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(Circulation Research. 1995;77:519-529.)
© 1995 American Heart Association, Inc.

Articles

Epidermal Growth Factor Receptor–Targeted Cytotoxin Inhibits Neointimal Hyperplasia In Vivo

Results of Local Versus Systemic Administration

Christopher J. Pastore, Jeffrey M. Isner, Patricia A. Bacha, Marianne Kearney, J. Geoffrey Pickering

From the Departments of Medicine (Cardiology), Pathology, and Biomedical Research (C.J.P., J.M.I., M.K., J.G.P.), St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass; Seragen Inc (P.A.B.), Hopkinton, Mass; and the John P. Robarts Research Institute and Department of Medicine (Cardiology) (J.G.P.), University Hospital, University of Western Ontario, London, Canada.

Correspondence to Jeffrey M. Isner, St. Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135.

	Abstract

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Abstract Smooth muscle cell accumulation is a key feature of restenosis that may be inhibited by the delivery of receptor-targeted cytotoxins. DAB₃₈₉EGF is a recombinant fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced by human epidermal growth factor (EGF). We investigated the effectiveness of DAB₃₈₉EGF to inhibit neointimal hyperplasia in the balloon-injured rat carotid artery. Incubation of rat carotid arteries with ¹²⁵I-labeled EGF revealed extensive EGF binding sites in the neointima of balloon-injured arteries. Sixty rats subsequently received either saline or DAB₃₈₉EGF (total dose, 0.15 mg) delivered immediately following balloon injury either systemically, via 14-day continuous osmotic pump infusion, or locally, via 30-minute intraluminal incubation. The effect of both treatment strategies was measured 2 weeks after injury by cross-sectional morphometric analysis of intimal area, the ratio of intimal/medial area (I/M), and the percent luminal narrowing (%LN). In addition, proliferative activity was assessed by immunostaining for the presence of the proliferating cell nuclear antigen (PCNA). Compared with controls, systemic delivery of fusion toxin significantly reduced intimal area, I/M, and %LN by 40%, 40%, and 29%, respectively. However, these rats exhibited 2% weight loss, indicating mild systemic toxicity. Local, intraluminal administration of DAB₃₈₉EGF yielded a more pronounced reduction in intimal area, I/M, and %LN by 74%, 79%, and 72%, respectively. This inhibitory effect was preserved at 3 weeks postinjury, and PCNA immunostaining of locally treated arteries revealed a virtual absence of proliferative activity in the neointima and media at this timepoint. In contrast to systemically treated rats, rats receiving fusion toxin locally gained weight at a rate similar to controls, indicating avoidance of systemic toxicity. We conclude that DAB₃₈₉EGF is a potent inhibitor of neointimal hyperplasia in vivo and that whereas an inhibitory effect may be achieved by systemic delivery, local delivery appears to be more potent, avoids systemic toxicity, and thus represents a feasible strategy to preempt restenosis.

Key Words: vascular smooth muscle • epidermal growth factor • restenosis • fusion protein

	Introduction

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The enduring limitation of balloon angioplasty and all other methods of percutaneous revascularization is the frequent occurrence of restenosis.^{1 2} A key feature of restenosis in many patients is the accumulation of intimal vascular smooth muscle cells (SMCs), which is believed to occur as the result of migration and proliferation of activated SMCs.^{3 4 5} Although several pharmacological agents designed to inhibit SMC proliferation have shown promise in animal models,^{6 7 8} few to this date have proven beneficial in human clinical trials. This may be due to a lack of potency and specificity toward the target cell population.

Recent studies have established that SMCs stimulated to proliferate in culture or in vivo express a higher number of receptors for mitogens such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) than nonactivated SMCs.^{9 10} This differential expression of receptors provides a rationale for the use of receptor-specific cytotoxic agents to target the activated, proliferating SMCs that typically constitute a restenotic lesion.^{5 11} Earlier work in our laboratory established that an EGF receptor–targeted cytotoxin, DAB₃₈₉EGF, completely inhibits the outgrowth of vascular SMCs from fragments of human atherosclerotic plaque explanted into a growth-stimulating environment.¹² DAB₃₈₉EGF is a genetically engineered fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced with human EGF.^{13 14} The mechanism of cytotoxicity is identical to that of native diphtheria toxin,¹⁴ which kills cells by arresting protein synthesis.^{15 16 17}

To further assess the potential utility of DAB₃₈₉EGF for the prevention of restenosis, we performed an in vivo study employing the rat common carotid artery balloon-injury model of accelerated lesion formation. We first confirmed that the expression of binding sites for EGF was a feature of the response to balloon injury and that these sites were localized to the developing intimal lesion. We then investigated the safety and the efficacy of DAB₃₈₉EGF as a means of abrogating lesion formation in vivo, using both systemic and local delivery strategies.

