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Circulation. 1996;94:1989-1995

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(Circulation. 1996;94:1989-1995.)
© 1996 American Heart Association, Inc.

Articles

Recombinant Mitotoxin Basic Fibroblast Growth Factor–Saporin Reduces Venous Anastomotic Intimal Hyperplasia in the Arteriovenous Graft

Changyi Chen, MD; Samer G. Mattar, MB, ChB; John D. Hughes, MD; Glenn F. Pierce, MD, PhD; Jennifer E. Cook, MS; David N. Ku, MD, PhD; Stephen R. Hanson, PhD; Alan B. Lumsden, MB, ChB

the Departments of Surgery (C.C., S.G.M., J.D.H., D.N.K., A.B.L.) and Medicine (S.R.H.), Emory University School of Medicine, Atlanta, Ga, and Department of Preclinical Development, Prizm Pharmaceuticals, San Diego, Calif (G.F.P., J.E.C.).


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background The plant cytotoxin saporin (SAP) is a potent ribosome-inactivating protein. When conjugated to basic fibroblast growth factor (FGF2), it selectively kills proliferating cells that have upregulated FGF receptors. In this study, we evaluated the effect of the recombinant chimeric mitotoxin rFGF2-SAP on venous anastomotic intimal hyperplasia, a major cause of failure of arteriovenous (AV) grafts.

Methods and Results Recently designed expanded polytetrafluoroethylene–based local infusion devices were implanted bilaterally as femoral AV conduits in six dogs. The venous anastomoses were the sites of continuous delivery of rFGF2-SAP (2.7 µg·kg-1·d-1) to one side and vehicle (4.6 µL·kg-1·d-1) as control to the contralateral side for 14 days. All animals survived, and all grafts were patent. Liver enzyme levels and histological analyses of liver, kidneys, and brain were normal, indicating the absence of systemic toxicity. Morphometric measurements and measurements of cell proliferation by bromodeoxyuridine index analysis were performed at both arterial and venous anastomoses. There were no significant differences between the treated grafts and the control grafts in intimal hyperplasia and intimal cell proliferation at the arterial anastomoses. In contrast, rFGF2-SAP reduced intimal thickness by 32%, intimal area by 40%, and cell proliferation index by 33% at the treated venous anastomoses compared with the control venous anastomoses (P<.05).

Conclusions These data demonstrate that local infusion of rFGF2-SAP significantly reduces venous anastomotic intimal hyperplasia and cell proliferation without systemic toxicity. This study suggests a new strategy for reducing intimal hyperplasia by the selective killing of proliferating smooth muscle cells with a potent chimeric mitotoxin through a novel local infusion device.


Key Words: growth substances • saporin • hyperplasia • grafting


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Intimal hyperplasia develops to various degrees after all vascular reconstructive procedures, including balloon angioplasty, atherectomy, surgical endarterectomy, and the insertion of vascular grafts. It is the primary cause of restenosis after revascularization and causes the failure of prosthetic grafts, including those used in the creation of AV grafts. For example, creation of an AV fistula with ePTFE is the most popular method for hemodialysis of renal failure patients, but patency rates are usually measured in months.1 2 3 The majority of failures are due to the rapid development of an intimal hyperplastic lesion at the venous anastomosis.1 4 5

Although several growth factors are thought to be involved in the formation of intimal hyperplasia after vascular injury, FGF2 appears to be a major factor.6 7 FGF2 lacks a signal peptide normally required for vectorial translation into the endoplasmic recticulum and secretion. Whether FGF2 is actually released through another, perhaps novel, mechanism remains to be established.8 Injury results in release of FGF2, which then stimulates SMC proliferation.6 Furthermore, injury also upregulates the expression of the FGF2 receptors on the SMCs, which in turn promotes the FGF2 effect.9 10 FGF2 binding to its cellular receptor transduces signals to the nucleus to initiate DNA replication and cell division via a mechanism of internalization and nuclear translocation of surface-bound growth factor.11 Infusion of FGF2 enhances SMC proliferation after vessel injury, whereas neutralizing antibodies to FGF2 inhibit DNA synthesis.6 7 12

SAP is a powerful ribosome-inactivating protein isolated from the seeds of the plant Saponaria officinalis.13 It has been used to make potent and effective immunotoxins and ligand toxins.14 15 16 17 FGF2 and SAP have been conjugated and characterized as a mitotoxin to selectively kill proliferating cells that express the FGF receptors.18 FGF2-SAP enters target cells via the FGF receptor, inhibits protein synthesis, and elicits cell death.19 20 Furthermore, Casscells and coworkers9 have shown that FGF2-SAP inhibits intimal proliferation without apparent effect on the uninjured artery in a rat model.

