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(Circulation. 1997;96:1291-1298.)
© 1997 American Heart Association, Inc.

Articles

Rat Arterial Wall Retains Myointimal Hyperplastic Potential Long After Arterial Injury

Martin G. Sirois, PhD; Michael Simons, MD; David J. Kuter, MD; Robert D. Rosenberg, MD, PhD; ; Elazer R. Edelman, MD, PhD

From Harvard-MIT Division of Health Sciences and Technology (M.G.S., E.R.E.) and Department of Biology (D.J.K., R.D.R.), Massachusetts Institute of Technology, Cambridge, Mass, Brigham and Women's Hospital (E.R.E.) and Beth Israel Hospital Departments of Medicine (M.S., R.D.R.), Harvard Medical School, Boston, Mass.

Correspondence to Martin G. Sirois, PhD, Research Center, Room S-5450, Montreal Heart Institute, 5000 Belanger St, Montreal, Quebec, Canada.

	Abstract

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Background Many novel molecular and pharmacological modalities have been proposed for the treatment of accelerated vascular diseases. Yet the fundamental question remains of whether the vessel wall can be treated once only or whether single-dose therapy simply delays the inevitable processes that lead to intimal hyperplasia. Since platelet adhesion and aggregation are critical events in vascular healing, we sought to determine whether the injured blood vessel would retain its myointimal potential after reversal of even prolonged periods of thrombocytopenia.

Methods and Results A novel nonimmune method sustained thrombocytopenia and suppressed postinjury neointimal hyperplasia by 88%. Infusion of fresh platelets, even 14 days after initial denuding injury, restored the full neointimal hyperplastic potential. Platelet depletion presumably removed factors chemotactic for vascular smooth muscle cells but had no effect on the overexpression of the platelet-derived growth factor receptor-ß (PDGFR-ß) subunit after vascular injury. In native vessels, 26.5±2.5% of medial smooth muscle cells expressed PDGFR-ß. In all animals, medial PDGFR-ß expression doubled 2 weeks after endothelial denudation and was evident in up to 74.5±2.5% of the cells forming the neointima.

Conclusions Thus, though the hyperplastic potential of the injured blood vessel can be delayed with removal of growth stimuli, it is not lost forever, and if the media is not made quiescent, neointimal hyperplasia is simply delayed rather than prevented. These results may have a profound effect on our understanding and treatment of accelerated proliferative vascular diseases.

Key Words: busulfan • platelet-derived factors • restenosis • cells, muscle, smooth • thrombocytopenia

	Introduction

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Myointimal hyperplasia remains a critical limitation to the mechanical relief of obstructive vascular disease. Despite continued advances in the design and use of endovascular catheters and devices, disease recurs in 30% to 50% of patients who undergo any form of angioplasty.^{1 2} The great paradox in this field is the failure of promising chemotherapeutic agents to alter the course of these accelerated arteriopathies. Although a wealth of positive tissue culture and animal experiments exists for compounds that might suppress myointimal growth, there has not been one single clinical trial with demonstrable benefit. One possible explanation for the discrepancy between preclinical and human trials is a difference in the mode and target of therapy. Virtually all preclinical trials provide a continuous level of drug for the duration of the animal's life after injury. As such, the treatment is naturally directed against the response to injury, including phenomena such as cell proliferation and migration. Drug administration is by necessity different in the human. Compounds can be administered only intermittently and for a defined period of time, which raises the question of whether such limited therapy is sufficient to suppress forever the intimal hyperplasia. To date, few investigations have addressed the question of whether therapeutic failure might result from the reemergence of myointimal potential once transient pharmacological suppression is released.