	Materials and Methods

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Rat Model of Balloon Injury
The model of balloon injury was based on that described by Clowes et al.¹⁸ Male Sprague-Dawley rats weighing 400 to 500 g were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg, Abbott Laboratories). The bifurcation of the left common carotid artery was exposed through a midline incision and the left common, internal, and external carotid arteries were temporarily ligated. A 2F embolectomy catheter (Baxter) was introduced into the external carotid and advanced to the distal ligation of the common carotid. The balloon was inflated with saline and drawn toward the arteriotomy site three times to produce a distending, deendothelializing injury. The catheter was then withdrawn and the proximal external carotid artery was ligated. For rats receiving continuous infusion of DAB₃₈₉EGF, vinyl tubing was attached to an osmotic minipump (Alza Corp) and inserted into the jugular vein contralateral to the injured artery. For rats receiving local therapy, the injured segment of the common carotid artery was isolated by temporary ligatures prior to permanent ligation of the external carotid. The treatment or control solution was then infused into the segment via a cannula introduced through the external carotid. The experimental protocol was approved by an institutional animal care and use committee and complied with the standards stated in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985).

Evaluation of EGF Binding Sites
At designated intervals after balloon injury, arteries were harvested and assessed for the presence and location of EGF binding sites.^{19 20} Five-millimeter arterial segments were incubated at room temperature for 90 minutes with ¹²⁵I-EGF (10 ng/mL, Becton-Dickinson) in Hanks' balanced salt solution supplemented with 20 mmol/L HEPES and 1 mg/mL albumin. Adjacent fragments from the same artery were incubated in the same medium but in the presence of a 250-fold excess of unlabeled EGF (2.5 µg/mL, Upstate Biotechnology Inc). Samples were then washed extensively with phosphate buffered saline (PBS), fixed in formalin, and embedded in paraffin. Cut sections were deparaffinized and dipped in radiographic emulsion (NTB-3, Kodak). After 2 to 6 weeks of exposure in the dark, slides were developed and counterstained with hematoxylin. With each assay, a fragment of rat skin was studied in an identical fashion and served as a positive control; in this control tissue, clusters of exposed silver grains were localized to the epidermis and the cells surrounding hair follicles.

Delivery of DAB₃₈₉EGF Fusion Toxin
DAB₃₈₉EGF was prepared and purified as previously described.^{12 14} The dosage used was based on preliminary toxicity studies using either daily intravenous injections²¹ or continuous intravenous delivery. Briefly, these preliminary studies utilized four total dosages (per 500-g rat) in 16 rats: 0.30 mg, 0.25 mg, 0.15 mg, and 0.11 mg. The highest dose shown to minimize weight loss and achieve 100% survival rate was a total dose of 0.15 mg DAB₃₈₉EGF protein; both systemic and local delivery groups therefore received this same total dose of 0.15 mg DAB₃₈₉EGF. Rats were weighed before the initial injury and again before they were killed for study, so that changes in weight could be employed as a measure of systemic toxicity.

DAB₃₈₉EGF was administered by constant infusion into the systemic circulation of 10 balloon-injured rats. A total dose of 0.15 mg DAB₃₈₉EGF fusion toxin was delivered intravenously at a mean rate of 4.5x10^-4 mg/h for 14 days via the osmotic minipump. The control group for systemic administration of DAB₃₈₉EGF consisted of 10 balloon-injured rats in which a constant infusion of saline was administered in an identical fashion. Rats were killed 2 weeks postinjury.

The local delivery treatment group consisted of 10 rats in which a 30-minute intraluminal incubation of 0.15 mg DAB₃₈₉EGF was performed immediately after balloon injury. After the 30-minute incubation, the cannula was withdrawn and the toxin was allowed to drain from the artery via the arteriotomy site, thus avoiding the entrance of toxin into the systemic circulation. The external carotid artery was then permanently ligated, and the rats were allowed to recuperate. The control group for local delivery consisted of 10 balloon-injured rats receiving an otherwise identical 30-minute application of saline.

