-PDGF receptor antibody was injected intravenously on study days 1, 4, 8, 15, and 22 at the dose of 10 mg/kg and heparin infused intravenously at a rate of 0.13 mg/kg per hour. Plasma and serum samples were obtained immediately before each subsequent antibody injection. On day 28 after the second injury, the animals received 3 injections of BrdU as previously described. At study day 29, the injured artery was surgically removed and processed for tissue analysis. Fifteen male animals (Papio cynocephalus), 7 to 12 kg in weight, were entered into the study. Ketamine (10 mg/kg) was used as a preanesthetic, and the animals were maintained on isoflurane during surgery. The operations were performed at Biosupport (Redmond, Wash), an AALAC-approved animal facility. The protocol was approved by the Animal Use and Care Committee at Biosupport, and all procedures followed Good Laboratory Practices as defined in current FDA, 21 CFR Part 58.
Study 2 used a sequential injury model to evaluate the ability of the chimeric antibody, in combination with heparin, to inhibit SMC replication at 4 days after balloon injury to the saphenous artery. After the first injury, the animals received a single bolus intravenous injection of antibody vehicle control (day 1) and infusion of saline through Alzet pumps into the femoral vein. On day 3, the animals received 3 injections of BrdU before tissue collection. On day 4, blood was drawn for serum collection, and the injured artery was surgically excised. The harvested artery was cut into multiple sections and processed for tissue analysis. Three pieces were placed into 10% formalin fixation for BrdU staining and morphometric analysis. The remaining segments were microdissected to separate the adventitia from the media, and the 2-vessel wall components were frozen separately for subsequent analysis. Adjacent noninjured saphenous artery and sections of carotid and axillary artery were also collected and similarly processed for analysis.
On day 4, before injuring the contralateral saphenous artery but after removal of the first injured artery, the animals received a single bolus intravenous injection of the chimeric anti–PDGF-β receptor antibody (10 mg/kg). The femoral vein was cannulated and heparin infused through Alzet pumps. At day 3 after the second injury, the animals received 3 injections of BrdU. On day 4, serum was collected and the second injured saphenous artery, along with noninjured saphenous, carotid, and axillary arteries, was excised and processed as described above. Five animals (Papio cynocephalus), weighing between 8 and 10 kg, were entered into study 2. The animals were preanesthetized with ketamine (10 mg/kg) and maintained under general isoflurane during surgery. All surgical procedures were done at the University of Washington Primate Center under guidelines approved by the University of Washington Animal Use and Care Committee.
ELISA to Measure Chimeric Anti–PDGF-β Receptor Antibody Levels
Ninety-six–well microtiter plates were coated with IgG/PDGFR-β fusion protein12 at 200 ng/mL diluted in coating buffer (0.1 mol/L Na2HCO3, pH 9.6). The plates were incubated overnight at 4°C, washed with ELISA C buffer (PBS, 0.05% Tween 20), then blocked with ELISA B buffer (PBS, 0.1% BSA, 0.05% Tween-20). Serum sample dilutions were made in ELISA B buffer. Standard curves were generated with purified chimeric antibody diluted into control baboon serum. Samples were added to duplicate wells and incubated at 37°C for 2 hours. The plates were washed to remove unbound antibody, and a 1:600 dilution of goat anti-human IgG4 antibody conjugated to horseradish peroxidase (Zymed) was added to the wells for 1 hour at 37°C. The wells were washed with ELISA C buffer, then incubated with OPD substrate solution (12.5 mL 0.1 mol/L Na citrate, pH 5.0, 5 mg o-phenylenediamine, 5 μL 30% H2O2). The reaction was stopped by the addition of 1N H2SO4 and the plates read at absorbance 490 nm in a Dynatech ELISA plate reader (Molecular Devices).
Activated Partial Thromboplastin Time Analysis to Monitor Levels of Circulating Heparin
Plasma was collected at various times after implantation of the Alzet osmotic pumps, and the heparin levels were monitored by activated partial thromboplastin time (APTT) analysis with the HEPTEST Assay (Product # 803, American Diagnostica) in an MLA Electra 800 clotting machine.