The injured blood vessel is especially sensitive to platelets. Loss of endothelial integrity is followed almost immediately by the adhesion, aggregation, and activation of platelets on the exposed subendothelial connective tissue. The platelet -granules secrete a range of potent growth factors including IGF-1, EGF, TGF-ß, and PDGF.^{3 4 5 6} These compounds elicit a cascade of events that culminate in the activation, phenotypical transformation, migration, and proliferation of VSMCs.^{7 8 9} If these events are of a sufficient magnitude, they can create a neointima that obstructs blood flow. Previous experiments with immune-mediated thrombocytopenia demonstrated that daily injections of antiplatelet antibodies sufficient to reduce platelet counts to <5x10³/µL led to inhibition of intimal thickening in the injured rabbit aorta.¹⁰ If the thrombocytopenia was limited to the first 24 hours after injury, early VSMC migration was inhibited without effecting proliferation, and although intimal hyperplasia was reduced compared with controls at 4 and 7 days, there was no effect at experiment termination 14 days after injury.¹¹

The use of antiplatelet antibodies is, however, constraining. The effects of isolated injections are transitory, and platelet counts return to baseline within 24 hours. Daily antiplatelet antibody injections are required to sustain thrombocytopenia but at an increased risk of anaphylactic shock and death.¹⁰ Busulfan, a bone marrow precursor cell-specific toxin, offers a more flexible alternative. This agent induces a nonimmune thrombocytopenia that eliminates the need for repeated injections, reduces dramatically the incidence of anaphylaxis, and enables immediate reversal of the platelet-depleted state with platelet transfusion. Under normal conditions, neointima formation occurs within the first 2 weeks after vascular arterial injury in a process that is from an early stage dependent on the presence of platelets. We wondered whether the absence of platelets for the first 2 weeks after vascular injury would be sufficient to prevent neointima formation and vascular healing or if the media would remain responsive to platelets once reintroduced into the bloodstream. For this purpose, we used the busulfan treatment model to explore the role of vascular wall sensitivity to platelet-generated stimuli in a rat carotid injury model.

	Methods

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Induction of Intimal Hyperplasia
Balloon denudation of common carotid arterial endothelium was performed in male Sprague-Dawley rats (350 to 425 g; Charles River Breeding Laboratories, Kingston, Mass) as previously described.¹² Animals were euthanatized at different periods after injury (Fig 1) with an overdose of ketamine and xylazine and were then exsanguinated and perfused with 50 mL of Ringer's lactate solution. The treated segment of the common carotid artery was removed, cut into two equal segments, and fixed in 10% formalin solution. The segments were embedded in paraffin, and eight sections of 6 µm were obtained by microtome along the length of the specimen. Sections were stained with Masson's trichrome and the areas of the intima, media, and adventitia, the intima-media area ratio, and the percent luminal occlusion were calculated for each arterial segment using computerized digital planimetry with a dedicated video microscope and customized software. The nature of specimen treatment was kept blinded during the data analysis. Expression of PDGFR-ß was followed immunohistochemically with a rabbit anti-human PDGFR-ß IgG (UBI), which possesses cross-reactivity for the rat PDGFR-ß, as determined by dot-blot and Western blot analyses. The antibody was diluted (1:1000) and applied for 90 minutes. An equivalent concentration of a nonspecific rabbit IgG (Vector Laboratories Inc) was used as a control antibody and did not stain the arterial specimens. A biotinylated goat anti-rabbit IgG (1:400) (Dako) was applied for 45 minutes. Peroxidase labeling was achieved with incubation using avidin-peroxidase complex (Vector Laboratories), and antibody visualization was established after a 5-minute exposure to 0.05% 3,3'-diaminobenzidine (Sigma) in 0.05 mmol/L Tris-HCl at pH 7.6 with 0.003% hydrogen peroxide. The arteries were counterstained by rapid immersion (25 seconds) in Gill's hematoxylin No. 3 solution and rinsed in distilled water.

