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Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:1730-1737

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:1730-1737.)
© 1998 American Heart Association, Inc.

Original Contributions

Effects of ß3-Integrin Blockade (c7E3) on the Response to Angioplasty and Intra-Arterial Stenting in Atherosclerotic Nonhuman Primates

Jonathan S. Deitch; J. Koudy Williams; Michael R. Adams; Christopher A. Fly; David M. Herrington; Robert E. Jordan; Marian T. Nakada; Joseph A. Jakubowski; ; Randolph L. Geary

From the Departments of Surgery (J.S.D., C.A.F., R.L.G.) and Comparative Medicine (J.K.W., M.R.A.) and the Section of Cardiology (D.M.H.) of Wake Forest University School of Medicine, Winston-Salem, NC; Centocor Corp (R.E.J., M.T.N.), Malvern, Pa; and Eli Lilly Corp (J.A.J.), Indianapolis, Ind.

Correspondence to Randolph L. Geary, MD, Division of Surgical Sciences, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail rgeary{at}bgsm.edu


*    Abstract
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 *Abstract
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Abstract—Because the ß3-antagonist abciximab (c7E3 Fab) has significantly improved late outcomes after coronary angioplasty, the ß3 integrins have been implicated in the arterial response to injury. However, the mechanisms underlying this benefit are unknown. The observation that c7E3 binds ß3 integrins on vascular cells ({alpha}vß3) with affinity equal to that for the platelet glycoprotein IIb/IIIa integrin has led to the hypothesis that c7E3 may act directly on the artery wall to prevent restenosis after angioplasty. To test this hypothesis, we studied the effects of c7E3 on structural changes within the artery wall after angioplasty or stent angioplasty in 23 male cynomolgus monkeys with established atherosclerosis. Animals were randomly assigned to receive either a bolus of c7E3 (0.4 mg/kg IV, n=11) followed by a 48-hour infusion (0.2 µg · kg-1 · min-1) or an equal volume of vehicle (n=12). Animals received weight-adjusted aspirin and heparin and then underwent unilateral iliac artery experimental angioplasty and subclavian artery stent angioplasty (Palmaz). Iliac artery lumen diameter (LD) was determined by angiography at baseline (LDPre), after angioplasty (LDPost), and 35 days later (LDDay35). Arteries were then fixed by perfusion and removed for analysis. Lumen, intima, media, and external elastic lamina (EEL) areas were measured in iliac artery cross sections. Values from each injured iliac artery were normalized to the contralateral uninjured iliac artery to control for interanimal variability in baseline artery size and atherosclerosis extent. Intimal area was also measured in subclavian stent cross sections. c7E3 blocked platelet aggregation and prolonged the bleeding time from 2.8±1.1 to 19.8±2.5 minutes, P<0.001. Experimental angioplasty increased LDPost an average of 28%, and the initial gain was similar in both groups (P=NS). Despite an anti-platelet effect, c7E3 did not inhibit iliac lumen narrowing (LDDay35-LDPost: c7E3, -0.69±0.17 versus vehicle, -0.99±.17 mm, P=0.35); intimal hyperplasia (neointima area: c7E3, 1.12±.28 versus vehicle, 1.22±.20 mm2, P=0.77); or decrease in artery wall size (EEL area [percent of uninjured control]: c7E3, 101±7% versus vehicle, 121±7%). Stent intimal hyperplasia was also unaltered by c7E3 treatment (neointimal area: c7E3, 1.09±0.16 versus vehicle, 1.28±0.11 mm2, P=0.36). These results suggest that the benefits of c7E3 treatment in coronary angioplasty were not from inhibition of intimal hyperplasia or improved artery wall remodeling. Alternative mechanisms should be explored to explain improved late outcomes after angioplasty in patients treated with c7E3.


Key Words: ß3 integrins • atherosclerosis • restenosis • stents • cynomolgus monkeys