Morphometric Analysis and BrdU Staining of Arterial Tissue Sections
For study 1, the excised ballooned arteries were perfusion-fixed at 100 mm Hg ex vivo with 10% formalin. Pieces of artery were embedded in paraffin, and 5-μm sections were cut from each tissue block for morphometric analysis as described by Geary et al.11 Tissue sections from study 2 fixed in formalin were similarly embedded in paraffin, and sections were cut from each block for analysis. Sections from both study 1 and study 2 were stained with a monoclonal antibody against BrdU (Boehringer Mannheim Corp) with an immunoperoxidase method as previously described11 to measure the level of cell replication.
Extraction of Baboon Aortic Tissues
Frozen baboon arterial segments were placed in a glass tissue homogenizer and 1 mL of TNEN buffer (20 mmol/L Tris base, pH 8.0, 100 mmol/L NaCl, 5 mmol/L EDTA, 0.5% nonidot-40, 1 mmol/L PMSF, 50U/mL aprotinin) added per 0.25 g of tissue. The tissues were homogenized on ice over a 10-minute period, and the homogenate was transferred to an Eppendorf tube and spun for 5 minutes in a microfuge. The supernatant was harvested and frozen at −80°C until assayed.
Immunostaining to Detect Chimeric Antibody in Tissues
Histological cross sections of paraffin-embedded tissue were mounted on positively charged microscope slides (Superfrost Plus, Curtin Matheson). Slides were deparaffinized in xylene and rehydrated in graded alcohol solutions. Endogenous peroxidase was blocked with 3% H2O2, followed by enzymatic pretreatment with 0.1% trypsin in 0.05 mol/L Tris buffer (pH 7.6) for 30 minutes at 37°C. A peroxidase-conjugated mouse monoclonal antibody to human IgG4 (Zymed Laboratories, Inc) was applied to the slides overnight at 4°C at a dilution of 1:10 in 1% BSA in PBS buffer (pH 7.3). Bound antibody complexes were visualized with the use of diaminobenzidine as the chromogen.
Study Protocol Design
The addition of anti–PDGFR-β antibody 220.127.116.11 to baboon SMCs in culture causes a dose-dependent decrease in [3H]thymidine incorporation, with 25 μg/mL of antibody able to inhibit >50% of the mitogenic activity in baboon serum (data not shown). The addition of 10 μg/mL of heparin alone to these cells suppresses mitogenic activity by 31%. When the antibody is added to the cells in combination with heparin, a cooperative inhibitory effect is observed, such that a dose of 1 μg/mL of the antibody, in the presence of 10 μg/mL of heparin, has inhibitory activity equal to 25 μg/mL of antibody alone (data not shown). These results suggest that coadministration of heparin with the anti-PDGF receptor antibody would enhance the ability of the antibody to inhibit smooth muscle cell activation in the in vivo studies.
The in vivo studies described in this report were designed to approximate a clinical study for the evaluation of PDGFR-β blockade by antibody 18.104.22.168. Because of the cooperative effect between heparin and the anti–PDGFR-β antibody to inhibit SMC replication, we designed the study to deliver both antibody and low-dose heparin. A chart describing study 1 is presented in Figure 1⇓.
To minimize animal-to-animal variation in lesion development, we used sequential injuries within each animal for evaluation of the test compounds. A concern before the initiation of the study was what effect the excision of the first injured saphenous artery would have on lesion development in the contralateral injured artery. To address this question, a preliminary study was run in which 2 control animals received sequential balloon injuries to their saphenous arteries 29 days apart. The arteries were removed, the vessels perfusion-fixed ex vivo, and the tissues processed for morphometric analysis. Evaluation of intima/media ratios, as a measurement of lesion development, from cross sections of the paired arteries showed that there was no significant difference between the first and second injured arteries (data not shown).
Circulating Levels of Chimeric Antibody
Circulating chimeric antibody levels for study 1 were determined for serum samples collected on study days 8, 15, 22, and 29, immediately before the next antibody injection. The average circulating antibody level was 60.9±8.7 μg/mL (mean±SD) at day 8, with mean antibody levels decreasing to 36.1±26.5, 15.4±23.3, and 11.5±23.7 μg/mL on days 15, 22, and 28, respectively. The decrease in circulating antibody level with time appeared to be directly related to the extent of the immune response generated by the baboon toward the chimeric antibody (data not shown).