Two additional groups of balloon-injured rats were studied as negative controls. The first (n=8) received DA_glu53B₃₈₉EGF, a fusion protein which binds to the EGF receptor but lacks an active toxic portion. The second (n=6) received DAB₃₈₉EGF_ala41, a fusion protein which contains an intact toxic component but lacks the ability to bind to the EGF receptor. In both cases, the control proteins were administered using the local delivery protocol, and the rats were killed for study 2 weeks postinjury.

The efficacy of single local administration over a longer time period was studied in 10 balloon-injured rats treated with DAB₃₈₉EGF and 10 treated with saline; all were killed and studied 3 weeks postinjury.

Finally, to specifically determine the effect of DAB₃₈₉EGF on hepatic function, 20 rats (n=5 per group) received vehicle (control) or total dose of 0.15 mg, 0.30 mg, or 0.60 mg DAB₃₈₉EGF, delivered systemically via an osmotic pump over a period of 7 days. On day 8, these animals were weighed and killed. The major organs were examined grossly, and serum samples were analyzed by clinical chemistry.

Vessel Harvesting and Morphometric Analysis
One hour before sacrifice, rats received an intravenous injection of 0.5 mL 0.5% Evans blue stain (Sigma Chemical Co) via the tail vein to identify the segment of deendothelialized artery. Under general anesthesia, rats were perfused with PBS at a pressure of 90 mm Hg via a cannula inserted into the left ventricle. Venous drainage was established through bilateral jugular venotomies, and perfusion with PBS continued until the effluent became grossly clear of blood, after which an additional 5 minutes of perfusion with 100% methanol was performed. A 1-cm segment of the deendothelialized (blue) carotid artery, together with the corresponding segment from the contralateral uninjured artery, was excised. In addition, a 1-cm² segment of the ileum was retrieved to serve as a positive control for proliferating cell nuclear antigen (PCNA) immunohistochemistry (see below). Tissues were fixed further by immersion in 100% methanol overnight, then embedded in paraffin, and cut into 5-µm cross sections onto silane-coated slides.

Neointimal lesion development was assessed in terms of cross-sectional neointimal area, ratio of intimal/medial area, and percent luminal narrowing. All measurements were made from hematoxylin and eosin– or elastin trichrome–stained cross sections projected onto a digitizing palette (Summagraphics Corp). Values for medial, intimal, and luminal area were calculated by a technician blinded to treatment regimen.

Proliferative Activity
The extent of proliferative activity in injured vessels at the end of the treatment period was evaluated by histochemical analysis for PCNA as previously described,⁵ using an avidin-biotin immunostaining technique (Elite Avidin-Biotin Detection System, Signet Laboratories). Following an overnight incubation of the primary antibody, sections were rinsed with PBS and incubated for 20 minutes with biotinylated anti-mouse IgG (Signet Laboratories) in rat serum and PBS/1% bovine serum albumin (BSA). The sections were then washed with PBS and incubated with avidin-biotin complex prepared using the manufacturer's instructions. After the sections were washed with PBS, 3-amino-9-ethylcarbazole substrate was applied for 10 minutes and then rinsed with dH₂O to yield a brown reaction product. After light counterstaining of cell nuclei with hematoxylin, sections were mounted with coverslips using aqueous mounting media. All intimal cells and all PCNA-positive cells were counted at high magnification. The extent of proliferative activity in histological cross sections was measured as the percentage of intimal cells that were positively stained with PCNA. PCNA staining of the rat ileum served as the positive control.

Statistical Analysis
Results were expressed as mean±SEM. Differences between means were evaluated using two-tailed, unpaired Student's t test. Differences were considered significant if P<.05. The Scheffé's F test/ANOVA was used to compare multiple local delivery (including treatment and control) groups at the 2-week timepoint.

	Results

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EGF Binding
EGF binding sites were readily detectable in the intima of balloon-injured rats, as indicated by a high concentration of exposed silver granules compared with the background signal in sections from arteries coincubated with ¹²⁵I-EGF and excess unlabeled EGF (Fig 1). In uninjured arteries, there was no visible difference in signal compared with that of arteries coincubated with radiolabeled EGF or excess unlabeled EGF. Seven days after injury, however, specific binding was noted throughout the intima. Fourteen days after injury, binding was localized primarily to the cell layers adjacent to the lumen (Fig 1).