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Abciximab, the mouse-human chimeric monoclonal Fab fragment (c7E3) directed against platelet ß3 integrins (IIb/IIIa), has improved clinical outcomes after coronary angioplasty by significantly reducing mortality, myocardial infarction, and subsequent target-vessel revascularization (Evaluation of IIb/IIIa Platelet Receptor Antagonist 7E3 in Preventing Ischemic Complications [EPIC] trial).1 2 In a follow-up to EPIC, EPILOG (Evaluation of PTCA to Improve Long-Term Outcome by c7E3 GP IIb/IIIa Receptor Blockade) used an altered c7E3 and heparin dosing strategy to reduce the risk of bleeding. EPILOG confirmed the benefit of c7E3 on the composite study end point but failed to achieve an equivalent decrease in late target-vessel revascularization.3 Although the short-term effects of c7E3 treatment are likely due to platelet inhibition, the 26% reduction in clinical restenosis observed at 6 months in the EPIC trial may be due in part to platelet-independent effects on the artery wall.2

c7E3 is a nonspecific ß3-integrin antagonist and binds with equal affinity to the {alpha}vß3 integrin expressed by endothelial cells and vascular smooth muscle cells and to the platelet IIb/IIIa receptor.4 In addition to platelet inhibition, blocking ß3 integrins within the artery wall could improve the injury response, because these receptors mediate vascular cell adhesive interactions crucial in migration,5 6 7 replication,8 9 apoptosis,10 and extracellular matrix reorganization.11 12 13 This concept is supported by animal studies in which intimal hyperplasia was inhibited by nonselective ß3-integrin antagonists14 15 16 but not by platelet-specific integrin antagonists.17 18 As in EPIC, clinical trials of platelet-specific integrin antagonists19 20 have reduced acute thrombotic complications of angioplasty, but (in contrast to EPIC) late outcomes were not improved.

Despite these observations, the mechanisms underlying the protective effects of c7E3 remain unexplained. We initiated a study to test the hypothesis that ß3-integrin blockade would improve angioplasty outcomes in part by preventing lumen narrowing from impaired artery wall remodeling or intimal hyperplasia. Because c7E3 exhibits substantial specificity for primate ß3-containing integrins, we studied its effects on the response to experimental angioplasty and stenting of arteries in cynomolgus monkeys with established atherosclerosis.


*    Methods
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 *Methods
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Animal Model
Twenty-three male cynomolgus monkeys (Macaca fascicularis) were selected from the breeding colony of the Comparative Medicine Clinical Research Center of Wake Forest University School of Medicine. All animals had been fed an atherogenic diet (0.28 mg cholesterol/kcal) for 3 to 5 years to induce complex atherosclerotic lesions.21 22 23 Animals were then randomized into treatment (n=11, c7E3) and control (n=12, vehicle) groups stratified by duration of diet and the total plasma cholesterol (TPC) to HDL cholesterol (HDL-C) ratio to control for baseline atherosclerosis extent.23 Aspirin (5 mg/kg) was administered 24 hours before angioplasty and stenting.

Animals were anesthetized with ketamine (10 mg/kg IM) and butorphanol (0.05 mg/kg IM), and the left femoral artery was exposed by using sterile techniques. Baseline hematology and coagulation studies were performed (see below), and cefazolin (25 mg/kg IV) and heparin (100 U/kg IV) were administered, followed by either c7E3 (0.4 mg/kg IV bolus) or an equal volume of vehicle (0.15 mol/L NaCl with 0.01 mol/L NaPO4 and 0.001% Tween 80). A 5F sheath was then inserted into the left common femoral artery, and hematology and coagulation studies were repeated. Baseline angiography was performed in the iliac arteries as previously described,24 and a 3F Fogarty balloon catheter (Baxter) was then inserted, inflated, and retrieved across the left common iliac artery 3 times under moderate tension.22 The catheter was then removed and cineangiography repeated. A Palmaz coronary stent (J & J Interventional Systems) was then deployed in the proximal left subclavian artery by using a 3.0-mm angioplasty catheter inflated to 6 atm for 30 seconds. The catheter and sheath were then removed, and the femoral puncture site was repaired with 7-0 prolene sutures (Ethicon).

An osmotic pump (Alzet 2-mL-1, Alza Corp) was implanted at the time of angioplasty to infuse either c7E3 (0.2 µg · kg · -1min · -1 IV) or vehicle for 48 hours. Pumps were preprimed overnight at 37°C in sterile saline and inserted subcutaneously into the left flank after the angioplasty procedure. Pump tubing was tunneled into the groin wound, inserted into the femoral vein, and secured with sutures (Prolene, Ethicon Corp). Wounds were closed in layers and the animals were then returned to single cages to recover.

Two days after angioplasty and stenting, the animals were anesthetized (as above), and hematology and coagulation studies were repeated. The infusion pumps were then removed. Thirty-five days after angioplasty and stenting, the animals were anesthetized for repeated hematology and coagulation studies. Iliac artery angiography was also repeated to document the final iliac lumen diameter (LD). Animals were then deeply anesthetized (pentobarbital, 100 mg/kg IV), heparinized (200 U/kg IV), and exsanguinated while lactated Ringer's solution was infused via a left ventricular canula at 100 mm Hg pressure. Stented left subclavian arteries and both iliac arteries were then fixed by perfusion with 10% buffered formalin at 100 mm Hg for 30 minutes. Arteries were removed en bloc and placed into fresh formalin for 36 hours before paraffin embedding (see below).