View larger version (18K): [in this window] [in a new window]   Figure 1. Flow diagram of the experimental protocol. In control animals balloon injury (BI) of the common carotid arteries was performed and the animals sacrificed 7, 14, or 28 days later (group A). In three different experimental groups (groups B through D), rats were injected intraperitoneally with busulfan on two separate occasions 3 days apart (20 mg/kg per injection), inducing a thrombocytopenic state. Balloon injury was performed on the 13th day after the first busulfan injection. In group B, rats remained thrombocytopenic after BI and were euthanatized at day 7 or 14 after injury. In group C, rats were infused with concentrated platelets 6 hours before BI and every other day thereafter to maintain a platelet count >5x105 platelets/µL. These animals were euthanatized 7 or 14 days after injury. In group D, the rats remained thrombocytopenic for the first 14 days after BI, and only then were concentrated platelets infused every other day as required to maintain a platelet count above 5x105 platelets/µL. These animals were euthanatized 28 days after injury. Six rats were examined in each group at each time point.

Induction of Sustained Thrombocytopenia
To evaluate the duration of medial responsiveness after endothelial denudation, we developed a model of nonimmune thrombocytopenia in the rat. As described previously,¹³ busulfan was prepared at a concentration of 100 mg/mL in polyethylene glycol (PEG) (Sigma) and stirred for 2 hours at 20°C. This suspension was then brought to a final concentration of 10 mg/mL in PEG and stirred for 2 hours at 75°C. Rats were anesthetized with ether inhalation and injected intraperitoneally with busulfan on two separate occasions 3 days apart (20 mg/kg per injection). While the rat was under ether-anesthesia, 250 µL of caudal venous blood was collected into a 25-gauge needle base for cell counts. Platelet and white blood cell counts were performed using the Unipette collection system (Becton-Dickinson) and determined with a hemacytometer.¹⁴ Balloon catheter arterial injury was performed when the platelet count was between 2x10⁴ and 4x10⁴ platelets per microliter. Some thrombocytopenic rats had prolonged bleeding and anemia after the surgery. To eliminate anemia as a potential modulator of neointima formation by keeping the hematocrit value >30%, rats were transfused with concentrated red blood cells from surgically untreated thrombocytopenic animals. Animals were euthanatized at 7 and 14 days after the carotid vascular injury and tissue harvested (Fig 1; group B).

Two sets of controls were performed. First, to determine whether busulfan had an effect on neointima formation, busulfan-treated thrombocytopenic rats were transfused with concentrated platelets. In brief, blood was obtained from donor rats (500 g) that had not been exposed to busulfan. Rats were anesthetized and then exsanguinated by cardiac puncture. Nine parts blood were collected into one part sodium citrate (3.8%; final concentration, 0.38%), and the anticoagulated blood was immediately centrifuged for 8 minutes at 500g. The supernatant platelet-rich plasma was then carefully removed. The remaining pelleted blood cells were twice resuspended to the original volume with Hanks' balanced salt solution without calcium or magnesium and centrifuged as before. The supernatants were removed and added to the original platelet-rich plasma. The pooled platelet suspension was centrifuged for 15 minutes at 3000g at 4°C, and the platelet pellet (containing 19.5x10⁹ to 25x10⁹ platelets) was resuspended in Hanks' balanced salt solution (15x10⁹ platelets/mL). From anticoagulated blood, 70% of the total platelets and <1% of the total white blood cells were recovered by this method. Platelet injections (15x10⁹ platelets/mL) were started 6 hours before arterial injury and repeated 2, 4, 6, 10, and 12 days after surgery (Fig 1; group C). This procedure maintained platelet counts at >5x10⁵ platelets/µL. The medial responsiveness of injured arteries was examined in another set of busulfan-treated thrombocytopenic rats with thrombocytopenia that was reversed 14 days after injury with the transfusion of fresh platelets from rats unexposed to busulfan. Platelet transfusions were continued every other day until the platelet count rose to >5x10⁵ platelets/µL, and rats were euthanatized 2 weeks later at day 28 after initial vascular injury (Fig 1, group D). It is important to note that busulfan was administered no less than 10 days before vascular injury, and because this compound possesses a serum half-life of 2 to 3 hours,¹⁵ it could not be present at the time of intervention. Moreover, no prolonged effect or immunoreactivity from these infusions was noted, since platelet infusions reproducibly and consistently elevated platelet counts.

