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(Circulation. 2001;103:1135.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Effect of Selective or Combined Inhibition of Integrins _IIbß₃ and _vß₃ on Thrombosis and Neointima After Oversized Porcine Coronary Angioplasty

Timothy J. A. Chico, MRCP; Janet Chamberlain, PhD; Julian Gunn, MD; Nadine Arnold, VN; Sherron L. Bullens, AB; Thomas R. Gadek, PhD; Sheila E. Francis, PhD; Stuart Bunting, PhD; Michael Horton, PhD; Lynda Shepherd; Michael T. Lipari, MSc; Clifford Quan, PhD; Jochen Knolle, PhD; Hans Ulrich Stilz, PhD; Anusch Peyman, PhD; David C. Crossman, MD

From the Cardiovascular Research Group, Division of Clinical Sciences (NGHT), University of Sheffield, Clinical Sciences Centre, Northern General Hospital, Sheffield, UK (T.J.A.C., J.C., J.G., N.A., S.E.F., L.S., D.C.C.); Genentech Inc, South San Francisco, Calif (S.L.B., T.R.G., S.B., M.T.L., C.Q.); the Department of Medicine, University College London Medical School, UK (M.H.); and Hoechst Marion Roussel Deutschland GmbH, Frankfurt, Germany (J.K., H.U.S., A.P.).

Correspondence to Timothy J.A. Chico, MRCP, Cardiovascular Research, Genentech Inc, 1 DNA Way, South San Francisco, CA 94080. E-mail timchico{at}gene.com

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Thrombosis and neointima formation limit the efficacy of coronary angioplasty (PTCA). Clinical trials have implicated the adhesion molecules integrin _IIbß₃ and integrin _vß₃ in these processes. The roles of these molecules in vascular smooth muscle cell adhesion, platelet aggregation, and the thrombotic and neointimal response to oversize porcine PTCA was investigated by use of a selective _IIbß₃ antagonist (lamifiban), a selective _vß₃ antagonist (VO514), and a combined _IIbß₃/_vß₃ antagonist (G3580).

Methods and Results—In vitro, both _vß₃ inhibitors caused dose-dependent inhibition of porcine vascular smooth muscle cell adhesion to vitronectin but not to collagen type IV, fibronectin, or laminin, whereas selective _IIbß₃ inhibition had no effect. Intravenous infusions of either _IIbß₃ inhibitor in swine profoundly inhibited ex vivo platelet aggregation to ADP, whereas selective _vß₃ inhibition had no effect. In a porcine PTCA model, intravenous infusions of the integrin antagonists were administered for 14 days after oversized balloon angioplasty injury. After PTCA, there was regional upregulation of integrin _vß₃ in the developing neointima, as assessed by immunohistochemistry. Six hours after PTCA, obstruction of lumen by thrombus was reduced significantly by _IIbß₃ inhibition compared with either control or _vß₃ inhibition (mean control, 18.7%; VO514, 18.5%; lamifiban, 6.4%; G3580, 7.9%). Twenty-eight days after PTCA, there was a significant reduction of neointima with inhibitors of either integrin (mean intima/media ratio: control, 3.08; VO514, 1.33; lamifiban, 0.97; G3580, 1.32).

Conclusions—We conclude that both integrin _IIbß₃ and integrin _vß₃ participate in neointima development after experimental angioplasty.

Key Words: platelets • cell adhesion molecules • angioplasty • thrombosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Neointima development is characteristic of experimental and therapeutic arterial injury¹ and is an important component of restenosis after percutaneous transluminal coronary angioplasty (PTCA).² The role of platelets in restenosis is controversial.³ Platelet-derived mitogens promote smooth muscle proliferation and migration in vitro, processes thought to be central to neointima formation,⁴ and antagonists of these mitogens reduce neointima formation.^{5 6} The integrin _IIbß₃ (platelet glycoprotein IIb/IIIa) mediates binding of activated platelets to fibrinogen,⁷ the "final common pathway" of aggregation. _IIbß₃ inhibitors therefore represent powerful tools with which to test the hypothesis that platelets contribute to neointima development.⁸ The first clinical trial of an _IIbß₃ antagonist, the EPIC trial, showed a reduction both in ischemic complications and in clinical restenosis, apparently supporting this hypothesis.⁹ The humanized monoclonal antibody (abciximab) used in the EPIC trial, however, binds to the integrin ß₃ chain, thus inhibiting both _IIbß₃ and _vß₃.¹⁰ _vß₃ is more widely distributed than _IIbß₃ and has diverse functions, including mediating migration and proliferation of vascular smooth muscle cells (VSMCs).^{11 12} _vß₃ expression is upregulated after arterial injury¹³ and thus may play a role in neointima formation. Effects on one or both of these integrins could have mediated the reduction in restenosis reported in the EPIC trial.