The analysis of plasma samples from study 1 showed a background clot time of 24.3±2.2 seconds (mean±SD). Heparin infusion, 0.13 mg/kg per hour, caused an increase in APTT that ranged from 2- to 4-fold and was maintained throughout the length of the study. Specific APTT times were 55.8±13.4, 46.9±17.2, 42.6±15.9, and 53.8±18.5 seconds (mean±SD) on study days 8, 15, 22, and 29, respectively. No bleeding complications were observed in the animals. The circulating levels of heparin ranged from 0.25 to 0.5 U/mL, corresponding to a level of 2 to 3 μg/mL of heparin.
Morphometric Analysis of Tissue Sections
Tissue sections from study 1 were obtained from multiple blocks of each test artery. Absolute intimal and medial areas were determined for each tissue section, and the data for all the blocks were averaged to give mean values for each animal. The data from 3 animals were eliminated from analysis because of the presence of occluding thrombi at the site of balloon angioplasty in either one or both arteries. A summary of the intima (I) and medial (M) areas and I/M ratios for the 12 remaining animals are given in Table 1⇓. The intimal areas were decreased significantly (53%) in the antibody-treated arteries (P=0.005). Analysis of the I/M ratios, to minimize differences in the absolute size of the arteries, also demonstrated a highly significant decrease (40%) in the antibody-treated animals (P=0.005). A photomicrograph of representative cross sections of injured arteries is shown in Figure 2⇓.
Cell Proliferation in Injured Vessels
Intimal and medial SMC proliferation, as determined by BrdU labeling (Tables 2⇓ and 3⇓), was not significantly different between the antibody-treated and vehicle control–treated arteries at either 4 or 29 days after injury. However, the total nuclear number was significantly decreased (P=0.007) in the intima of the antibody-treated arteries at 29 days.
Detection of Chimeric Antibody 163.3.1 in the Vessel Wall
High levels of the chimeric antibody were detected in the artery wall extracts as determined by ELISA (Figure 3⇓). The level of antibody in the medial portion of the injured saphenous artery was ≈8-fold higher than the level detected in the noninjured artery (952 ng/mg vs 124 ng of antibody/mg of total protein, respectively). The antibody level in the adventitia was also increased in the injured artery compared with the noninjured artery.
Frozen sections obtained from the saphenous artery of the antibody-treated animals were stained to localize the chimeric antibody in the artery wall. Strong staining for the chimeric antibody was observed in the media of the injured artery segments, whereas a lower level of staining was detected in the adventitia (Figure 4⇓). No staining was detected in adjacent noninjured tissue. The elevated levels of antibody in the adventitia measured by ELISA (Figure 3⇑), as compared with the lower levels detected by immunostaining, probably are due to the presence of trapped blood in the vasa vasorum, which would contain high antibody levels and would be included in the tissue extracts. Processing of the frozen tissue sections for immunostaining would eliminate trapped blood.
The studies reported in this article are unique in that it is one of the first times that inhibition of intimal hyperplasia has been demonstrated in a nonhuman primate model of vascular injury. This is a key point because the bulk of restenosis studies have been done in lower mammals including rats, rabbits, and pigs.15 16 17 18 Although many compounds have proven effective in these models to inhibit intimal hyperplasia, all of those further tested in humans have failed to maintain their efficacy.15 16 The published observations that heparin is an effective inhibitor of intimal hyperplasia in the rat19 but having no effect in the baboon11 further supports the concept that primates may respond differently to acute vascular injuries. Although it has been demonstrated that PDGF-β receptor blockade by either antisense or a selective tyrosine kinase inhibitor has been effective in blocking intimal hyperplasia in the rat and pig,1 20 it has not been clear that such a response would occur in a primate.
We believe that the baboon response most closely approximates the response that one will see in humans. Although the baboon model we used is not one of preexisting atherosclerotic injury, it is a model in which significant intimal hyperplasia occurs. From a clinical perspective, this most closely approximates the response seen after the placement of vascular stents. Restenosis occurring because of stent placement in humans is the result of true intimal hyperplasia in the absence of vessel wall remodeling associated with vasoconstriction. With the increased use of stents and the continued problem with in-stent restenosis, a specific inhibitor of intimal hyperplasia should have clinical utility. Our data suggest that an anti–PDGF-βreceptor antibody may be such a drug.
