Effect of Platelet-Derived Growth Factor Receptor-α and -β Blockade on Flow-Induced Neointimal Formation in Endothelialized Baboon Vascular Grafts
Abstract—The growth of neointima and neointimal smooth muscle cells in baboon polytetrafluoroethylene grafts is regulated by blood flow. Because neointimal smooth muscle cells express both platelet-derived growth factor receptor-α and -β (PDGFR-α and -β), we designed this study to test the hypothesis that inhibiting either PDGFR-α or PDGFR-β with a specific mouse/human chimeric antibody will modulate flow-induced neointimal formation. Bilateral aortoiliac grafts and distal femoral arteriovenous fistulae were placed in 17 baboons. After 8 weeks, 1 arteriovenous fistulae was ligated, normalizing flow through the ipsilateral graft while maintaining high flow in the contralateral graft. The experimental groups received a blocking antibody to PDGFR-α (Ab-PDGFR-α; 10 mg/kg; n=5) or PDGFR-β (Ab-PDGFR-β; 10 mg/kg; n=6) by pulsed intravenous administration 30 minutes before ligation and at 4, 8, 15, and 22 days after ligation. Controls received carrier medium alone (n=8). Serum antibody concentrations were followed. Grafts were harvested after 28 days and analyzed by videomorphometry. Serum Ab-PDGFR-α concentrations fell rapidly after day 7 to 0, whereas serum Ab-PDGFR-β concentrations were maintained at the target levels (>50 μg/mL). Compared with controls (3.7±0.3), the ratio of the intimal areas (normalized flow/high flow) was significantly reduced in Ab-PDGFR-β (1.2±0.2, P<0.01) but not in Ab-PDGFR-α (2.2±0.4). Ab-PDGFR-α decreased significantly the overall smooth muscle cell nuclear density of the neointima (P<0.01) compared with either the control or Ab-PDGFR-β treated groups. PDGFR-β is necessary for flow-induced neointimal formation in prosthetic grafts. Targeting PDGFR-β may be an effective pharmacological strategy for suppressing graft neointimal development.
Neointimal formation in prosthetic vascular grafts narrows the lumen and affects long-term patency.1 Graft neointima is composed of extracellular matrix and vascular smooth muscle cells. It arises because of the migration of vascular smooth muscle cells into the graft lumen; once within the developing neointima, these cells proliferate and deposit a significant quantity of extracellular connective tissue matrix.2 The extent of neointimal formation in aortoiliac polytetrafluoroethylene (PTFE) grafts in baboons depends on blood flow.3 4 Grafts, under high-flow conditions (created by placement of a distal femoral arteriovenous fistula), form smaller neointimal layers than those that heal under normal flow.4 Ligation of the distal femoral arteriovenous fistula 2 months after graft insertion results in a ≈4-fold neointimal expansion within 28 days.3 5 6 Because blood viscosity and graft diameter remain constant in this model, graft blood flow directly influences the shear stress present at the endothelialized graft surface. Previous studies have shown that pressure-dependent wall stress is not affected by flow reduction in this model.3 4 The change in shear stress induced by fistula ligation initiates a subendothelial neointimal response marked by early smooth muscle cell proliferation (day 4).3
Whereas platelet-derived growth factor (PDGF)–B is nearly undetectable in the neointima of these grafts 4 days after normalization of flow, PDGF-A mRNA and protein increase and can be localized to the region of maximal smooth muscle cell proliferation (inner third of the intima, cells closest to the luminal surface).6 Healing baboon PTFE grafts perfused ex vivo release mitogenic activity that can be blocked with a polyclonal antibody to PDGF.7 8 In contrast to the changes in ligand expression, mRNA levels for both PDGF receptor-α and -β (PDGFR-α and -β) are increased after normalization of flow for 4 days. The role of these 2 receptors is presently being defined. This study tests the hypothesis that inhibition of PDGFR-α or PDGFR-β with specific mouse/human chimeric antibodies will modulate smooth muscle cell migration and proliferation in vitro and flow-induced neointimal formation in vivo.
Materials and Methods
Antibody Production, Administration, and Monitoring
Blocking murine/human chimeric antibodies to both PDGFR-α (Ab-PDGFR-α) and PDGFR-β (Ab-PDGFR-β) were generated by Celltech Therapeutics, Ltd.