View larger version (0K): [in this window] [in a new window]   Figure 1. Autoradiographs of sections of balloon-injured rat carotid arteries incubated with 125I-EGF with (a) and without (b) a 250-fold excess of unlabeled EGF. Specific EGF binding is evident in the cells adjacent to the lumen. N indicates neointima; M, media; and A, adventitia. Bars=0.1 mm.

Lesion Morphometry: Saline-Treated Controls
In untreated, balloon-injured rat carotid arteries examined 2 weeks postinjury, highly cellular neointimal lesions were observed, consisting predominantly of SMCs, which were identified by staining with a monoclonal antibody to SMC -actin (HHF-35). The internal elastic lamina at the site of lesion formation typically remained intact. Arteries occluded by organized thrombus were excluded from morphometric analysis. The extent of neointimal thickening was similar for control groups receiving either local or systemic administration of saline.

Lesion Morphometry: DAB₃₈₉EGF Fusion Toxin
Lesion formation was significantly reduced in rats treated by either local or systemic administration of DAB₃₈₉EGF fusion toxin and examined at 2 weeks postinjury (Fig 2). Neointimal thickening was significantly reduced by all three indices of morphometric analysis for both treatment regimens. The inhibitory effect of DAB₃₈₉EGF was most pronounced in those rats treated by local delivery of the fusion protein.

View larger version (0K): [in this window] [in a new window]   Figure 2. Photomicrographs of sections of balloon-injured rat carotid arteries 2 weeks postinjury. a, Representative example of an untreated balloon-injured artery stained with elastic trichrome. b, Same arterial segment shown in panel a, stained with hematoxylin and eosin. Significant reduction in lesion formation was achieved by systemic delivery of fusion toxin, as shown in panel c, a representative elastic-trichrome stained injured artery treated with systemic delivery of DAB389EGF. d, Same systemically treated artery stained with hematoxylin and eosin. The inhibitory effect of fusion toxin on lesion formation was even more pronounced when delivered locally to injured arteries, as shown in representative sections stained with elastic trichrome (e) and hematoxylin and eosin (f). Bars=0.4 mm.

Absolute cross-sectional area of the neointima in arteries retrieved from rats receiving systemic administration of DAB₃₈₉EGF (n=10) was reduced by 37% compared with balloon-injured arteries from 10 untreated control rats (0.12±0.02 mm² vs 0.19±0.01 mm², P=.0012) (Fig 3A). The cross-sectional area of the neointima in arteries of rats treated by local fusion toxin administration (n=10) was reduced at 2 weeks postinjury by 61% compared with controls (n=10) (0.07±0.02 mm² vs 0.18±0.03 mm², P=.0021) (Fig 3B). Local delivery of fusion toxin was thus 57% more effective than systemic delivery in achieving a reduction in neointimal cross-sectional area.

View larger version (27K): [in this window] [in a new window]   Figure 3. Systemic and local delivery of DAB389EGF fusion toxin in rats. Computer-aided morphometric characterization of neointimal lesions in control arteries and arteries treated with systemic delivery of fusion toxin 2 weeks postinjury (A) revealed a significant reduction in lesion formation as measured by intimal area, ratio of intimal/medial area (I/M), and percent luminal area narrowing. The reduction in neointimal lesion formation achieved by local administration of fusion toxin (B) was highly significant as measured by morphometric analysis of cross-sectional intimal area, ratio of intimal/medial area (I/M), and percent luminal area narrowing when compared to controls 2 weeks postinjury.

Both treatment regimens also produced a highly significant reduction in the I/M ratio. Systemic delivery of DAB₃₈₉EGF reduced I/M by 40% compared with controls (0.88±0.12 vs 1.47±0.09, P=.0009) (Fig 3A). Local delivery achieved a 75% reduction in I/M compared with controls (0.33±0.10 vs 1.33±0.20, P=.0003) (Fig 3B). The reduction in I/M observed in locally treated arteries thus again exceeded (69% greater) that observed with systemic treatment.