Animal care and procedures were performed at the Comparative Medicine Clinical Research Center of Wake Forest University School of Medicine in accordance with state and federal laws. Animal protocols were approved by the institutional Animal Care and Use Committee and conformed to guidelines set forth in the "Principles of Laboratory Animal Care" (formulated by the National Society for Medical Research) and by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 86-23, revised 1985).

Plasma Lipids, Hematology, and Coagulation Studies
Plasma lipids were determined 3 times during the period of atherosclerosis induction, including TPC, HDL-C, LDL-C, triglycerides, and lipoprotein(a) [Lp(a)]. The ratio of TPC to HDL-C was determined and the average ratio for each animal used for randomization into treatment and control groups. Hematology and coagulation studies were performed before and immediately after angioplasty and stenting, then 2 and 35 days later, and included the following measurements: hematocrit (HCT), platelet count (PLT), partial thromboplastin time (PTT), prothrombin time (PT), bleeding time (BT), and platelet aggregation in response to 20 µmol/L ADP.

BTs were measured in the forearm by using a neonatal sphygmomanometer cuff inflated to 40 mm Hg in the upper arm. A standardized laceration was created by using a BT kit (Surgicutt, ITC, Inc), and the BT was determined according to the manufacturer's directions. Blood was wicked from the cut with filter paper after 30 seconds and then at 30-second intervals until the bleeding had stopped, at which point the time was recorded.

Platelet aggregation studies were performed before drug administration, 30 minutes after the bolus, 2 days after the bolus/infusion, and at necropsy. Blood was drawn from the lesser saphenous vein with a 19-gauge needle and syringe containing 0.1 mol/L sodium citrate anticoagulant. Within 30 minutes of collection, blood was centrifuged at 180g for 10 minutes at room temperature. The platelet-rich plasma supernatant was counted in a Coulter counter and diluted with platelet-poor plasma to a final concentration of 2.5±0.25x108 platelets/mL. Platelet-poor and platelet-rich plasmas (0.5 mL) were then pipetted into aggregometer cuvettes containing a magnetic stir bar (1200 rpm). A stable baseline was obtained with a standard aggregometer (Chronolog Lumi-Aggregometer) at 37°C for 1 minute. Aggregation was then initiated by adding 20 µmol/L ADP and allowed to proceed for 5 minutes. Aggregation was characterized by recording the change in percent light transmittance and the change in the initial slope of aggregation after platelet shape change (Figure 1Down).



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  		Figure 1. Line chart recording shows complete inhibition of platelet aggregation in response to 20 µm ADP after c7E3 bolus (0.4 mg/kg IV, above) and normal platelet aggregation in a vehicle-treated animal (below). Platelet aggregation was normal at baseline in both study groups but completely inhibited after administration of c7E3. Inhibition continued throughout the 2-day infusion of c7E3 and normalized before necropsy 35 days after angioplasty.

Plasma c7E3 levels were measured at each time point by enzyme immunoassay using monoclonal antibodies specific for c7E3 Fab.25 In brief, an anti-c7E3 capture antibody (Centocor Corp) was coated onto microtiter plates, which were then washed 3 times with PBS and blocked with PBS and 1% BSA to inhibit nonspecific binding. Serial dilutions of c7E3 standards (0.98 to 1000 ng/mL) or plasma test samples (1:5 or greater dilution) were then added for 1 hour at 37°C. Plates were rinsed with PBS and incubated for 1 hour at 37°C with a second anti-c7E3 Fab antibody conjugated to biotin (Centocor), which recognizes a different c7E3 epitope than the capture antibody. The biotinylated antibody was reacted with horseradish peroxidase and then with o-phenylenediamine (Sigma Chemical Co) as a chromogen. Color development was stopped with 4N H2SO4 and quantified by absorbance with a microtiter plate reader at 480 nm with 650 nm subtraction. Plasma c7E3 concentrations were then determined by using a standard curve generated from the c7E3 standards included with each assay.