In Vitro Effects of Ab-PDGFR-α and Ab-PDGFR-β
Quiescent baboon vascular smooth muscle cells were prepared as previously described.9 Incorporation of [3H]thymidine (1 μCi/mL; ICN) with and without Ab-PDGFR-α or Ab-PDGFR-β (3, 6, 12, 25, 50, and 100 μg/mL) was measured after stimulating the cells with serum (10% FCS) or PDGF-BB (10 ng/mL). Maximal stimulation was expressed as the fold increase over starved cells (DMEM only), and the effects of Ab-PDGFR-α and Ab-PDGFR-β were then expressed as a percentage of the stimulated control. Cell proliferation in response to serum (10% FCS) was assessed in the absence or in the presence of 4 concentrations of Ab-PDGFR-α or Ab-PDGFR-β (3, 6, 12, and 25 μg/mL). Cell counts were determined on days 1, 3, and 5 using a Coulter counter (Coulter Electronics Inc). Duplicate cell counts were averaged for 3 experiments. Mean cell counts were determined for each group for each day.
Migration Assay. Smooth muscle cell migration was assayed by a Boyden chamber method using a 48-well microchemotaxis chamber, as previously described.10 11 Twenty-five microliters of DMEM with PDGF-BB (10 ng/mL) was placed in the lower compartment. Two concentrations each of Ab-PDGFR-α or Ab-PDGFR-β (25 and 50 μg/mL) were applied to the upper compartment along with the cells. A nonspecific IgG antibody (25 and 50 μg/mL) was used as an additional control. Migration was determined as the mean of cells that had migrated per ×400 field and expressed as a percentage of DMEM control. All experiments were performed in triplicate.
In Vivo Effects of Ab-PDGFR-α and Ab-PDGFR-β at 29 Days
Seventeen male juvenile baboons (Papio cynocephalus), weighing ≈10 kg, received bilateral aortoiliac unwrapped PTFE bypass grafts (W.L. Gore & Associates, Inc) with ligation of the intervening native vessels and bilateral arteriovenous fistulae in the superficial femoral vessels, as previously described.3 Eight weeks after implantation, unilateral ligation of an arteriovenous fistula was performed. This intervention converted a graft with a high-flow state to one with normal flow, as previously described. The contralateral graft was maintained under high flow as a control. Control animals received vehicle whereas the remainder received either Ab-PDGFR-α (10 mg/kg) or Ab-PDGFR-β (10 mg/kg) by intravenous pulsed administration 30 minutes before the operative procedure and 4, 8, 15, and 22 days after the intervention.12 Serum samples were drawn preoperatively and at each time point before the booster injection thereafter. An ELISA system was used to quantify the trough level of Ab-PDGFR-α and Ab-PDGFR-β in the serum at each time point as previously described.12 On day 29 after fistula ligation, animals were euthanized, and tissue was obtained for study. Animal care complied with the Guide for the Care and Use of Laboratory Animals issued by the Institute of Laboratory Animal Resources.13
Standard morphometric measurements were performed on histological cross sections stained with hematoxylin and eosin. The mean luminal and neointimal area for each graft was determined by averaging the areas from 6 cross sections taken at equal distances along the graft, excluding the perianastomotic regions. The number of endothelial cell nuclei per mm circumference and the number of smooth muscle cell nuclei per mm2 identified in the neointima were counted in a minimum of 8 high-power fields (2 in each quadrant of the cross section).
Immunohistochemical procedures were performed according to the avidin-biotin-peroxidase method (Vector Laboratories). Endothelial cells were identified by von Willebrand factor staining (DAKO Corp), smooth muscle cells by α-actin (Boehringer Mannheim), macrophages by CD68 (DAKO Corp), and T lymphocytes by CD3 (DAKO Corp). A murine IgG monoclonal antibody was used as a negative control for all experiments. Levamisole was added to block endogenous alkaline phosphatase activity, and immune complexes were localized using the chromogenic alkaline phosphatase substrate Vector Red (Vector Laboratories). Bromodeoxyuridine (BrdU) labeling of proliferating smooth muscle cells in specimens was evaluated by staining tissue sections with a monoclonal antibody to BrdU (Boehringer-Mannheim). BrdU-stained smooth muscle cell nuclei were counted, and the BrdU labeling index (%) was calculated. Labeling of apoptotic smooth muscle cells in the same specimens was evaluated using terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling (TUNEL; Boehringer-Mannheim). Stained smooth muscle cell nuclei were counted, and the TUNEL labeling index (%) was calculated.