Statistical Analysis
Data are mean±SE. Statistical comparisons were determined by ANOVA followed by an unpaired Student's t test with Bonferroni's correction for multiple comparisons. Data were considered to be significantly different if P<.05 was observed.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Induction of Thrombocytopenia by Busulfan Treatment
Busulfan (20 mg/kg IP) was injected twice within 72 hours. Platelet counts were reduced to <2% of normal (2x10⁴ platelets/µL) within 13 days after the first busulfan injection and were sustained for an additional period of 12 to 13 days (Fig 2). By day 14 after injury (27 days after busulfan injection), platelet counts in non–platelet transfused animals had risen to 2.32x10⁵±0.71x10⁵/µL and by day 28 after injury (41 days after busulfan injection) to 9.77x10⁵±0.99x10⁵/µL. In the control animals unexposed to busulfan platelet counts before vascular injury, platelet counts at baseline and days 14 and 28 after injury were 13.81x10⁵±0.85x10⁵/µL, 12.90±1.21x10⁵/µL, and 12.7±0.89x10⁵/µL, respectively. At the busulfan concentration and dosing regimen noted, leukocyte counts remained at 10 255±439/µL throughout the course of the experiments from the day of injection until last harvest and were statistically indistinguishable from control values of rats untreated with busulfan (10 239±1358/µL). In the absence of vascular injury, the erythrocyte count was not affected by busulfan treatment (data not shown). Blood vessels treated with busulfan continued to express constitutive receptors such as PDGFR-ß and demonstrated none of the classic cytotoxic effects, such as pyknosis, fibrosis, or vacuolization (Figs 3A and 5A).

View larger version (17K): [in this window] [in a new window]   Figure 2. Induction of sustained thrombocytopenia in rats by a nonimmune approach. Intraperitoneal injections of busulfan (20 mg/kg for each injection) steadily reduced platelet counts within 13 days after the first busulfan injection and maintained the thrombocytopenic state for an additional 12 to 13 days. Values are mean±SE for 5 or 6 animals.

View larger version (158K): [in this window] [in a new window]   Figure 3. Effects of thrombocytopenia and platelet transfusion on restenosis development. Vascular injury was performed with the passage of an inflated balloon catheter, and intimal thickening was assessed on Masson-Trichrome–stained specimens. Representative sections are presented for the common carotid artery in its native uninjured state (A) and 14 days (B) and 28 days (C) after denudation. In another group of studies, rats were treated with busulfan to induce a thrombocytopenic state as in Fig 2. Once thrombocytopenic, the common carotid artery was ballooned, and the rats were euthanatized 14 days later (D). To determine whether busulfan treatment had an adverse effect on the capacity of the medial cells to induce intimal thickening, another group of thrombocytopenic rats was transfused with platelets 6 hours before vascular injury and every other subsequent day; the animals were euthanatized 14 days later (E). In the last group, the busulfan-treated rats were kept thrombocytopenic for the first 14 days after vascular injury and only then were transfused with platelets every other day for the next 14 days; these rats were euthanatized 28 days after the initial injury (F). Original magnification x100.

View larger version (153K): [in this window] [in a new window]   Figure 5. Expression of PDGFR-ß in the media and intima of injured carotid artery after various treatments. PDGFR-ß expression in common rat carotid arteries was revealed with standard immunohistochemistry techniques. The cross reactivity of rabbit anti-human PDGFR-ß IgG with rat PDGFR-ß was determined by dot-blot and Western blot analyses. Positive expression of PDGFR-ß was revealed by brown staining of medial and intimal cells on treatment of the samples with anti–PDGFR-ß antibodies; the nonspecific PDGFR-ß staining was evaluated by replacing the primary antibodies with nonspecific rabbit IgG antibodies (B, left panel). The uninjured artery served as control for basal PDGFR-ß expression (A). The expression of PDGFR-ß in both media and intima at day 14 and 28 after injury (B [right panel] and C). PDGFR-ß expression was also revealed in busulfan-treated rats. For the group of busulfan-treated rats kept thrombocytopenic for the full 14 days after vascular injury, the media formation was minimal or absent and PDGFR-ß expression was observed in the media (D). Transfusion of platelets 6 hours before vascular injury and every other day for 14 days induced intimal thickening. PDGFR-ß expression was observed in the media and the neointima (E) when platelet transfusion was delayed for the first 14 days after injury and then infused every other day from day 14 until day 28; intimal thickening developed and PDGFR-ß was expressed in both media and intima compartments (F). Concentration of the anti–PDGFR-ß antibodies was 1:1000 for all specimens. Black arrow indicates internal elastic lamina in all sections. Original magnification x400.