The disparity between the effectiveness of local and systemic delivery was further evidenced by the analysis of cross-sectional luminal area narrowing. The native lumen of untreated control arteries narrowed by 50% in cross-sectional area 2 weeks after balloon injury. Systemic delivery of fusion toxin decreased %LN by 29% compared with controls (39.19±5.30% vs 55.20±2.83%, P=.0159) (Fig 3A). Although statistically significant, this reduction in %LN was considerably less than the 71% reduction in %LN achieved by local delivery of the fusion toxin (13.79±3.74 vs 47.93±6.49%, P=.0002) (Fig 3B). Compared with systemic delivery, local delivery was 113% more effective in reducing %LN.

Further examination of locally treated arteries harvested at either 2 or 3 weeks after injury revealed that the degree to which neointimal lesion formation was inhibited by fusion toxin at 2 weeks was preserved at 3 weeks (Fig 4). This finding was consistent, regardless of which morphometric index (intimal area, I/M, or %LN) was considered.

View larger version (12K): [in this window] [in a new window]   Figure 4. Neointimal thickening in locally treated arteries after 2 vs 3 weeks. Morphometric analysis of locally treated arteries harvested at 2 weeks or 3 weeks postinjury revealed that both the ratio of intimal/medial area (I/M) and percent luminal area narrowing (%LN) increased in control arteries from 2 to 3 weeks postinjury (P=.015 and P=.036, respectively; *P<.05). Cross-sectional intimal area (INTIMA) also appeared to increase in control arteries from 2 to 3 weeks at a rate approaching statistical significance (P=.053). In contrast, the degree to which neointimal lesion formation was inhibited by DAB389EGF fusion toxin at 2 weeks was preserved at 3 weeks.

Lesion Morphometry: DAB₃₈₉EGF_ala41 and DA_glu53B₃₈₉EGF
DAB₃₈₉EGF_ala41 contains an intact toxic component yet lacks the ability to bind to the EGF receptor. Among six rats treated by local administration of DAB₃₈₉EGF_ala41 and examined 2 weeks later, neointimal thickening was similar to that seen in saline-treated controls (Intima=0.17±0.03; I/M=1.31±0.11; %LN=36.59±5.06) (Table).

View this table: [in this window] [in a new window]   Table 1. Lesion Morphometry of Local Delivery: 14 Days Postinjury

DA_glu53B₃₈₉EGF is able to bind to the EGF receptor but lacks an intact toxic component. Arteries from eight rats which received local administration of DA_glu53B₃₈₉EGF were examined two weeks postinjury. Lesion formation was again similar to that seen in rats receiving local delivery of saline (Intima=0.25±0.04, I/M=1.58±0.20, %LN=56.72±5.66) (Table).

PCNA Expression
The low level of proliferative activity inferred from examination of conventionally stained histological sections of treated arteries was confirmed by immunohistochemical staining for PCNA in both the intima and media (Fig 5). Compared with controls, intimal cell proliferation was significantly reduced in locally treated sections 2 weeks postinjury (22.4±3.7% vs 0.2±0.2%, P=.0006). The percentage of intimal cells staining positively for PCNA in control sections decreased significantly after 2 weeks (22.4±3.7% to 9.8±1.8%). PCNA expression at 3 weeks postinjury in control sections was limited to neointimal cells at the luminal interface. In sections from treated arteries, intimal proliferative activity remained virtually absent at the 3-week timepoint, and the reduction in cell proliferation with fusion toxin treatment was again highly statistically significant when compared with controls (9.8±1.7% vs 0.6±0.6%, P=.0012).

View larger version (0K): [in this window] [in a new window]   Figure 5. Photomicrographs showing proliferative activity as assessed by immunostaining for PCNA. Panel a shows a representative PCNA-stained cross section of an untreated balloon-injured rat carotid artery harvested 2 weeks postinjury (bar=0.25 mm). PCNA-positive cells account for 20% of the total number of neointimal cells, and almost all positive staining is localized to superficial neointimal cell layers adjacent to the lumen. Arrows point to the internal elastic membrane. The open arrow points to a cell stained positive for PCNA, which is shown in panel b at high magnification (open arrow) (bar=0.05 mm). Panel c shows an artery treated locally with fusion toxin and immunostained for the presence of PCNA (bar=0.25 mm). PCNA-positive cells are totally absent in both the neointima and media. In 30% of locally treated arteries, negative staining for PCNA in the neointima and media was internally controlled by the presence of positive staining in scattered cells outside the external elastic membrane in the adventitia, as is shown in panel c (open arrow) and at higher magnification in panel d (open arrow) (bar=0.05 mm).