Data and Statistical Analysis
After determination of intimal area of each graft, the ratio of the neointimal areas in the normalized-flow and high-flow grafts was calculated. Similarly, data on nuclear density, BrdU, and TUNEL labeling were also converted to ratios of the normalized flow to the high-flow grafts. Ratio of normalized to high-flow grafts were used to decrease the influence of interanimal variability. Data are expressed as the mean±SEM, and statistical differences between these groups of data were tested with ANOVA with a post hoc Dunnett test. A P value <0.05 was regarded as significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
In Vitro DNA Synthesis, Cell Proliferation, and Migration
In baboon smooth muscle cells starved for 48 hours, addition of serum (10% FCS) or PDGF-BB resulted in 4.8±1.4-fold and 7.5±1.4-fold increases, respectively, in DNA synthesis compared with unstimulated cells. In the presence of Ab-PDGFR-α, there was a concentration-dependent decrease in serum or PDGF-BB–mediated DNA synthesis with IC50 25 and 13 μg/mL, respectively (Figure 1⇓). In the presence of Ab-PDGFR-β, there was also a concentration-dependent decrease in serum and PDGF-BB–mediated DNA synthesis with IC50 of 100 and 40 μg/mL, respectively (Figure 1⇓). Incubation with either Ab-PDGFR-α and Ab-PDGFR-β decreased cell proliferation in response to serum in a concentration-dependent manner (Figure 2⇓). Smooth muscle cell migration toward the chemoattractant PDGF-BB through a thin Matrigel membrane in a Boyden chamber was inhibited by Ab-PDGFR-β but not Ab-PDGFR-α in the upper chamber (Figure 3⇓).
In Vivo Effects of Ab-PDGFR-α and Ab-PDGFR-β at 28 Days
Circulating Blood Antibody Levels
Serum specimens from control animals were used as negative control samples for ELISA determination of Ab-PDGFR-α and Ab-PDGFR-β concentrations. No detectable antibody levels were found in the serum of control animals receiving vehicle alone. On the basis of previous in vitro and in vivo studies, the target serum concentrations of the Ab-PDGFR-α and Ab-PDGFR-β were determined to be 50 μg/mL. The average trough concentration of PDGFR-α antibody in the treated group was above the desired 50 μg/mL level at 4 and 8 days. Thereafter, they dropped to undetectable levels by day 15 (Table 1⇓). The average trough concentrations of PDGFR-β antibody in the treated group was above the desired 50 μg/mL level at all but the last time point during the study (Table 1⇓). No relationship existed between the degree of inhibition of neointimal thickening and each animal’s average serum antibody concentration for the 4-week period.
In the control group at 28 days, normalization of flow by ligation of the ipsilateral fistula induced a 3.1-fold increase in neointimal formation compared with the contralateral side (P=0.002; Table 2⇓, Figure 4⇓). In the Ab-PDGFR-α–treated group, there was a 2.2-fold increase in the neointima of the normalized flow graft after 28 days after fistula ligation (Table 2⇓, Figure 4⇓). In Ab-PDGFR-β–treated animals, the normalized-flow grafts showed only a 1.2-fold increase in the neointima over the contralateral high-flow graft in the same animals (Table 2⇓, Figure 4⇓). Compared with the control group, the ratio of neointimal area in the high- and normalized-flow groups was significantly decreased by the presence of Ab-PDGFR-β (P<0.01) but not Ab-PDGFR-α.
Using standard immunohistochemical techniques, graft cross sections were stained for the presence of endothelial cells, smooth muscle cells, macrophages, and T lymphocytes. The graft neointima in each group was completely endothelialized with a neointima made up almost entirely by smooth muscle cells. This is consistent with previous studies. Macrophages and T lymphocytes were present within the graft matrix of all grafts. No significant numbers of either cell type (<1 per high-power field) were seen within the neointima of any group. In addition, no discernible differences in the presence of either macrophages or T lymphocytes in the matrix were observed between the antibody-treated groups and the control group.