Animal studies of platelet inhibition or depletion on neointimal responses to arterial injury have shown conflicting results, whereas most studies with selective _vß₃ antagonists or peptides with wider integrin specificity have shown reductions in neointima development.^{13 14 15 16 17} These studies differ in model, specificity of agents, dose and duration of treatment, and level of platelet inhibition achieved. It is not possible to compare separate studies of antiplatelet agents and anti-_vß₃ therapies. To resolve these issues, we undertook a comparative study using a porcine PTCA model. A selective _IIbß₃ antagonist (lamifiban),¹⁸ a selective _vß₃ antagonist (VO514),¹⁹ and a combined _IIbß₃ and _vß₃ antagonist (G3580)²⁰ were used to attempt to discern the relative contributions of these molecules to the coronary arterial response to PTCA. Additional experiments were performed to assess the effects of these compounds on porcine VSMC adhesion and to examine the expression of _vß₃ in the porcine coronary artery after PTCA.

	Methods

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Reagents
The structures of lamifiban¹⁸ and G3580²⁰ have been described previously. Lamifiban is a peptidomimetic agent selective for _IIbß₃ (IC₅₀ for fibrinogen binding to recombinant human _IIbß₃, 1.4 nmol/L; IC₅₀ for vitronectin binding to recombinant human _vß₃, >1 000 000 nmol/L). G3580 is a cyclic peptide (IC₅₀ for fibrinogen binding to _IIbß₃, 1.5 nmol/L; IC₅₀ for vitronectin binding to recombinant human _vß₃, 8 nmol/L). VO514 is a peptidomimetic selective for _vß₃ (IC₅₀ for fibrinogen binding to recombinant human _IIbß₃, >10 000; IC₅₀ for vitronectin binding to recombinant human _vß₃, 2 nmol/L).²¹

VSMC Adhesion
Six-well plates (Iwaki) were coated with fibronectin 2 µg/cm², laminin 2 µg/cm², collagen type IV 10 µg/cm², or vitronectin 125 ng/cm² (Sigma) by overnight incubation at 4°C. Porcine VSMCs were derived by explantation and used as confluent cultures (passages 2 to 5) suspended in serum-free medium. Cells (1x10⁵) in 1 mL of medium were added to each well after serial dilutions of antagonist or vehicle (10-µL volume). Plates were incubated at 37°C for 2 hours, then washed 3 times with PBS. Adherent cells were quantified by counting 10 random high-power fields (x200) and expressed as percentage of control. Experiments were carried out in triplicate.

Porcine PTCA and Intravenous Infusions
Experiments were carried out in accordance with Home Office regulations. Juvenile male Sus scrofa were anesthetized with ketamine (33 mg/kg) and enflurane (3% to 5%). A dual-lumen Hickman line (Bard) was placed in the right external jugular vein, and an 8F arterial sheath (Bard) was placed in the right common carotid artery. Animals were heparinized (200 IU/kg). At time 0, an intravenous bolus of saline, lamifiban (0.2 mg/kg), G3580 (1.5 mg/kg), or VO514 (100 µg/kg) was administered, followed by an intravenous infusion via a CADD-1 infusion pump (Sims Deltec). Doses of _IIbß₃-inhibiting agents were adjusted according to ex vivo platelet aggregation: lamifiban 0.12 to 0.21 mg · kg^-1 · h^-1 and G3580 0.875 to 1.5 mg · kg^-1 · h^-1. VO514 was infused at a constant rate (0.6 mg · kg^-1 · h^-1), a dose suggested from earlier experiments as likely to produce complete _vß₃ inhibition (data not shown). The target level of inhibition of platelet aggregation with lamifiban and G3580 was 80%. Quantitative coronary angiography was performed (IDIS) to identify a segment in the right coronary artery and left anterior descending coronary artery that would produce a 1.3:1-sized inflation with a standard 20-mm coronary angioplasty balloon. Each artery was dilated twice for 30 seconds at 8 atm between 30 and 60 minutes.