Recently we designed and characterized an ePTFE-based local infusion device that can deliver therapeutic agents such as FGF2-SAP directly through the wall of the graft, thereby achieving high drug concentrations in the blood boundary layer along the graft wall and at downstream anastomotic sites.21 22 By delivering an agent via this device, we can achieve maximum therapeutic effects while minimizing systemic toxicity. This study demonstrates the feasibility of a femoral AV ePTFE loop graft used with this approach in a canine model and evaluates the effect of a locally infused recombinant chimeric mitotoxin, rFGF2-SAP, on venous anastomotic intimal hyperplasia and cell proliferation.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Local Infusion Device
The design of the ePTFE-based local infusion device has been described.21 22 Briefly, the device consisted of a silicone rubber cuff reservoir glued in position between two rings of a ringed-ePTFE graft (6-mm ID, 10 cm long; Gore-Tex, WL Gore and Associates, Inc). The infusion reservoir was located near one end of the graft, generating a short arm and a long arm (Fig 1Down). The short arm was anastomosed to the femoral vein and the long arm to the femoral artery during the surgical procedures (see below). An implantable osmotic pump (model 2ML2, Alza Co) was connected to the infusion device. These osmotic pumps reliably deliver infusates at a predictable rate (5 µL/h) over a period of 14 days. Pumps for the treated anastomoses were loaded with rFGF2-SAP, whereas pumps for the control were loaded with vehicle (see below). Pumps were primed as directed by the manufacturer's instructions.



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  		Figure 1. Infusion device consisting of a silicone rubber cuff reservoir glued in position between the two rings of a ringed 6-mm-ID ePTFE tube (thin wall, 30-µm internodal distance). The hub is attached to a 1-mm-ID silicone rubber catheter tubing, which is then attached to an osmotic pump.

Recombinant Mitotoxin (rFGF2-SAP)
The rFGF2-SAP used in this study is a genetically conjugated fusion protein that is a potent recombinant chimeric mitotoxin (Prizm Pharmaceuticals). Biochemical characteristics and activity have been described.23 rFGF2-SAP was provided at a concentration of 0.5 mg/mL. This agent was formulated in 10 mmol/L citrate, 0.14 mol/L NaCl, and 0.1 mmol/L EDTA at pH 6.0 (vehicle). To test the stability of rFGF2-SAP before its use in animals, it was incubated at 37°C for 14 days and was shown to have retained its full biological activity by an in vitro cellular cytotoxicity assay.23 One milligram of rFGF2-SAP (2 mL) was loaded into an osmotic pump and delivered to the venous anastomosis via an ePTFE-based infusion device at the rate of 71 µg/d, which was equivalent to an average rate of 2.7 µg·kg-1·d-1. Two milliliters of vehicle was loaded into another pump and infused to the venous anastomosis on the contralateral side, which served as an internal control with a delivery rate of 120 µL/d, the same rate as the rFGF2-SAP infusion.

Animal Model
Six adult male mongrel dogs weighing 26±4 kg each were used in this study. All animal procedures and care were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH publication No. 80-23, revised 1985). Anesthesia was induced with thiopental sodium (10 to 20 mg/kg IV). The animals were intubated endotracheally, and anesthesia was maintained with 1% to 2.5% isoflurane. Under sterile conditions, the common femoral artery and vein were exposed bilaterally. After systemic heparin anticoagulation (100 U/kg), an ePTFE-based infusion device was implanted bilaterally between the femoral artery and vein. End-to-side anastomoses were performed at the femoral locations with running 6-0 polypropylene suture. On completion of the anastomoses, an osmotic pump preloaded with rFGF2-SAP was connected to the infusion device on one side, and another osmotic pump preloaded with vehicle was connected to the infusion device on the contralateral side as a control. These pumps were implanted in subcutaneous pockets (Fig 2Down). The incisions were closed with 3-0 polyglactin suture.