Endothelial Cell Layer
Normalization of flow in the control grafts did not induce any significant change in endothelial cell nuclear density in the control group. Similarly, there was no change in endothelial cell nuclear density between the Ab-PDGFR-α–treated or the Ab-PDGFR-β–treated groups (Table 3⇓). Endothelial cell proliferation and apoptosis were similar in all groups (Table 4⇓).
Smooth Muscle Cell Layer
The smooth muscle cell nuclear densities of the neointima in the control grafts increased significantly, with normalization of flow for 28 days (Table 3⇑). Similarly, total nuclear number was also increased by 8-fold (Table 3⇑). In the Ab-PDGFR-α–treated group, smooth muscle cell nuclear densities and total nuclear number were lower than the control group, regardless of flow conditions (Table 3⇑). The cell densities of the neointima in the Ab-PDGFR-β–treated groups were unchanged compared with the control groups, regardless of flow conditions (Table 3⇑). However, the total nuclear number was significantly reduced in the normalized flow group, as would be expected given the reduction in neointimal area (Table 3⇑). Both the high-flow and normalized-flow grafts exposed to Ab-PDGF-β showed significant increases in smooth muscle cell proliferation indices at 28 days. Apoptosis indices in the neointima of the Ab-PDGFR-α– and the Ab-PDGFR-β–treated grafts were similar to those of the control grafts (Table 4⇑).
PDGF Receptors and Response to Injury
PDGF consists of 2 polypeptide chains (A and B) that associate into 3 dimeric isoforms (AA, AB, and BB). In addition, there are 2 separate PDGF receptors, α and β. Dimerization of the receptors is required for high-affinity ligand binding, such that the α/α receptor binds all 3 PDGF dimers (AA, AB, and BB), the α/β receptor binds AB and BB, and the β/β receptor binds only PDGF-BB.14 Of the PDGF isoforms, PDGF-BB appears to play the most active role in intimal lesion formation after injury. PDGF-BB is a potent in vitro mitogen and induces a strong migratory response in vascular smooth muscle cells.15 16 Although weaker than PDGF-BB, PDGF-AA is also a potent mitogen for cultured baboon and rat smooth muscle cells. However, in a Boyden chamber used to measure cell migration, PDGF-AA inhibits PDGF-BB– and fibronectin-induced smooth muscle cells migration.15 17 Activation of the PDGFR-α by either PDGF-AA and PDGF-BB (in the presence of Ab-PDGFR-β) generates an inhibitory signal in baboon smooth muscle cells.15 The Ab-PDGFR-β can inhibit the migratory response to PDGF-BB and is an effective antiproliferative agent in response to either serum or PDGF-BB. The Ab-PDGFR-α cannot inhibit the migratory response to PDGF-BB but is an effective antiproliferative agent in response to either serum or PDGF-BB.
PDGF is an essential growth factor in the development of intimal hyperplasia.19 Administration of an anti-PDGF antibody in the rat carotid injury model inhibits intimal thickening by ≈40%.18 Neutralizing antibodies to PDGF-A have no observable effect after injury.19 Insertion of the PDGF-BB gene, the dominant PDGF isoform, into balloon-injured porcine arteries results in the increased development of intimal hyperplasia,20 whereas infusion of PDGF-BB has been shown to accelerate smooth muscle migration and intimal thickening but to have little effect on proliferation in the rat arterial injury model.21 The signaling mechanisms of PDGFR-α and PDGFR-β in vascular smooth muscle cells are distinctly different in that PDGFR-α can promote both cellular hypertrophy and hyperplasia, whereas PDGFR-β mediates a mitogenic and migratory response.16 Although quiescent arterial smooth muscle cells express 10 times more PDGFR-β than PDGFR-α, after serum stimulation, cell surface PDGFR expression decreases (loss of PDGFR-α is greater than loss of PDGFR-β).22 In control and 2-day rat injured carotid arteries, mRNA for PDGFR-α is readily detectable, but PDGFR-β expression remains very low. Between 2 and 7 days after the injury, PDGFR-α expression is slightly increased (35%), reaching a maximum at day 7. In contrast, PDGFR-β expression doubles over the same time period. From 7 to 14 days, there is a further increase in PDGFR-β expression, whereas PDGFR-α expression decreases.23 24 Both antibodies to PDGFR-β and antisense oligonucleotides to PDGFR-β significantly reduce intimal hyperplasia.12 24 25