Neointimal Hyperplasia and Medial Responsiveness to Platelets
As expected, no intimal thickening was observed in rat common carotid arteries whose endothelial surface was left intact (Fig 3A). The extent of neointima formed at days 7, 14, and 28 in animals subject to balloon deendothelialization served as control data for all subsequent experiments (Figs 3B, 3C, and 4). At these times, intima-media area ratios of 0.89±0.09, 1.37±0.15, and 1.71±0.15 were observed (Fig 4). Busulfan-treated thrombocytopenic rats developed virtually no neointima after vascular injury. In these animals, the intima-media area ratios were 0.1±0.04 and 0.17±0.06 at 7 and 14 days after injury, representing reductions of 89% and 88% from control values (P<.001, Figs 3D and 4). Inhibition of neointima was mediated by the absence of platelets rather than by busulfan. When busulfan-treated thrombocytopenic rats received fresh platelets from normal rats 6 hours before vascular injury and every other day afterward, intima-media area ratios were 0.69±0.18 and 1.00±0.2 at 7 and 14 days after injury. These values were not statistically different from those observed in control rats (Figs 3E and 4). Most importantly, the media retained its ability to respond to infused platelets even 2 weeks after injury. If the busulfan-treated rats were kept thrombocytopenic for the first 14 days after vascular injury and only then reinfused with platelets for 2 weeks until they were euthanatized at day 28, the intima-media area ratio was 0.95±0.18 (Figs 3F and 4). The area of this neointima was no different from the value observed at 14 days after injury in control rats with intact platelets (1.37±0.15) (Figs 3B and 4). Medial areas were no different in any treated groups, including the control and thrombocytopenic rats (Fig 5). We could not determine the effect of a busulfan-thrombocytopenic state for 28 days after injury, since the platelet count in busulfan-treated rats started to rise spontaneously at 14 days after injury and returned to normal levels within the next 2 weeks. Finally, none of the animals in any group subjected to denuding injury exhibited endothelial regeneration.

View larger version (30K): [in this window] [in a new window]   Figure 4. Effects of thrombocytopenia and platelet transfusion on restenosis development. Black columns represent the intima-media area ratio after carotid arterial injury with a balloon catheter in normal rats not exposed to busulfan treatment. Grey columns represent the data for busulfan-treated thrombocytopenic rats transfused with fresh platelets every other day after vascular injury. The hatched columns represent the intima-media area ratio of busulfan-thrombocytopenic rats that were not transfused with platelets after a vascular injury. The white column represents the intima-media area ratio in rats that were thrombocytopenic for the first 14 days after vascular injury and then transfused with fresh platelets every other day for 2 weeks. Values are mean±SE of 5 or 6 animals. *P<.05; **P<.01 compared with respective busulfan-untreated rats (black column).