Quantitative Iliac Artery Angiography
Iliac cineangiography was recorded onto 35-mm film with a calibration ruler included in each image. A computer-assisted edge-detection protocol was then used as previously described26 to define the average LD of injured and contralateral uninjured common iliac arteries. Image magnification was calibrated from the ruler, and mean common iliac LD (in millimeters) was determined before injury, immediately after injury, and 35 days after injury by technicians blinded to treatment assignment. Anatomic landmarks were used to ensure that the same portion of the iliac artery was measured at each of the 3 time points.

Histology and Morphometry
Perfusion-fixed common iliac arteries were divided into 5 rings of equal length ({approx}4 mm) for paraffin embedding. Sections of 5-µm thickness were cut from each ring and stained with Verhoeff–van Gieson's stain for cross-sectional morphometry. A videomicroscopic image of each cross section was captured and digitized by using a Power Macintosh and video frame grabber.23 Digitized images were then analyzed with NIH-Image software (public domain from National Institutes of Health; zippy.nimh.nih.gov). Areas bounded by the external elastic lamina (EEL), internal elastic lamina (IEL), and lumen were determined and the medial and intimal areas then calculated by subtraction.22 23 Values for each of the 5 cross sections of individual iliac arteries were averaged.27 We have previously shown a strong correlation within individual atherosclerotic monkeys between right and left iliac artery size (EEL area), plaque area, and lumen area.22 Therefore, injured iliac arteries retrieved 35 days after angioplasty were compared with the contralateral uninjured control iliac arteries to normalize for interanimal variation in baseline iliac lumen, plaque, and artery wall area. In this way, the change from baseline was estimated within each animal, and normalized values were then compared between c7E3-treated and control groups. Neointima was delineated from preexisting atherosclerosis by immunostaining the cross sections for von Willebrand factor by using a polyclonal antibody (Dako) as previously described.28 Neointima is readily distinguished from underlying atherosclerosis by its uniform, intense staining at 1 month after angioplasty. The depth of artery wall injury was graded in each cross section as previously described23 : 0, no fracture; 1, plaque fractured; 2, IEL disrupted; 3, media fractured; 4, EEL disrupted; and 5, wall rupture.

Stented subclavian artery segments were divided at the stent articulation, and the distal stent half was embedded under vacuum in methyl methacrylate and sectioned into 4 rings (EXAKT system), which were mounted onto plastic slides, polished to 20-µm thickness, and stained for morphometry. Images were captured from each cross section (see above) and the neointimal area determined by subtracting the lumen area from that bounded by the stent struts and preexisting atheroma. Neointimal thickness (in millimeters) was determined by dividing intimal area (square millimeters) by stent perimeter length (millimeters) to normalize neointimal area for variations in artery size. Mean neointimal area and thickness were determined by averaging values from 4 sections per stented artery segment. A single observer blinded to animal treatment group determined all area measurements and injury grades.

Statistical Analysis
Unpaired comparisons were made between c7E3-treated and control animals for hematology, coagulation, angiography, and morphometry outcomes, whereas paired comparisons were made within treatment groups for changes in hematology and coagulation studies over time. Student's t test (2-tailed) was used with significance assigned at the P<0.05 level. All values are reported as mean±SEM, n=11 for c7E3-treated animals, and n=12 for control animals unless specified otherwise.


*    Results
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 up arrowMethods
 *Results
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Plasma Lipids and Extent of Preexisting Atherosclerosis
The atherogenic diet increased TPC levels to 13.1±1.1 mmol/L (507±42 mg/dL) for all animals (n=23), and randomization resulted in similar lipid values among c7E3-treated and vehicle-treated animals (Table 1Down). For comparison, animals fed a low-cholesterol chow diet (15% of calories from fat) have TPC levels of 3.6±0.6 mmol/L (120 mg/dL, n=170). Animal weights were similar for c7E3 and vehicle groups (4.9±0.4 versus 5.2±0.4 kg, respectively, P=0.52). Resulting baseline atherosclerosis extent was also similar in c7E3 and vehicle groups, as determined by measuring plaque area in the uninjured right common iliac artery of each animal (intimal area: c7E3, 0.85±0.16 versus vehicle, 0.83±0.15 mm2, P=0.92).


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  		Table 1. Baseline Plasma Lipids

Effects of c7E3 on Hematology and Coagulation Parameters
Baseline blood work (HCT, PLT, PT, PTT, and BT) was similar for c7E3 and control animals (Table 2Down). Immediately after administration of c7E3, platelet aggregation was completely inhibited (Figure 1Up) and the BT prolonged (Table 2Down). The PT was unchanged and the PTT was increased transiently in both groups by heparin. The anti-platelet effects of c7E3 persisted throughout the 2-day treatment period but later normalized before necropsy. The BTs of treated animals had partially recovered at 2 days and were normal at necropsy (Table 2Down). The experimental angioplasty and stent procedure resulted in modest blood loss in both c7E3 and vehicle groups, with an associated fall in HCT that was not increased by c7E3 treatment (P=0.51). There were no bleeding complications related to c7E3 administration. PLT counts were similar in treated and control animals at baseline and during the 2-day treatment period but increased slightly in treated animals while decreasing slightly in controls by day 35 (Table 2Down).