The decrease in intimal hyperplasia obtained with PDGF-β receptor blockade could be due to a variety of factors. The most likely explanation is inhibition of migration of SMCs from the media to the intima rather than inhibition of SMC replication. This is supported by the decrease in intimal cell number measured in the antibody-treated arteries (Table 2⇑), with no corresponding decrease in replication rate. A similar finding was made by Ferns et al,3 who used an anti-PDGF ligand antibody in a rat balloon injury model. This conclusion is further supported by the observation that systemic administration of PDGF-BB after injury to the rat carotid artery preferentially stimulates cell migration and not cell replication.4
When circulating levels of anti–PDGF-β receptor antibody were compared with intimal lesion size measured at 29 days, no correlation was observed (data not shown). This was true for all of the serum collection time points, suggesting that the level of circulating antibody present in the early stages of the study was sufficient to inhibit neointimal lesion formation measured at 29 days. Therefore, a shorter period of antibody administration than the 4 weeks used in this study might be equally effective. From the immunostaining studies, it is clear that the balloon injury provided for freer movement of the anti-PDGFR antibody out of the blood and into the artery wall, when compared with noninjured artery sections (Figure 4⇑). This allows the antibody to localize in the artery wall, thereby providing for sustained high concentrations of antibody at the site of the developing intimal lesion.
Although inhibition of PDGF-β receptor for 2 to 4 weeks is sufficient to give inhibition of lesion development at 29 days, it is not known if this inhibition persists at later times. The finding that a 12-hour administration of antibody 7E3 in humans after angioplasty therapy leads to a decrease in the need for repeat revascularization over a 1-year time period21 suggests that blockade of initiating events after acute vascular injury may have prolonged benefits regarding changes in vessel wall architecture.
When the size of the lesion at 29 days in the control artery was correlated with the difference in lesion size between the control and treated arteries (control intima minus treated intima) within each animal, a significant correlation was observed (r2=0.908) (Figure 5⇓). These results suggest that there may be a subgroup of hyperresponders within the baboon population, and it is those animals with the largest lesions in the control arm of the study that showed the biggest benefit from the antibody treatment. If this is the case, then it would be similar to the response seen in humans, in whom only 40% of the individuals undergoing balloon angioplasty exhibit restenosis.
One question that remains to be addressed from these studies is the contribution of heparin to the decrease in intimal thickening. In a previous study conducted in baboons, heparin alone at a 4-fold higher dose had no effect on intimal thickening.11 Because of this finding, we believe that the anti–PDGF-β receptor antibody−not the heparin−was responsible for the inhibition observed. The lack of efficacy by heparin to inhibit vascular restenosis has similarly been observed in human clinical trials.22 23 Although our cell culture studies showed a cooperative effect between the antibody and heparin to inhibit smooth muscle cell proliferation, we believe that the presence of heparin only allowed us to use lower doses of antibody. In data not presented in this article, we have determined that heparin has no direct effect on inhibiting the binding of PDGF to the PDGF-β receptor, nor has it any effect on the binding of antibody 22.214.171.124 to the PDGF-β receptor or on the ability of the antibody to block the binding of PDGF. It will be important to follow up these studies with a separate study looking at the effects of the antibody alone in the absence of heparin. With the use of a synthetic vascular graft model in the baboon,24 we have obtained preliminary data showing that the chimeric antibody alone is able to inhibit intimal hyperplasia (C.E. Hart and A.W. Clowes, unpublished observations, 1998).
In summary, the data obtained from this study indicate that PDGF plays a key role in regulating the extent of intimal hyperplasia at sites of acute vascular injury in the baboon. These findings have increased significance because they are one of the first studies to demonstrate an effective pharmacological strategy to inhibit intimal lesion development in a nonhuman primate model.
These experiments were supported in part by grants HL-30946 and RR-00166 from the National Institutes of Health, USPHS. We would like to acknowledge Annaleen Vermeulen, Jon Berry, Karinne Fuji, and Jim West for technical assistance.
The experiments described in this report were paid for in part by commercial funding from ZymoGenetics and Celltech Therapeutics. In addition, ZymoGenetics provided financial support to Dr Clowes’ laboratory.
- Received February 10, 1998.
- Revision received September 3, 1998.
- Accepted September 17, 1998.
- Copyright © 1999 by American Heart Association
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