PDGFR-ß Expression
Platelets are a primary source of PDGF-BB, a critical chemotactic factor for VSMCs. To determine whether thrombocytopenia mediated by busulfan treatment had an effect on PDGFR-ß level, we quantified PDGFR-ß expression immunohistochemically with a rabbit anti-human PDGFR-ß IgG that cross reacts with rat PDGFR-ß. Expression in medial VSMC doubled after vascular injury, rising within 14 days from 26.5±2.5% in control noninjured vessels (No Injury) to 51.2±5% (P<.001), and 74.5±2.5% of the neointimal cells expressed the subunit (BID14; Figs 5A, 5B, 6A, and 6B). Four weeks after injury, medial PDGFR-ß expression returned to basal levels (27.0±2.4%) and intimal values fell to 32.6±3.2% (BID28; Figs 5C, 6A, and 6B). In thrombocytopenic rats, the inhibition of intimal hyperplasia after a vascular injury stemmed from the absence of platelets and not from the suppression of medial PDGFR-ß expression. Medial PDGFR-ß expression 14 days after balloon denuding injury in busulfan-treated animals rose 2.2-fold to 57.6±4.5% [Plts (-) D14; Figs 5D and 6A], an increase indistinguishable from expression in control animals not exposed to busulfan (BID14). Busulfan alone did not change PDGFR-ß expression or effect VSMC proliferation or migration. Platelet transfusions initiated 6 hours before vascular injury and repeated every other day afterward in busulfan-treated animals [Plts (-/+) D14 restored the neointimal thickening after vascular injury (Figs 3E and 4). Moreover, PDGFR-ß expression observed in both the media (42.4±5.2%; Figs 5E and 6A) and the intima (63.2±3% Figs 5E and 6B) were statistically indistinguishable from control animals untreated with busulfan (BID14). In busulfan-treated rats that were kept thrombocytopenic for the first 14 days after the injury and only then transfused with platelets every other day until day 28 (Plts (-/+) D28), the PDGFR-ß expression returned to basal level in the media (26.0±5.0; Figs 5F and 6A) and intima (34.0±5.2; Figs 5F and 6B) in a manner indistinguishable from that observed in the control rat unexposed to busulfan treatment (BID28) (Figs 5C, 6A, and 6B).

View larger version (39K): [in this window] [in a new window]   Figure 6. Expression of PDGFR-ß in the media (A) and intima (B) of injured carotid artery after various treatments. Total cell counts were determined from nuclear localization of the counterstain. PDGFR-ß–expressing cells were distinguished from cells that stained brown with nonspecific rabbit IgG antibody. The latter was exceedingly rare and counted for <1% of the cells. The expression of PDGFR-ß was then presented as a percentage of the total cells. Four subsections of each artery were evaluated in 5 or 6 rats per group. Determinations were made only if at least 100 cells were present in the media or intima. In the absence of injury (No Injury), 26.5±2.5% of all medial cells stained positively (brown staining), which represents the basal expression of PDGFR-ß. 14 days after a balloon-denuding injury (BID14), the PDGFR-ß expression doubled on medial VSMCs and was evident in 75% of the cells forming the neointima. By day 28 after the injury (BID28), the medial expression of PDGFR-ß returned to the basal level; it decreased to 32% in the intima by day 28 (BID28). In busulfan-treated animals kept thrombocytopenic for a full 14 days after the injury [Plts (-) D14], the medial PDGFR-ß expression was statistically indistinguishable from the balloon-injured control rats (BID14), whereas no significant neointima was formed. Platelet transfusion from the day of the surgery until day 14 [Plts (-/+) D14] restored myointimal proliferation in these busulfan-treated animals and maintained the overexpression of the receptor subunit in both vascular compartments. In busulfan-treated rats that received platelet transfusions from days 14 to 28 [Plts (-/+) D28], PDGFR-ß expression was reduced to values observed in rats not treated with busulfan and 28 days after vascular injury. *P<.05 and ***P<.001 compared with noninjured rats (No injury); P<.001 compared with normal rats subject to balloon-arterial denudation (BID14).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
One of the fundamental questions in vascular biology is whether treatment of an injured artery for a brief period will forever halt the natural progression of events leading to luminal obstruction or have only a limited temporal effect that simply delays the inevitable hyperplastic response to injury. The data reported here support the latter and portend gravely for our ability to deal fully with the burden of proliferative vascular disease with transient interventions. It is possible that complete and permanent inhibition of intimal hyperplasia may not be achieved without complete restoration of vascular homeostasis.