The present study has demonstrated that human/murine chimeric antibodies to human PDGFR-α and PDGFR-β alter flow-induced neointimal formation in baboon prosthetic grafts. Ab-PDGFR-β inhibits its formation, whereas Ab-PDGFR-α alters its structural composition (a decrease in nuclear number). Each antibody-treated group represents the unopposed action of 1 receptor and the loss of function of the other. Administration of the Ab-PDGFR-α implies loss of PDGFR-α actions with unopposed PDGFR-β responses, whereas administration of the Ab-PDGFR-β implies loss of PDGFR-β activity with unopposed PDGFR-α responses. All Ab-PDGFR-α– and Ab-PDGFR-β–treated animals had adequate antibody concentrations during and immediately after flow reduction for the first 7 days. Previous work has demonstrated that the baboon initiates a systemic immune response after exposure to a chimerized antibody; this immunologic response probably accounts for the drop in Ab-PDGFR-α concentrations.12 Ab-PDGFR-β inhibited flow-induced neointimal formation. This result is in keeping with previous studies of balloon-injured arteries in the primate.12 25 26 Ab-PDGFR-α did not inhibit flow-induced neointimal formation but did significantly decrease smooth muscle cell density within the newly formed neointima. This report is the first experimental study to demonstrate a possible role for the PDGFR-α in the formation of neointimal formation. The mechanisms whereby these 2 antibodies achieved their effect are not clearly defined.
PDGF Receptors and Vessel Wall Development
During the development of the neointima, it appears that the smooth muscle cells revert to an early embryonic phenotype. Therefore, the study of the PDGF system in the embryo should yield clues to the role of PDGF receptors in the developing neointima. PDGF-AA, PDGF-BB, PDGFR-α, and PDGFR-β are independently regulated in the embryo and are required for organ development.27 Deletion of the PDGF genes in mice is lethal. Cumulative findings suggest that PDGF-A is crucial for alveolar myoblast ontogeny and required for cardiovascular development,28 whereas PDGF-B is required for renal mesangial cell ontogeny.29 PDGF-B–deficient mice have aberrant vascular development.30 PDGF-BB acting on PDGFR-α and PDGFR-β promotes lung growth, whereas PDGF-AA acting through PDGFR-α is necessary for lung branching.27 With respect to the kidney, PDGF-B and PDGFR-β promote mesangial development.29 PDGFR-α knockout mice have dysmorphic cardiovascular development with normal endothelium and reduced smooth muscle cell numbers in the wall.31 In the Ab-PDGFR-α–treated animals in the present study, we documented a similar phenomenon; inhibition of Ab-PDGFR-α during the acute phase of neointimal expansion after acute flow reduction resulted in a significant reduction in smooth muscle cell numbers and density.
PDGF, PDGF Receptors, and Blood Flow
Given the ability of the endothelium to act as a mechanical transducer, it is likely that the endothelium mediates in whole or in part the flow-induced neointimal response. A shear stress-responsive element has been identified for both PDGF-BB and PDGF-AA.32 Endothelial cell PDGF secretion is abluminal, allowing the endothelium to target adjacent smooth muscle cells, which are known to express the relevant receptor, PDGFR-β. In rat carotids, a reduction in blood flow produces an increase in endothelial cell proliferation, peaking at 72 hours.33 PDGF-A and PDGF-B increase without detectable changes in expression of either PDGFR-α or PDGFR-β within 48 hours.33 The stimulus for neointimal formation in this model is an alteration in shear stress on the luminal surface of the grafts. In these endothelialized grafts, there is no denuding injury or deposition of platelets, both of which are required for intimal hyperplasia after arterial injury.3 PDGFR-α and PDGFR-β mRNA expression in the neointima was increased after flow reduction.34 There was a significant 3-fold increase in PDGF-A mRNA and protein in the grafts, whereas PDGF-B mRNA was detected only in small amounts and did not appear to be affected by flow reduction.34 PDGF-B protein and mRNA are detectable in the matrix of the graft and appear to be mainly localized to macrophages. It is not known whether flow reduction results in increased PDGF-B production by these macrophages.6