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  		Table 2. Coagulation and Hematology Variables

In treated animals, plasma c7E3 levels increased appropriately after the initial bolus, remained elevated during the 2-day treatment period, and were then undetectable at 35 days. Plasma drug levels after the initial bolus of c7E3 (550±120 ng/mL) were comparable to those achieved in human beings 1 hour after the standard clinical dose of 0.25 mg/kg IV, and levels achieved after the 2-day continuous infusion (233±124 ng/mL) were similar to those in patients after the standard infusion of 10 µg/min for 12 hours.25

Iliac Angiography
Angiographic assessment of iliac artery LD was completed successfully at each time point (preinjury, postinjury, and at 35 days) for 10 c7E3- and 9 vehicle-treated animals. Preangioplasty lumen caliber was similar for left and right iliac arteries in both treated and control animals (LD: c7E3, 2.35±0.17 versus 2.36±0.17 mm; vehicle, 2.42±0.07 versus 2.40±0.11 mm; left versus right, respectively, P=NS). The degree of lumen dilation after experimental angioplasty averaged 28.4±3.3% and was not significantly different among the c7E3 and vehicle animals. LD returned to near baseline in both groups 35 days after experimental angioplasty, and c7E3 treatment did not prevent lumen narrowing (LDDay35 as a percent of LDPre: c7E3, 93±6% versus vehicle, 95±5%, P=0.84). The average loss of iliac artery LD after angioplasty (LDDay35-LDPost) was -0.69±0.17 mm in the c7E3-treated group versus -0.99±0.17 mm in the vehicle group (P=0.35).

Effects of c7E3 on Iliac Artery Morphometry
Morphometry data derived from each injured iliac artery (day 35) were normalized to the contralateral uninjured iliac artery within each animal to control for interanimal variability in baseline artery size and plaque size. Previous studies by our group have shown that the artery wall, lumen, and plaque size are similar within the right and left common iliac arteries of individual atherosclerotic monkeys (r=0.98 for intimal area and r=0.90 for lumen area, n=109 male monkeys).22 23

Baseline uninjured iliac artery size (EEL area), plaque area, and lumen area were similar for both groups (P=NS). Experimental angioplasty resulted in fracture of preexisting atherosclerotic plaques and injury to the underlying artery wall (Figure 2Down).22 23 The depth of artery wall injury was similar for c7E3- and vehicle-treated animals (injury grade 0 to 5: c7E3, 2.7±0.5 versus vehicle, 3.2±0.3, P=0.33). Resulting intimal hyperplasia led to the accumulation of neointima that increased the intimal area in injured iliac arteries (Figures 2 through 4DownDownDown). Although the neointima was slightly smaller among c7E3-treated animals, this effect was not statistically significant (Figures 3Down and 4Down).



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  		Figure 2. Composite photomicrograph of iliac and subclavian artery cross sections from a c7E3-treated animal. Lesions are typical of arteries from both treatment groups. Atherogenic diet induced complex intimal lesions shown in uninjured iliac artery (A). Contralateral iliac artery was removed 35 days after experimental angioplasty (B). Angioplasty has fractured the preexisting plaque (p; arrowheads) and injured the overlying media, with neointimal ingrowth (n). Palmaz stents were deployed in the proximal left subclavian artery, and after 35 days, neointimal ingrowth has covered the underlying plaque and stent struts (C). Original magnification: A and B, x40; C, x100. All panels, Verhoeff–van Gieson's stain.



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  		Figure 3. Bar chart shows mean cross-sectional area measurements (mm2) from injured iliac arteries removed 35 days after experimental angioplasty in animals treated with c7E3 (solid bars) or vehicle alone (crosshatched bars). There were no significant differences between groups for each of these end points. EEL indicates area bounded by external elastic lamina, an indication of overall artery size.