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  		Figure 2. ePTFE-based local infusion device implanted bilaterally between the femoral artery and vein. An osmotic pump preloaded with rFGF2-SAP was connected to one device, and another osmotic pump preloaded with vehicle was connected to the contralateral device as an internal control. These pumps were implanted in subcutaneous pockets. Treatment was "blinded."

BrdU (Sigma Chemical Co), in a dose of 50 mg/kg dissolved in 50 mL of normal saline, was administered intraperitoneally 24 hours before euthanasia at 14 days. The animals were anesthetized as described above, and the femoral arteries, veins, and grafts were exposed. Blood flow through the AV grafts was measured by an ultrasonic flowmeter (model T201, two-channel, Transonic Systems, Inc). A sternotomy was performed, and Ringer's solution was infused at 120 mm Hg pressure through a wide-bore needle into the left ventricle while the animal was synchronously exsanguinated via a cannula placed in the right atrium. Once blood was cleared from the circulatory system, the arteries, veins, and implanted devices were perfusion-fixed in situ with 2.5% glutaraldehyde. Grafts with 3-cm segments of attached femoral artery and vein were harvested and fixed in 10% buffered formalin for 4 hours and then placed in 70% ethanol (Baxter Diagnostics Inc). Portions of major organs such as brain, liver, pancreas, kidneys, spleen, skeletal muscle, and small intestine were also excised for histological analysis.

Blood samples drawn before surgery and at 3 days, 7 days, and 14 days after surgery were analyzed by a dedicated veterinary laboratory (Antech Diagnostics) for hematological parameters, such as hematocrit and leukocyte count, and serum biochemical parameters.

The investigators were "blinded" at all stages of the experiments as to which side of the grafts had received rFGF2-SAP.

Histology and Morphometry
After fixation, cross sections of specimens were taken perpendicular to the vessel long axis. ePTFE grafts embedded in paraffin were sectioned at a distance of 2 mm between the heel and toe of each anastomosis and at 5-mm intervals along the entire graft length and attached native vessels (Fig 3ADown). The tissue blocks were processed through graded ethanol, infiltrated with xylene and paraffin, and then embedded in paraffin blocks. Five-micrometer sections were cut and stained with hematoxylin-eosin and Verhoeff-Masson's stain. Tissue ingrowth overlying the luminal surface of the graft adjacent to the anastomosis was considered to be graft intimal hyperplastic tissue (Fig 3BDown). Morphometric measurements of the area of graft intima were performed by computer image analysis software (Optimas, Bioscan, Inc) on a magnified image relayed from a microscope-mounted video camera to a digitizing pad and video monitor (Thomas Optical) as previously described.22 All specimens were sectioned in the same fashion in that four evenly divided blocks were generated between the heel and toe of each anastomosis. From the four tissue blocks, mean values based on morphometric measurements of graft intimal areas were obtained for statistical comparisons. The thickness of graft intima was calculated from the measurements of graft intimal area and distance of tissue ingrowth from the suture line at each anastomosis (average intimal thickness=intimal area÷length of ingrowth). Therefore, the morphometric measurements were made at identical regions of the grafts in all animals.




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  		Figure 3. Specimen sectioning and morphometric measurement. A, Specimens were sectioned at a distance of 2 mm between the heel and toe of each anastomosis and at 5-mm intervals along the entire graft length and attached native vessels. Four tissue blocks were generated from each anastomosis, and the slides generated from these tissue blocks were subjected to morphometric analyses. B, Tissue ingrowth overlying the luminal surface of the graft adjacent to the anastomosis was considered to be graft intima. The areas of graft intima were measured with a computer-assisted morphometric system. The morphometric boundaries were defined as indicated by thicker lines. The thickness of graft intima was calculated from measurements of graft intimal area and distance of tissue ingrowth from the suture line (average intimal thickness=intimal area÷length of ingrowth).

Immunocytochemistry
The avidin-biotin complex immunoperoxidase procedure (LSAB Kit, Dako Co) was used to identify determinants characterizing intimal cell types and proliferating cells as previously described.22 24 Briefly, immunostaining for {alpha}-actin– and factor VIII–related antigens was performed to identify SMCs and endothelial cells, respectively. Proliferating cells were identified with anti-BrdU monoclonal antibody (Dako). BrdU-positive cells were quantified manually with a cell-counting technique on a calibrated micrometer grid under microscope (x400). In each field, all cells were counted, and the number of positively stained cells was expressed as a percentage of total cells to arrive at the BrdU index. A minimum of 10 fields were quantified per section.