Platelets play a critical role in many aspects of the vascular response to injury, and induction of thrombocytopenia with subsequent reintroduction of platelets presents a convenient way to examine the issue of vascular responsiveness. Previous studies demonstrated that platelets adhere rapidly to the denuded arterial wall.¹¹ Single injections of antiplatelet antibodies inhibited platelet adhesion for the duration (24 hours) of the thrombocytopenic state. During the next 24 hours, platelets returned to normal levels and adhered to the denuded carotid area. Intimal hyperplasia was reduced within the first week of injury, but the lesion size was indistinguishable from controls at 2 weeks.¹¹ Only the sustained thrombocytopenia that followed repeated antibody injections for the duration of the experiments prevented intimal hyperplasia in the long term.¹⁰ It is generally accepted that the depletion of circulating platelets inhibits development of the intimal lesion by suppressing migration but not proliferation of medial VSMCs.¹¹ It was also concluded that platelet adhesion to subendothelial connective tissue any time within the first 2 days after injury will still elicit the cascade that culminates in neointima formation and that thrombocytopenia extending through the peak period of VSMC migration and proliferation would eliminate this effect.^{16 17}

However, no study has yet examined whether the blood vessel could still respond to a platelet challenge after sustained thrombocytopenia; for example, the 2-week period during which the hyperplastic response to injury is completed under normal conditions.¹⁸ In our study, busulfan, a chemotherapeutic agent that inhibits platelet production, was used to induce a thrombocytopenic state.¹³ Within 13 days after the first injection of busulfan, the rats became thrombocytopenic and remained so for an additional 14 days. After that period, platelet synthesis was restored spontaneously, and platelet counts returned to normal levels within the next 2 weeks. The prolonged action of this drug eliminated the need for repeated antibody administration that can lead to anaphylactic shock and death,^{10 11 18} and its specificity for platelets at the doses used allowed platelet reinfusion at any time. At the same time, the short half-life (2 to 3 hours) of busulfan¹⁵ ensured that it would be fully eliminated by the time vascular injury was performed. As a result, busulfan treatment had no demonstrable effect on leukocytes or erythrocytes, the bioactivity of existing or transfused platelets, or the migration or proliferation of VSMCs at any time after platelet transfusion. Blood vessels treated with busulfan demonstrated none of the typical cytotoxic effects, such as pyknosis, fibrosis, or vacuolization, and continued to express PDGFR-ß in response to injury.

The sustained thrombocytopenic state virtually eliminated neointima formation 14 days after vascular injury. Transfusions of fresh platelets from the day of vascular injury reversed the thrombocytopenic state and permitted intimal thickening equivalent to controls. Most significantly, neointima could still be formed even when platelet transfusions were delayed for 2 weeks after injury. The neointima that formed after platelet infusion from day 14 up to day 28 was virtually identical to the extent of the lesion observed in control animals or in thrombocytopenic rats infused with platelets from day 0 to day 14 after vascular injury (Figs 3 and 4). Thus, the thrombocytopenic media retained its platelet responsiveness beyond the theoretical peak limit of VSMC activation observed in control conditions.¹⁶

If the myointimal hyperplastic potential requires the presence of specific ligands, receptors, and injured environment, then platelet transfusions even 2 weeks after injury in the previously thrombocytopenic animals enabled all of these components to be present. Adhesive platelets to subendothelial matrix secrete a variety of growth factors, including all three isoforms of PDGF (-AA, -AB, and -BB),¹⁹ although activated rat platelets primarily release PDGF-BB.^{20 21} Both, PDGF-AA and PDGF-BB are mitogenic for cultured VSMCs through the activation of the appropriate PDGF receptor.^{22 23 24} However, while activation of PDGFR-ßß stimulates migration and proliferation, activation of PDGFR- inhibits migration.²⁵ The same effects were noted in vivo when PDGF-BB stimulated both VSMC migration and proliferation after vascular injury, though its chemoattractant effect was estimated to be at least 10-fold greater than its mitogenic potential.^{26 27} Furthermore, receptor subunit expression is minimal in quiescent VSMCs of native arterial wall, falls within 24 hours of vascular injury, and then rises considerably during the next 2 weeks.²⁸ Consequently, it might be important to measure and regulate the expression and stimulation of PDGFR-ßß.