In the control grafts, overall smooth muscle cell nuclear density increased with normalization of flow. In the Ab-PDGFR-β group, nuclear density was similar to that of the control group. The presence of the antibody significantly reduced the development of the neointima. We have demonstrated that the antibody can inhibit both cell proliferation and migration in vitro. We did not produce a reduction in neointimal formation with the Ab-PDGFR-α in the grafts after normalization of flow but did decrease smooth muscle cell density. Several possibilities exist to explain this effect. First, the marked reduction in serum trough concentrations, which resulted in a transient blockade of PDGFR-α, may explain the lack of inhibition. Second, PDGFR-α phosphorylation can occur in response to both mechanical forces (cyclic strain and shear stress) and is independent of ligand exposure and cannot be inhibited by antibodies against PDGFR-α.35 If the PDGFR-α can be activated by either a ligand or a mechanical force, there is a possibility that each induces a different cellular response and may point to the lack of a correlation between the in vitro and in vivo data. No direct evidence is available to support this hypothesis. Third, a decrease in neointimal smooth muscle cells may imply an increase in the synthesis of or a decrease in the degradation of the extracellular matrix in the neointima. The neointima is predominantly composed of versican, and we have demonstrated that its accumulation is flow dependent.36 37 Fourth, one can speculate that if PDGF-A/PDGFR-α acts as a survival system for smooth muscle cells in the neointima, the level of apoptosis in the early time period may have been significantly greater than that recorded in control grafts. In support of this hypothesis, the present data demonstrate a decrease in nuclear density in the normal- and high-flow grafts treated with Ab-PDGFR-α.
The present study examines 1 late time point in the continuum of the developing neointima after ligation of the fistula. Thus, any conclusions regarding differences in effect of PDGF-α and -β receptor blockade in this model must be highly qualified as representing a static as opposed to a kinetic view of neointima formation in these animals. The presumed action of Ab-PDGFR-β in decreasing migration and proliferation will require verification in shorter-term in vivo studies. One concern is the markedly elevated smooth muscle cell proliferation rates seen at the 28-day time point. This may reflect a loss of Ab-PDGF-β efficacy and the beginning of a “catch-up” phenomenon. We were surprised by the increase in the neointimal area of the high-flow grafts in the presence of both antibodies; although the changes did not reach statistical significance, there is a trend present. We have no explanation for this trend. The data on PDGFR-α are significantly complicated by the rapid clearance of the antibody from the circulation within 7 days. The reduction in neointimal cell density at the late time point, 21 days after the disappearance of the antibody, is both intriguing and perplexing; obviously, this phenomenon must be explored at earlier time points. We plan to conduct additional studies to define this unique observation with Ab-PDGFR-α.
In a primate model, pulsed administration of the Ab-PDGFR-β maintains effective serum trough concentrations of the antibody and has a sustained biologic effect—inhibition of flow-induced neointimal formation. This effect is probably due to the marked inhibition of PDGFR-β–mediated cell migration and the added effects of unopposed activated PDGFR-α (which inhibits smooth muscle cell migration). Although there was an early and dramatic decline in serum Ab-PDGFR-α concentrations, the administration of this antibody was associated with an unexpected biological response. Further studies to define the role and signaling mechanisms of PDGFR-α in altering smooth muscle cell nuclear density are obviously required. Targeting PDGFR-β may be an effective pharmacological strategy for suppressing neointimal development, and clinical trials of the Ab-PDGFR-β are planned.
This study was supported by US Public Health Service Grants HL30946, RR00166, and HL07828 and by ZymoGenetics, Inc. Antibodies against PDGFR-α and PDGFR-β were provided by Celltech Therapeutics; PTFE grafts were provided by W.L. Gore & Associates, and polypropylene suture by Davis & Geck. M.G.D. is a recipient of NIH Cardiovascular Training Grant Fellowship HL07828. D.P.M. was supported by the Pacific Vascular Foundation, and P.K.T. was supported by the Swedish Institute, the Swedish American Foundation, and the Swedish Heart Lung Foundation. We thank Debra Gilbertson for technical assistance with the chimeric antibody characterization.
- Received September 16, 1999.
- Accepted January 11, 2000.
- © 2000 American Heart Association, Inc.
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