Statistical Analysis
Statistical analysis was performed on a Macintosh Quadra 650 computer with Excel 4.0 statistical software. The paired (two-tailed) Student's t test was used to determine the significance of differences in intimal thickness, intimal area, and cell proliferation rates between the rFGF2-SAP–treated grafts and the control grafts. Results were considered significant at a value of P<.05. The code as to which side had received rFGF2-SAP was broken only after all analyses were done.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Anastomotic Intimal Hyperplasia
All of the animals survived, and all of the grafts were patent at 14 days. Blood flow through the control grafts was 563±134 mL/min, and blood flow through the treated grafts averaged 554±125 mL/min (P>.5). Measurable intimal hyperplasia had developed at both the arterial and venous anastomoses. SMCs were the major type of anastomotic intimal cells identified by {alpha}-actin immunostaining. Endothelial cells covered the surfaces of all of the anastomotic intima, as demonstrated by factor VIII–related antigen immunostaining. The venous anastomoses of the AV grafts on one side received rFGF2-SAP, while the venous anastomoses on the contralateral side received vehicle as internal control. At the treated venous anastomoses, rFGF2-SAP reduced intimal thickness and intimal area by 32% and 40%, respectively (Table 1Down), compared with controls (Fig 4Down). The arterial anastomoses of both the treated and control grafts received neither rFGF2-SAP nor vehicle. There were no significant differences in intimal thickness or intimal area at the arterial anastomoses between the treated grafts and the control grafts (Table 2Down). These data demonstrate that if the infusion device is attached at a position in proximity to the venous anastomosis, rFGF2-SAP treats only the venous anastomosis and significantly reduces intimal hyperplasia.


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  		Table 1. Intimal Hyperplasia and Cell Proliferation at Venous Anastomoses




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  		Figure 4. Intimal hyperplasia at the venous anastomoses of AV ePTFE grafts. A, Marked intimal hyperplasia was present at the vehicle-treated (control) venous anastomosis. B, Reduced intimal hyperplasia was seen at the rFGF2-SAP–treated venous anastomosis. Verhoeff-Masson's stain, magnification x40. L indicates lumen; I, intimal hyperplasia.


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  		Table 2. Intimal Hyperplasia and Cell Proliferation at Arterial Anastomoses

Cell Proliferation
Cell proliferation was assayed by BrdU labeling index. There was no significant difference in cell proliferation at the arterial anastomoses between the treated and the control grafts (Table 2Up). In contrast, rFGF2-SAP reduced intimal cell proliferation by 33% at the venous anastomoses compared with the control grafts (Table 1Up). These data show that rFGF2-SAP significantly inhibits intimal cell proliferation at the treated anastomoses.

Graft Endothelialization
Endothelium-like cells covered anastomotic intimal hyperplastic tissue at both venous and arterial anastomoses. There were no significant differences in extent of graft endothelialization between the treated and the control venous anastomoses (Table 3Down), although the treated side showed less endothelialization. However, rFGF2-SAP inhibited anastomotic endothelial cell proliferation by 59% (Table 3Down) compared with the control side. No significant differences of either extent of endothelialization or endothelial cell proliferation were seen between the treated and the control arterial anastomoses (Table 3Down).


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  		Table 3. Effects of rFGF2-SAP on Graft Endothelialization and EC Proliferation at Anastomoses

Systemic Toxicity
There was no significant loss of body weight in the dogs during the experiment (data not shown). Hematocrit, total leukocyte count, platelet count, and activated partial thromboplastin time were within normal range at the time of euthanasia. Tissues from the major organs of these animals, including brain, heart, lung, liver, kidneys, pancreas, and skeletal muscle, showed no significant pathological lesions, and standard serum biochemical parameters were within the normal range (Table 4Down). Thus, no systemic toxicity of the mitotoxin (rFGF2-SAP) administered through an ePTFE-based local infusion device was observed.