PDGFR-ß expression was therefore used as a marker of vascular responsiveness and vessel wall quiescence (Figs 5 and 6). In our model, PDGFR-ß was present in 25% of the VSMCs in the uninjured media. Receptor expression rose significantly within the first 2 weeks after vascular injury and diminished to or near basal levels during the next 14 days. Thrombocytopenia had no effect on this pattern. Two weeks after injury and busulfan treatment, PDGFR-ß expression was evident in >60% of medial VSMCs. Thus, when platelets bearing and capable of releasing potent stimuli such as growth factors and chemoattractants are reintroduced to the still-injured blood vessel overexpressing the PDGFR-ß, myointimal hyperplasia becomes virtually inevitable. For hyperplasia to be reversed, vascular homeostasis and quiescence must be restored. It is important to note that by 28 days after injury, PDGFR-ß expression had returned to control values even in the group of rats kept thrombocytopenic for the first 14 days.

We and others have demonstrated that the most potent means of establishing vascular homeostasis after injury is with restoration of the denuded endothelium.^{29 30 31 32} It is interesting to note that the endothelial cell monolayer was not restored in thrombocytopenic animals, and this effect may play an important role in guiding their ability to develop a neointima even late after arterial injury. It is, however, not clear whether platelets are necessary for restoration of this monolayer. Finally, we note that while the rat model has limited applicability to human disease, it has served as a wonderful model for studying the biological events that control the vascular response to injury. Many of the studies in which PDGF biology has been examined and first elucidated were performed in the rat, and thus we felt compelled to perform our initial studies in this animal.

The clinical implications of these findings are of paramount performance. If pharmacological inhibition of proliferation or migration of VSMCs will successfully inhibit intimal hyperplasia only if maintained until quiescence is reinstated, then issues of dose and delivery are critical. We and others have shown, for example, that while the continuous release of heparin suppresses intimal hyperplasia after balloon angioplasty or endovascular stent implantation, intermittent dosing exacerbates disease³³ and a short course of therapy inhibits only a part of the process.^{12 34} The combined force of these reports and our current data might explain why the promise of a vast amount of laboratory research has not been realized in clinic trials. The goal may now need to be to attain vascular quiescence rather than simply inhibition of central cellular events. Only future studies will enable us to determine whether the vessel wall can ever be put fully to rest after injury.

In summary, when thrombocytopenia was produced with busulfan, the neointima that was expected to form after balloon denudation was reduced without cytotoxic effects on VSMCs. What was most surprising, however, was that restoration of platelets 2 weeks after thrombocytopenia and vascular injury still enabled formation of a neointima indistinguishable from the neointima formed in native animals after injury. Thus, therapy directed exclusively at inhibiting the myointimal hyperplastic response without restoring vascular quiescence may be bound for failure. Permanent reduction of intimal hyperplasia requires not only the immediate separation of growth stimuli from the injured blood vessel wall but removal of the growth potential. Even the most elegant intervention cannot be withdrawn before this occurs. Clinical trials based on preclinical animal experimentation need to keep this matter clearly in mind.

	Selected Abbreviations and Acronyms

 

EGF = epidermal growth factor IGF-1 = insulin-like growth factor PDGF = platelet-derived growth factor PDGFR-ß = platelet-derived growth factor receptor subunit PDGFR-ßß = platelet-derived growth factor receptor PDGF-BB = platelet-derived growth factor ligand TGF-ß = transforming growth factor-ß VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported in part by grants from the NIH, of the NHLBI, including GM/HL-49039 (E.R.E.), HL-33014 and HL-41484 (R.D.R.), HL-54838 (D.J.K.), HL-53793 (M.S.), and from the Burroughs-Wellcome Fund in Experimental Therapeutics (E.R.E.) and the Whitaker Foundation for Biomedical Engineering (E.R.E.). Dr Sirois is a recipient of a fellowship from the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada. We wish to thank Philip A. Seifert for his technical assistance with immunohistochemistry.

Received December 18, 1996; revision received February 18, 1997; accepted February 24, 1997.

	References

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