Effect on the Media
Neointimal cells were presumed to originate from the media because the rat artery contains no native intima.²⁵ Medial proliferation after injury resulted in a rise in medial cell number in untreated balloon-injured arteries compared with uninjured arteries. There was no evidence for an adverse effect of DAB₃₈₉EGF on medial integrity. Specifically, no evidence of medial necrosis was observed with fusion toxin treatment at this dose, and the medial elastic lamina typically remained intact. The area of the media in systemically and locally treated arteries was statistically similar to that measured for the respective controls (0.14±0.10 mm² vs 0.13±0.01 mm², P=NS, and 0.15±0.01 mm² vs 0.14±0.01 mm², P=NS).

Quantification of medial cell density in treated arteries revealed that inhibition of SMC proliferation did not compromise medial integrity. Balloon injury resulted in a significant increase in medial cell density in untreated arteries compared with uninjured arteries (3.5x10³ cells/mm² vs 2.0x10³ cells/mm², P=.0001). Medial cell number was reduced in systemically treated arteries compared with injured, non–toxin treated arteries (2.8x10³ cells/mm² vs 3.5x10³ cells/mm², P=.0013) but remained elevated compared with normal uninjured arteries (2.8x10³ cells/mm² vs 2.0x10³ cells/mm², P=.0011). A similar pattern was observed in locally treated arteries, in which medial cell number was reduced compared with injured, non–toxin treated controls (2.4x10³ cells/mm² vs 3.3x10³ cells/mm², P=.0036) but was similar to normal uninjured arteries (2.4x10³ cells/mm² vs 2.0x10³ cells/mm², P=NS) (Fig 6). In none of the experimental groups—injured versus uninjured, treated versus untreated—was any inflammatory cell infiltrate observed.

View larger version (0K): [in this window] [in a new window]   Figure 6. Photomicrographs of hematoxylin and eosin–stained medial cell layers at high magnification. Arrowheads point to the internal elastic membrane, which marks the boundary between the neointima (N) and the media (M). Small arrows point to the external elastic membrane, separating the media from the adventitia (A). Examination of the media 2 weeks postinjury in normal, uninjured arteries (a), untreated injured (control) arteries (b), systemically treated injured arteries (c), and injured arteries treated with local delivery of fusion toxin (d) revealed similarity in medial cellularity among panels a, c, and d, indicating no evidence of medial necrosis as a result of fusion toxin treatment, while cellularity in panel b increased. Quantification of medial cell number suggested that favorable effects of fusion toxin on neointimal lesion development were achieved without concurrent compromise of medial integrity. Bars=0.1 mm.

Toxicity of DAB₃₈₉EGF
Control rats gained 4.0±1.0% of their original body weight over the 2-week period after initial injury (Fig 7). Rats receiving fusion toxin delivered systemically exhibited a 2.0±1.0% loss of body weight, indicating mild systemic toxicity. In contrast, rats that received the same total dose of fusion toxin delivered locally gained weight at a rate similar to controls (4.5±1.5% vs 6.0±1.0%, P=NS), indicating the avoidance of systemic toxicity.

View larger version (14K): [in this window] [in a new window]   Figure 7. Percent change in body weight over the 2 weeks following balloon injury. Rats treated with local delivery of DAB389EGF fusion toxin avoided systemic toxicity, as assessed by their ability to gain weight at a rate similar to corresponding control rats. In contrast, rats treated with systemic delivery of fusion toxin exhibited 2% weight loss, indicating mild systemic toxicity, which differed significantly from the group of corresponding control rats, which gained weight following injury. *P<.05.