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  		Table 4. Summary of Organ Histology and Serum Biochemical Parameters From Dogs That Received rFGF2-SAP Through a Local Infusion Device


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
A recent approach, now in clinical trials for the treatment of certain cancers, involves the use of specific ligands to transport toxins into target cells.25 26 The strategy, originally applied in immunotherapy by conjugation of toxins to monoclonal antibodies, has been used to couple toxins to growth factors to specifically kill proliferating SMCs that have upregulated corresponding receptors.27 28 29 30 Based on observations that injury upregulates high-affinity receptors for FGF2 in the injured vessels,9 10 FGF2 receptor–mediated cytotoxicity for SMCs may provide a technique for developing therapies to treat intimal hyperplasia. It has been shown that a chemically conjugated mitotoxin, FGF2-SAP, through systemic administration inhibits intimal proliferation by targeting FGF receptors in the injured rat carotid artery.9 However, systemic toxicity was also observed in this rat model. In the present report, we provide evidence that a recombinant chimeric mitotoxin, rFGF2-SAP, through local administration treats only the venous anastomoses in a canine AV graft model and significantly inhibits anastomotic intimal hyperplasia (intimal thickness and area) and cell proliferation (BrdU index) without systemic toxicity. Transgraft local drug administration in this study makes mitotoxin therapy safer and more effective. Blinded analysis and the evaluation of untreated vessels from each study animal (internal control) render these results more reliable because possible artifacts, including individual variability and technique bias, are eliminated.

Standard clinical ePTFE grafts (30-µm internodal distance) are impervious to the leakage of blood elements because of their hydrophobic nature. However, solutes external to the graft will pass through the graft wall as the external pressure exceeds the intraluminal pressure. When the graft is placed between an artery and a vein, solutes enter the bloodstream in the highest concentration at the graft-blood interface and are transported downstream by blood flow, with slower mixing toward the center-stream blood flow occurring primarily through diffusion. If this approach is used for drug delivery, relatively high concentrations of infused agents will be maintained at the delivery site and along the distal graft wall reaching to the distal anastomosis. This concept has been supported by previous computational and experimental studies in vitro (unpublished data). This approach has previously been shown to be remarkably efficient for inhibition of thrombus formation in a porcine AV shunt model and a rabbit model of vena cava replacement with ePTFE.21 22 This strategy for local drug delivery has also been used in the present study. A major advantage of local drug administration is the potential to greatly reduce systemic side effects, since the absolute amount of compound administered is relatively small. Although Casscells and coworkers9 showed a reduction of intimal hyperplasia by giving FGF2-SAP (100 µg·kg-1·d-1) intravenously in a rat model, severe systemic toxicity was also observed. In contrast, we administered a very low dosage of rFGF2-SAP (2.7 µg·kg-1·d-1) via a local infusion device in a dog model and showed significant reduction of venous anastomotic intimal hyperplasia and cell proliferation without detectable systemic toxicity.

The use of an internal control was based on the prediction that the concentration of circulating rFGF2-SAP would be below the active range. The half-life of rFGF2-SAP in vivo is 12 minutes, and the minimal effective dose of rFGF2-SAP for SMCs in vitro is 5 ng/mL (unpublished data). In this study, the infusion rate of rFGF2-SAP was 50 ng/min. Given the dog plasma volume of {approx}40 mL/kg, the steady-state plasma rFGF2-SAP level calculated according to clearance kinetics is 0.13 ng/mL, a concentration 38-fold lower than the level of drug required for biological activity. Therefore, if the infusion device is attached at a position in proximity to the venous anastomosis, rFGF2-SAP treats only the venous anastomosis of treated grafts, and we do not anticipate any effect of circulating rFGF2-SAP on the arterial anastomoses of treated grafts or on venous and arterial anastomoses of the contralateral control grafts. The differences in measurements of intimal hyperplasia and cell proliferation at arterial anastomoses between the control and the treated sides were relatively smaller (not statistically significant) than those at the venous anastomoses (significant). The small differences in intimal thickness, intimal area, and cell proliferation at the arterial anastomoses on the treated and the control sides were presumably due to interanimal variabilities. Since rFGF2-SAP was infused into the venous circulation, if the systemic toxicity had occurred, the arterial anastomotic intimal growth on both the treated and the control sides should have been affected equally.

Much of our understanding of the cellular biology of intimal hyperplasia comes from studies performed in animal models. In particular, the model of balloon injury to the rat common carotid artery has been studied extensively. However, results from rat models may not be directly applicable to humans, since several treatments that showed efficacy in rats have failed in larger animal models and in human trials. For example, although ACE inhibitors limited intimal hyperplasia in a rat carotid artery injury model,31 a large animal model and two international clinical trials subsequently showed a lack of effectiveness of these treatments for reducing both vascular lesion development and restenosis.32 33 34 Large animals, such as pig,35 dog,36 and baboon,32 whose vessel structure and hemodynamics more closely resemble those of humans, have thus been preferred in studies of intimal hyperplasia, since these models may be more clinically relevant.