To assess the thrombotic response, 2 animals in each group were killed at 6 hours with intravenous pentobarbitone. Coronary arteries were dissected out, divided into 2- to 3-mm blocks, immersion-fixed in 10% formalin for 24 hours, embedded in paraffin, and sectioned. Perfusion fixing was avoided to prevent dislodgment of thrombus. To assess neointima formation, infusions continued for 14 days in the remaining animals. Animals were killed at 28 days with pentobarbitone, and hearts were removed, perfusion-fixed with 10% formalin, and sectioned as above.

Platelet Aggregation
Platelet-rich plasma was produced by centrifugation of citrated (0.38%) blood (11 seconds at 13 000 rpm) and aggregated in an Aggrecorder aggregometer (DIC) with 20 µmol/L ADP (Sigma) as agonist.

Arterial Morphometry
Sections with a breached internal elastic lamina (IEL) were analyzed. Arterial dimensions and areas were measured by a single observer blind to treatment using computerized image analysis (SeeScan). Thrombus area is expressed as percentage obstruction of lumen area (lumen area in vivo was derived from the perimeter of IEL). Neointima was assessed by intima/media area ratios, and injury score was determined by percentage breach in IEL.

Immunohistochemistry
Expression of _vß₃ was assessed by use of archival sections from porcine PTCA experiments conducted in the same manner. Sections were from animals killed at 1, 6, and 18 hours and 3, 7, 14, and 28 days after injury. Six injured arteries were examined from all time points except 28 days, at which point 38 injured arteries were examined. Sections were dewaxed, and nonspecific binding was blocked with 3% H₂O₂ and 1% dried milk/PBS. Anti-human _vß₃ monoclonal antibody 5H8 (Genentech Inc) was used to detect porcine _vß₃. Antibody specificity was validated immunocytochemically, biochemically, and functionally in tests using pig osteoclasts and other tissues (data not shown). 5H8 was applied in 1:100 dilution for 1 hour at room temperature. After application of a biotinylated secondary antibody and the Vector ABC kit, DAB solution was added and sections were counterstained with Carazzi’s hematoxylin.

Sections were graded by 2 blinded observers as - (negative), + (1% to 33% cells stained), ++ (34% to 66% positive), and +++ (>67% positive).

Plasma Levels
Plasma drug levels were taken from 2 animals per group. Whole blood was collected into EDTA (0.17%) and centrifuged at 13 000 rpm for 5 minutes to produce plasma. This was assayed for lamifiban, G3580, or VO514 by a previously described method.²²

Statistical Analysis
Data are expressed as mean±SEM. Statistical analysis was by 1-way ANOVA. Statistical calculations were performed with Instat (GraphPad).

	Results

Top Abstract Introduction Methods Results Discussion References

 
VSMC Adhesion
Selective _IIbß₃ inhibition (by up to 100 µmol/L lamifiban) had no effect on VSMC adhesion to any of the substrates tested (data not shown). In contrast, selective _vß₃ inhibition (with VO514) or combined _vß₃ and _IIbß₃ inhibition (with G3580) dose-dependently reduced adhesion of VSMC to vitronectin (Figure 1A and 1B). Neither agent affected VSMC adhesion to laminin, fibronectin, or collagen type IV in concentrations up to 100 µmol/L and 1 µmol/L for VO514 and G3580, respectively (data not shown). The IC₅₀ for adhesion to vitronectin was 5 µmol/L for G3580 and 25 nmol/L for VO514, higher than the quoted IC₅₀ for adhesion of recombinant human _vß₃ binding to vitronectin with these agents.^{21 22} This may be due to interspecies differences, the presence of uncoated nonspecific binding sites in the well, or the expression of additional vitronectin-recognizing receptors on VSMCs.