Examination of hepatic function in rats receiving total dosages of 0.15 mg, 0.30 mg, and 0.60 mg DAB₃₈₉EGF administered systemically via osmotic pump for 7 days disclosed considerable animal-to-animal variability with respect to toxicity. Toxicities observed in rats administered DAB₃₈₉EGF at a total dose of 0.60 mg (n=5) ranged from mild weight loss to death; bilirubin and alkaline phosphatase levels were elevated in this group. In addition, the one rat that died prematurely in this group had slightly increased hepatic transaminases. Only one rat in the group that received 0.30 mg DAB₃₈₉EGF (n=5) showed signs of significant toxicity, consisting of 22% weight loss and a fourfold increase in hepatic transaminases. None of the rats in the 0.15-mg group (n=5) showed any evidence of toxicity or abnormality with respect to liver function.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The goal of cytotoxic therapy using recombinant fusion proteins such as DAB₃₈₉EGF is to reduce vascular narrowing by targeting activated SMCs in the vascular wall. We have previously demonstrated that DAB₃₈₉EGF is highly cytotoxic to vascular SMCs proliferating in culture and that this effect is mediated by the EGF receptor.¹² It has also been demonstrated in vitro that the number of receptors for EGF on proliferating rat SMCs is higher than on nonproliferating cells⁹ ; immunohistochemical evidence of increased receptor expression may be seen within the first 30 minutes after balloon injury of the rat carotid artery (see Supplemental Figures). In the present study, we have demonstrated that EGF binding sites are expressed in the neointima after balloon injury to the rat carotid artery and that these sites are concentrated in the cell layers closest to the lumen. It has been previously documented in studies of thymidine incorporation that these layers typically represent the site where the most sustained proliferative activity occurs.¹⁸ These results were confirmed in the present study by immunostaining for PCNA. Thus, there are both in vitro and in vivo data to support the concept of exploiting EGF receptor expression to target those SMCs in the vessel wall that are actively contributing to accelerated lesion development.

The mechanism of cytotoxicity for DAB₃₈₉EGF is identical to that of native diphtheria toxin.¹⁴ Endocytosis of the receptor-bound DAB₃₈₉EGF molecule is followed by internalization into the endosomal compartment. The capacity of activated SMCs for lysosomal degradation of internalized DAB₃₈₉EGF may be overloaded by a high intake of DAB₃₈₉EGF due to high EGF receptor expression. Once internalized, the enzymatically active fragment A of diphtheria toxin translocates into the cytosol, where it catalyzes the covalent linkage of ADP-ribose to elongation factor–2. This step arrests protein synthesis, causing cell death. Halting protein synthesis is an attractive means of killing cells to prevent vascular lesion formation, because it will likely lead to a depletion of intracellular factors that could otherwise have a mitogenic effect on nearby cells following lysis of the targeted cell. The abrupt release of intracellular contents of vascular SMCs may be potentially mitogenic²² ; we speculate, however, that this is an unlikely consequence of DAB₃₈₉EGF treatment because of prior depletion of intracellular proteins and because the mechanism of cell death does not appear to involve acute disruption of cell membrane integrity. This notion is supported by the observation that the inhibitory effect of a single treatment with DAB₃₈₉EGF was not different when treated arteries were evaluated at 2 versus 3 weeks postinjury. Thus, the absence of "rebound" proliferation is consistent with the presumed non–membrane lysing mechanism of DAB₃₈₉EGF cytotoxicity. While it has been previously suggested that diphtheria toxin may kill cells by catalyzing a sequence of events reminiscent of programmed cell death, or apoptosis,²³ this issue remains controversial.²⁴ Further work will be required to determine the potential role of this pathway in the use of diphtheria-based fusion proteins.

Direct killing of activated SMCs may also avoid potential limitations of approaches designed to modulate SMC behavior by interfering with either the growth factor milieu or intracellular signaling events. The existence of multiple regulatory pathways for SMC proliferation provides inherent redundancy; consequently, the net effect of interfering with any single process may be blunted by this redundancy. In contrast, selective cell killing by DAB₃₈₉EGF should not be influenced by this multiplicity of control mechanisms.

Other approaches to receptor-targeted killing of SMCs have been investigated. Casscells et al¹⁰ targeted bFGF receptors with a cytocidal conjugate of bFGF with saporin and achieved a significant reduction in neointimal thickening in balloon-injured rat arteries. When delivered locally, however, extensive medial necrosis was noted. In the present study, local administration of DAB₃₈₉EGF appeared to inhibit injury-induced SMC proliferation in the media but did not adversely affect overall medial integrity, implying a higher specificity to intimal cells.