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  		Figure 4. Bar chart shows normalized cross-sectional area measurements from injured iliac arteries removed 35 days after experimental angioplasty in animals treated with c7E3 (solid bars) or vehicle alone (crosshatched bars). Measurements made in each injured iliac artery were normalized to the corresponding measurement from the contralateral uninjured iliac artery within each animal. Because iliac plaque area and lumen area are highly consistent from right to left in each animal, this strategy controls for interanimal differences in baseline artery size and lesion extent (refer to text). Data are expressed as percent of control uninjured iliac artery. In contrast to raw data presented in Figure 3Up, after normalization the decrease in artery size and lumen area 35 days after experimental angioplasty was more pronounced in treated animals than in controls. *P<0.05, c7E3 versus vehicle.

After experimental angioplasty, the initial increase in LD (by angiography, as described above) was lost by day 35 in both groups of animals (Figures 3Up and 4Up). c7E3 did not prevent lumen narrowing, and the slightly smaller average lumen area at day 35 in the c7E3-treated animals was accounted for by a small but significant (P<0.05) concomitant decrease in artery wall size (EEL area). Thus, c7E3 did not enhance compensatory artery wall remodeling but rather slightly increased wall shrinkage (Figures 3Up and 4Up).

Effects of c7E3 on Stent Intimal Hyperplasia
Stent intimal hyperplasia was evaluated in cross sections cut from successfully stented subclavian arteries in 11 c7E3-treated and 8 vehicle-treated animals (Figure 2Up). In the vehicle group, we were unable to cross the subclavian artery with stents in 3 animals and 1 stent thrombosed. Stent injury scores were similar between c7E3 and vehicle animals (mean strut depth 0 to 5; see scale in Methods: c7E3, 0.3±0.09 versus vehicle, 0.2±0.06 mm, P=0.73). Stent neointimal area was similar for both groups, and when neointimal area was normalized to stent circumference, mean neointimal thickness was also unaltered by c7E3 treatment (Figure 5Down).



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  		Figure 5. Bar chart shows mean neointimal area (mm2) accumulating within Palmaz stents 35 days after deployment into subclavian artery of c7E3- and vehicle-treated animals. Mean neointimal thickness (mm), determined by dividing neointimal area by stent circumference, is also shown for each group.


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The current study was undertaken to test the hypothesis that nonselective ß3-integrin blockade can inhibit restenosis after angioplasty by augmenting artery wall remodeling or by limiting intimal hyperplasia. Our goal was to gain insight into the mechanisms by which c7E3 dramatically improved patient outcomes after angioplasty in recent clinical trials and to extend the results of previous experimental studies documenting ß3-integrin regulation of vascular cell function in vitro and in vivo. The major findings of the current study were that c7E3, given at a dose comparable to that in EPIC but for a longer duration, significantly inhibited platelet function but did not prevent lumen narrowing after experimental angioplasty or Palmaz stent angioplasty in atherosclerotic monkeys.

Determinants of Postangioplasty Lumen Narrowing: Potential Targets for ß3-Integrin Blockade
In the monkey model, as in human beings, experimental angioplasty dilates the lumen by fracturing atherosclerotic plaque and by stretching or tearing the overlying media.22 23 Acute or subacute loss of the gain in lumen caliber results from recoil, thrombus formation, and vasospasm. A brief wave of replication then occurs throughout the artery wall, peaks at 4 days, and returns to basal levels within 7 to 14 days.22 Cells then migrate and accumulate at sites of injury, forming a neointima between 7 and 28 days.22 28 Despite increased intimal mass, lumen narrowing is explained largely by changes in artery wall size (EEL area) rather than by lumen encroachment from the neointima per se.23 Therefore, lumen narrowing after experimental angioplasty in the monkey model, as in human beings,29 is largely due to failed remodeling or artery wall "shrinkage."

Each of these components of the response to injury depends in part on adhesive interactions between cells and matrix, thus providing the potential for intervention at the level of cell surface integrins. Targets for c7E3 specifically include thrombus formation, by inhibiting platelet activation and adhesion (IIb/IIIa), and tissue factor–mediated thrombin generation, recently shown to involve both IIb/IIIa and {alpha}vß3.30 c7E3 could also inhibit vasospasm within treated artery segments, as RGD peptides promote vasorelaxation31 in part by blocking {alpha}vß3.32

Perhaps the most compelling evidence for participation of the ß3 integrins in the response to injury comes from studies of intimal hyperplasia. ß3 antagonists have successfully reduced neointimal formation after experimental angioplasty in rabbits and hamsters,14 15 16 and the individual components of intimal hyperplasia (cell replication, migration, and extracellular matrix elaboration) each depend in part on ß3 integrins, as demonstrated by in vitro blocking experiments.5 6 7 8 9 33 34 Inflammation at sites of injury may also be inhibited by c7E3 blockade of the leukocyte Mac-1 integrin, which mediates leukocyte binding to fibrinogen and intercellular adhesion molecule-1.35 Preventing the influx of activated leukocytes may reduce the elaboration of cytokines and growth factors implicated in intimal hyperplasia.