View larger version (50K): [in this window] [in a new window]   Figure 1. Dose-dependent inhibition of adhesion of VSMCs to vitronectin with (A) G3580 and (B) VO514.

_vß₃ Expression After Balloon Injury
As assessed by semiquantitative immunohistochemistry, integrin _vß₃ was detectable at moderate levels in the endothelium, media, and adventitia of both control and injured arteries. After injury, there was an increase in _vß₃-positive cells in the developing neointima, greatest at 28 days, the latest time point assessed in the study (Table and Figure 2).

View this table: [in this window] [in a new window]   Table 1. Semiquantitative Analysis of vß3 Expression After Oversized Balloon Injury in the Porcine Coronary Artery

View larger version (133K): [in this window] [in a new window]   Figure 2. Time course of expression of vß3 after arterial injury. Immunostaining vß3 with 5H8 antibody. a, Non-PTCA control; b, 1 hour after PTCA; c, 7 days after PTCA; and d, 28 days after PTCA. L indicates lumen. Arrowheads indicate representative vß3 positive cells. Arrows indicate breached IEL. Bars=25 µm.

In Vivo Experiments
Sixty-one animals were used. There were 4 procedural deaths (2 of the control, 1 lamifiban, and 1 G3580 groups) and 4 later deaths of unknown cause (1 G3580, 1 VO514, 2 control). Hickman line dislodgment or damage occurred in 12 animals (4 control, 3 lamifiban, and 5 G3580 animals). No deaths or dislodgment occurred in animals used to analyze thrombus formation. Control animals whose lines became dislodged remained in the trial, whereas active-treatment animals were withdrawn from analysis. Thus, data for platelet aggregation and arterial morphometry were available for 8 animals in the group to assess thrombus formation (2 per group) and 37 animals in the group to assess neointima (10 control, 9 lamifiban, 9 G3580, and 9 VO514). Only platelet aggregation data from animals subsequently analyzed for arterial morphometry are shown.

Effect of ß₃-Integrin Antagonists on Ex Vivo Platelet Aggregation
Both _IIbß₃ antagonists produced sustained and equivalent inhibition of ex vivo platelet aggregation to 20 µmol/L ADP, immediately after commencement (Figure 3A) and during the 14-day infusions (Figure 4). Selective _vß₃ antagonism did not affect platelet aggregation compared with control. The target of 80% mean inhibition of platelet aggregation was exceeded with both _IIbß₃ antagonists (mean percent baseline aggregation over 14 days: control, 113.8%; VO514, 108.7%; lamifiban, 14.9%; and G3580, 14.2%). There were no significant differences at any time point between control and VO514 treatment or lamifiban and G3580 treatment.

View larger version (24K): [in this window] [in a new window]   Figure 3. A, Ex vivo platelet aggregation (20 µmol/L ADP agonist) 0 to 300 minutes. Juvenile swine were treated with a bolus and 14-day infusion of saline control (n=10), lamifiban (n=9), G3580 (n=10), or VO514 (n=9). Oversized PTCA was performed on right coronary artery and left anterior descending coronary artery between 15 and 30 minutes; 2 animals per group were killed at 6 hours, and arteries were examined for thrombus (B). Data represent mean (SEM) for each group. B, Percentage lumen obstruction by thrombus 6 hours after PTCA. Degree of lumen obstruction was assessed histologically 6 hours after oversized PTCA. Number of arteries was 4 per group. Data represent mean (SEM).

View larger version (28K): [in this window] [in a new window]   Figure 4. Ex vivo platelet aggregation (20 µmol/L ADP agonist) 8 hours to 14 days. Juvenile swine were treated with a bolus and 14-day infusion of saline control (n=8), lamifiban (n=7), G3580 (n=8), or VO514 (n=7) after oversized PTCA. Data represent mean (SEM) for each group.