The inhibitory effect of fusion toxin on SMC proliferation was assumed to develop in the absence of complete reendothelialization, previously shown by Clowes et al¹⁸ to remain incomplete for up to 12 weeks following balloon injury. The sensitivity of SMCs and endothelial cells to DAB₃₈₉EGF is in part determined by their respective expression of EGF receptors. EGF receptors are expressed by endothelial cells in the normal vessel wall, but their numbers are decreased exponentially in the presence of transforming growth factor-ß²⁵ ; the latter appears to be upregulated both in human restenotic lesions²⁶ and in the injured rat carotid artery.²⁷ Arterial injury may therefore provide an environment in which endothelial cells express fewer EGF receptors than proliferating SMCs and are thus less sensitive to DAB₃₈₉EGF.

An additional consideration in this respect is that local delivery may be superior to systemic delivery of the fusion toxin for limiting any potential inhibitory effect on reendothelialization of the angioplasty site. Toxin delivered systemically may exert a toxic effect upon the intact endothelium adjacent to the site of denudation; it is the endothelial cells in these adjacent segments and/or branches from which reendothelialization must commence. In contrast, fusion toxin delivered locally to the site of arterial injury is less likely to exert an effect upon endothelial cells in these adjacent segments, thus reducing any inhibitory effect upon reendothelialization.

Previous animal trials using diphtheria toxin–based fusion proteins for noncardiovascular applications have resulted in varying degrees of weight loss in rodents (P.A. Bacha, unpublished data, 1993). In the current study, marked differences in weight gain between groups receiving local and systemic delivery were observed; we therefore conclude that in the rat model, local delivery was not only superior for inhibiting neointimal lesion development but also avoided the deleterious effects associated with systemic toxicity. Recent work has concentrated on identifying from a variety of methods the most efficient and practical local delivery system.^{28 29 30 31 32}

To our knowledge, DAB₃₈₉EGF is the only investigated agent that has been shown to completely inhibit the proliferation of human vascular SMCs in culture.¹² The purpose of the present study was to confirm the ability of DAB₃₈₉EGF to preempt neointimal hyperplasia in an in vivo model. Our results demonstrate that DAB₃₈₉EGF successfully reduces neointimal thickening in the balloon-injured rat carotid artery model when delivered systemically as well as locally. This effect was significant when judged by any or all of three conventionally employed indices of morphometric analysis. We thus conclude that DAB₃₈₉EGF is a potent inhibitor of neointimal hyperplasia in vivo and that while systemic delivery demonstrates a significant effect in this regard, local delivery of DAB₃₈₉EGF appears more potent, avoids systemic toxicity, and thus represents a feasible strategy to be tested for prevention of restenosis.

View larger version (0K): [in this window] [in a new window]   Figure 8. Supplemental Figure I. EGF receptor immunohistochemistry. A, Photomicrograph of an uninjured rat carotid artery immunostained with a monoclonal antibody specific for the epidermal growth factor (EGF) receptor. Note the presence of the endothelium (magnification x100). B, Photomicrograph of an injured rat carotid artery harvested 30 minutes postinjury and immunostained for the presence of the EGF receptor (magnification x100). C, The same arterial section shown in A at higher magnification (x200). D, The same arterial section shown in B at higher magnification (x200). E, The same uninjured rat carotid artery shown in A and C, at a different segment, immunostained with the monoclonal antibody specific for the EGF receptor (magnification x200). F, The same uninjured rat carotid artery shown in B and D, at a different segment, immunostained with the monoclonal antibody specific for the EGF receptor (magnification x200).

View larger version (0K): [in this window] [in a new window]   Figure 9. Supplemental Figure II. Control panel. A, Negative control for EGF receptor immunohistochemistry performed on a section of rat skin (magnification x100). B, The same section of rat skin as shown in A at higher magnification (x200). C, Positive control for EGF receptor immunohistochemistry performed on a section of rat skin (magnification x100). D, The same section of rat skin as shown in C at higher magnification (x200). E, Negative control for the EGF receptor immunohistochemistry performed on a section of injured rat carotid artery harvested 30 minutes postinjury (magnification x100). F, The same arterial section as shown in E at higher magnification (x200).

	Acknowledgments

 
This study was supported in part by grants HL-40518 and HL-02824 (Dr Isner) from the National Heart, Lung, and Blood Institute, National Institutes of Health, and a grant (MT-11715, Dr Pickering) from the Medical Research Council of Canada. The authors wish to thank Eleanor Sullivan for her expert assistance with the histological preparations, Susan Rossow for her skilled assistance with immunostaining, and Mickey Neely for manuscript preparation.

Received September 15, 1994; accepted February 24, 1995.

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