As mentioned above, remodeling has emerged as a key determinant of lumen caliber after angioplasty.29 A decrease in artery wall size (shrinkage) after angioplasty may be due to extracellular matrix reorganization analogous to that in healing wounds in other tissues.28 Wound contraction occurs from integrin-dependent cellular reorganization of matrix and matricellular proteins.11 12 13 If, as in cutaneous wounds, smooth muscle cells and myofibroblasts within the healing artery wall reorganize and compact extracellular matrix, artery wall tissue contraction may occur. In support of this concept, smooth muscle cells have been shown to contract collagen and fibrin gels via ß1 and ß3 integrins, respectively,11 36 37 as contraction is prevented by specific blocking antibodies. Theoretically, c7E3 could inhibit adhesive interactions between cells and the ß3 ligands4 abundant within the injured atherosclerotic artery wall, including vitronectin, thrombospondin, fibronectin, denatured collagen, fibrin(ogen), von Willebrand factor, and osteopontin.11 28 38 39 40 41 42

ß3 Integrins and Restenosis: Contrasts Among the Current Study, EPIC, and EPILOG
Despite the evidence cited above, the findings of the current study argue against a central role for ß3 integrins in restenosis. Intimal hyperplasia was unaltered in treated animals, both in stents and in injured iliac arteries, and the relative shrinkage of the iliac artery wall after experimental angioplasty was slightly greater in the c7E3 group than in the controls. These data suggest that the improved outcomes in the EPIC trial may not be due to changes in artery lumen caliber but rather to other forms of vessel wall "passivation."

The dose of c7E3 in the current study was selected to be comparable to that used in the EPIC trial25 but for a longer period of time. We extended the infusion from 12 hours (as in EPIC and EPILOG) to 2 days to maximize potential treatment effects. Free plasma levels of c7E3 at the end of the 2-day infusion were equal to those achieved in human beings treated with the EPIC dosing regimen.25 Free drug levels would have fallen shortly after removing the infusion pumps, but platelet inhibition continues for several days after the infusion in humans, and presumably, artery wall effects persist as well.25 This is an important point, because the induction of ß3 integrin ({alpha}vß3) after injury appears within this time frame.

Although {alpha}vß3 is largely limited to adventitial microvessels in the normal human and monkey artery wall, its expression is prominent in atherosclerotic vessels.28 The pattern of staining in monkey atherosclerosis with an {alpha}vß3-specific antibody (LM609) is similar to that in human coronary atherosclerosis.28 43 Expression increases further after experimental angioplasty. Beginning at 2 days, increased staining is seen in the injured media overlying plaque fracture sites, and by 4 to 7 days, medial staining is diffuse.28 Intense staining for {alpha}vß3 is also seen in {alpha}-actin–positive cells appearing in the neointima as early as 7 days, but at 1 month the mature neointima has only modest, diffuse staining.28 These data indicate that an appropriate target for c7E3 is present in the atherosclerotic artery wall and that plasma levels of free drug comparable to those in EPIC were available for >2 days after angioplasty. This time frame overlaps with the increased expression of {alpha}vß3 by medial and intimal cells participating in the initial injury response.

Although the current study suggests that restenosis may not have been reduced in EPIC, the lack of angiography data from that trial leaves this question unanswered. The clinical benefit in EPIC was achieved at the cost of increased bleeding complications.1 2 To address this issue, a follow-up trial, EPILOG, was initiated to determine whether an alternative heparin and c7E3 dosing strategy could maintain the benefit of EPIC without the increased risk of bleeding. EPILOG used a weight-adjusted dose for both c7E3 and heparin, in contrast to the fixed dosing for both drugs in EPIC.3 The result was striking, in that the increased risk of bleeding was eliminated while a significant reduction in the 30-day composite end point of death, myocardial infarction, and target-vessel revascularization was achieved. In contrast to EPIC, however, the need for target-vessel revascularization at 6 months was not reduced in the c7E3 treatment group.3 Although differences exist between these 2 trials (ie, 12% unplanned use of stents in EPILOG), the disparity in late outcomes remains unexplained.