Recently we carried out a time-course study of intimal hyperplasia in a canine model of femoral AV ePTFE grafts and found that the lesions produced in this animal model are very similar to those seen in humans.37 38 39 40 Intimal hyperplasia at the venous anastomoses developed more rapidly than that at the arterial anastomoses during a 3-month study, supporting the clinical evidence that venous anastomotic lesions are a main cause of AV graft failure. One possible reason for this propensity is that chronic platelet aggregation occurring in the midportion of the prosthetic graft leads to the release of SMC mitogens that bathe the distal anastomosis. A hyperplastic response then ensues. Some investigators have suggested that disturbed flow was a major factor in the development of venous anastomotic intimal hyperplasia in AV grafts.41 42 43 In our 2-week study, although measurable intimal hyperplastic tissues were produced at both the arterial and venous anastomoses, we did not see significant differences in intimal hyperplasia and cell proliferation between arterial and venous anastomoses in the control grafts, probably because of the relatively short duration of this study. However, this model is very useful to study biological mechanisms of stenotic lesion development and to evaluate therapeutic interventions.

Although the potent effect of recombinant human FGF2 has been demonstrated on canine tissues,44 and the approach taken in this study is promising, a potential limitation of this experimental model is that the recombinant human FGF2 genetically conjugated with the plant protein SAP may be antigenic in dogs. If animals raise neutralizing antibodies against rFGF2-SAP, prolonged local infusion therapy will not be effective. This problem has been raised in the immunotoxin treatment of human malignancies in animal models and in patients.45 Immune responses against both the toxin and the antibody moiety might be reduced by humanized antibodies46 or by immunosuppressive agents such as deoxyspergualin.47 Thus, the antigenic properties of rFGF2-SAP should be characterized. The duration of the study was 2 weeks, and we are therefore unable to comment on the prospects for long-term benefit with this local rFGF2-SAP therapy. In general, the type of ligand and toxin will need to be optimized to increase the specificity and decrease the nonspecific toxicity of these chimeric toxins. It is also possible that the destruction of targeted SMCs may result in adverse effects, such as the release of growth factors or cytokines. These factors will require further investigation in vitro and in vivo before clinical studies can be attempted. In addition, the sample size is small, and only one time point was studied in this report. Further studies are now warranted to fully characterize this novel approach, including expanding the time course and the sample size.

In summary, local infusion of rFGF2-SAP significantly reduces venous anastomotic intimal hyperplasia and cell proliferation without systemic toxicity in a canine AV loop graft model. There was no difference in blood flow between the treated and control grafts. This study suggests a promising strategy for reducing intimal hyperplasia by the selective killing of proliferating SMCs with a powerful chimeric mitotoxin through a local infusion approach. Recombinant chimeric mitotoxin interventions to limit cellular proliferation at specific vascular injury sites could yield new treatments for vascular proliferative diseases and provide insight into their pathogenesis. Further investigations, including time-course, dose-response, immunogenicity, and toxicity studies for the recombinant chimeric mitotoxin, are therefore warranted.


*    Selected Abbreviations and Acronyms
 
AV = arteriovenous
BrdU = bromodeoxyuridine
ePTFE = expanded polytetrafluoroethylene
FGF2 = basic fibroblast growth factor
PTFE = polytetrafluoroethylene
rFGF2-SAP = recombinant chimeric mitotoxin of FGF2 and SAP
SAP = saporin
SMC = smooth muscle cell


*    Acknowledgments
 
This study was supported in part by research grants HL-31469 and HL-48667 from the National Institutes of Health and by a grant from the American Heart Association. We thank Carolyn Suwyn for her expert technical assistance in tissue processing and histology procedures and Beverly Noe for her laboratory assistance.


*    Footnotes
 
Reprint requests to Alan B. Lumsden, MB, ChB, 1364 Clifton Rd NE, Box M-11, Atlanta, GA 30322.

Received December 11, 1995; revision received April 11, 1996; accepted April 16, 1996.


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

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