Our dosing strategy delivered more weight-adjusted c7E3 than in EPIC or EPILOG, but we gave only a single weight-adjusted bolus of heparin, as in EPILOG, and in contrast to both studies, we did not continue aspirin therapy after the procedure. Heparin and aspirin have not reduced restenosis rates in nonhuman primates44 or in human beings,45 but potential combined effects with c7E3 may need to be considered.

Our treatment protocol also differed from dosing strategies of other ß3 inhibitors that are effective at inhibiting intimal hyperplasia in nonprimate animal models. Choi et al14 infused an RGD-peptide inhibitor of ß3 integrins into the adventitial space for 2 weeks after rabbit carotid balloon injury, at which point intimal thickening was reduced compared with that in controls. Matsuno et al16 administered another RGD-containing peptide and found inhibition of intimal hyperplasia after carotid denudation in hamsters with a 7-day infusion when started before the injury. These RGD compounds have a broader specificity than does c7E3, in that they can inhibit non–ß3-containing {alpha}v integrins. In a third study by van der Zee et al,15 angioplasty was performed in rabbit iliac arteries and vessels treated by local or systemic delivery (daily injections for 48 hours) with the anti-ß3 antibody LM609. This study was similar to our protocol, in that treatment was continued for 48 hours and arteries were then removed for analysis at 4 weeks. In contrast to our findings, LM609 inhibited intimal hyperplasia in rabbits. Although the normalized change in iliac intimal area was slightly reduced in c7E3-treated animals ({approx}10%, Figure 4Up), this difference was not statistically significant. Given the observed variability in intimal area, many more animals would be required to determine whether this small decrease was due to c7E3 or to chance alone. Unfortunately, none of the previous animal studies reported changes in lumen area after angioplasty or the effects on artery wall remodeling. Differences in animal species, method of arterial injury, a lack of preexisting atherosclerosis, and the varied anti-ß3 agents used in each study preclude direct comparisons to the results presented herein.

Limitations of the Model
Cynomolgus monkeys are uniquely suited for modeling human atherosclerosis because the pathological characteristics of their atherosclerotic lesions show a striking resemblance to human lesions,21 and monkey lipid46 47 and coagulation48 profiles are similar to those of human beings. Human arteries generally remodel to accommodate atherosclerotic plaque growth and thereby prevent lumen narrowing,21 49 and the same is true for the monkey model.21 However, as with any animal model, there are shortcomings. Stenosis does develop spontaneously in the monkey model but infrequently during the first few years of atherosclerosis induction. Less than 10% of animals develop flow-limiting stenoses in iliac or coronary arteries within 3 years of consuming an atherogenic diet, and longer studies are impractical. Thus, the lumen narrowing measured in the current study is not truly restenosis but rather loss of the initial gain after angioplasty in an atherosclerotic artery.

The injury produced by an embolectomy catheter may also differ from that induced by a Greuntzig-type catheter. The embolectomy catheter injury is more likely to denude the endothelium and produce longitudinal shear. However, we have documented that preexisting atherosclerotic plaque is not removed by this procedure,22 and a striking similarity exists between the appearance of these injured arteries and those from human angioplasties, as documented by histopathological examination and intravascular ultrasound.29 50 51 The embolectomy catheter may also produce a more consistent injury from artery to artery because the degree of damage is not so critically dependent on accurate balloon sizing as with fixed-diameter catheters.

The perfect "model" remains the human patient. However, until minimally invasive techniques such as magnetic resonance angiography, duplex ultrasonography, or intravascular ultrasound achieve suitable resolution, the cynomolgus monkey model of atherosclerosis may provide the closest parallel.

In summary, c7E3 did not improve the structural response of the injured artery wall after experimental angioplasty or Palmaz stenting that resulted in lumen narrowing in atherosclerotic nonhuman primates. This result is in contrast to previous studies in nonprimate species.14 15 16 Our findings suggest that the durable benefits of c7E3 treatment evident at 6 months in the EPIC trial were not due to modulation of artery wall remodeling or intimal hyperplasia. The molecular and cellular bases for the protective effects of ß3-integrin antagonists after coronary artery reconstruction remain to be defined.


*    Acknowledgments
 
Funding was provided by a grant from Centocor and Eli Lilly (Dr Geary) and by NIH grants RO1-HL57557 (Dr Geary), PO1-HL45666 (Dr Williams), and HL49488 (Dr Herrington). The authors wish to thank Deanna Brown for her assistance with histopathology, Deborah Golden and Kelly Forest for assistance with animal care, and Michelle Gammons for preparing the manuscript.

Received February 3, 1998; accepted April 30, 1998.


*    References
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