Effect of ß₃-Integrin Antagonists on Thrombus Formation
Four injured arteries per group were harvested 6 hours after the start of the infusions. There were no significant differences in injury scores between the groups (data not shown). Treatment with either _IIbß₃ antagonist significantly reduced percentage luminal obstruction by thrombus (Figure 3B) from 18.7% in the control to 6.4% in the lamifiban group and 7.9% in the G3580 group (P<0.05 for both groups). The selective _vß₃ inhibitor (VO514) had no effect on thrombus formation (18.5% versus 18.7% control).

Effect of ß₃-Integrin Antagonists on Neointima Formation
When the remaining coronary arteries were examined at 28 days after PTCA, 9 of 58 were found to be uninjured, with no discernible site of balloon injury (4 control, 1 lamifiban, 2 G3580, and 2 VO514). Thus, 49 injured arteries were examined (12 control, 13 lamifiban, 12 G3580, and 12 VO514). There were no significant differences in the mean injury scores between groups (control 26.9%, lamifiban 22.5%, G3580 23.5%, and VO514 21.4%). There was a significant reduction in intima/media area ratios for all treatment groups compared with control: control, 3.06; lamifiban, 0.97 (P<0.01); G3580, 1.32 (P<0.05); and VO514, 1.33 (P<0.05) (Figure 5A). There were no significant differences in luminal or arterial areas between the groups (Figure 5B).

View larger version (19K): [in this window] [in a new window]   Figure 5. The 28 days post-PTCA neointima/medial area ratios (A) and lumen and arterial areas (B) in 4 groups. Intimal and medial areas were measured 28 days after oversized PTCA. Number of arteries: Control, 12; lamifiban, 13; G3580, 12; and VO514, 12. Data represent mean (SEM).

Plasma Levels
Mean (SEM) plasma levels of the 3 agents during the infusions were G3580, 6.69 µmol/L; lamifiban, 1.30 µmol/L; and VO514, 8.55 µmol/L. These levels, compared with the IC₅₀ of adhesion of VSMCs to vitronectin (see above) of each agent are shown in Figure 6. Plasma levels of G3580 approximated the IC₅₀, whereas those of VO514 were far in excess of its in vitro IC₅₀. It is likely, therefore, that the level of inhibition of _vß₃ in the G3580-treated group was significantly less than that in the VO514 group.

View larger version (36K): [in this window] [in a new window]   Figure 6. Plasma levels of G3580, VO514, and lamifiban during neointima experiments. Plasma was taken from 2 animals per group at different times. Data represent mean (SEM). In vitro IC50 of each agent for adhesion of porcine VSMCs to vitronectin is also shown.

As was seen in the VSMC adhesion experiments, plasma levels required for platelet inhibition with lamifiban or G3580 were higher than required for inhibition of previously published data of recombinant human _IIbß₃ binding to fibrinogen (see Methods).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We describe the first comparison of the effects of antagonists of different integrins on the arterial response to injury. These data indicate that continuous infusions of antagonists of either _IIbß₃ or _vß₃, started before and continued for 2 weeks after porcine PTCA, significantly reduce neointima formation at 28 days. _IIbß₃ inhibitors had marked effects on platelet aggregation and thrombus formation 6 hours after PTCA, whereas a selective _vß₃ inhibitor showed no effect. Equally, the _vß₃ inhibitors were able to inhibit VSMC adhesion, a property not shared by the selective _IIbß₃ inhibitor, although final reductions in neointima were comparable between the 3 treatment groups.

Both _IIbß₃ inhibition and combined _IIbß₃/_vß₃ inhibition induced rapid inhibition of ex vivo platelet aggregation, translating into concomitant reductions in adherent thrombus. Measurement of ex vivo platelet aggregation may overestimate the degree of platelet inhibition, and the relevance of this measurement to in vivo platelet function is unclear. Nevertheless, in this study there was a constant level of platelet inhibition that was equivalent between the 2 agents.

Both agents significantly reduced neointima (relative reductions: lamifiban, 65.6%; G3580, 57.6%). These reductions were of similar magnitude to the reductions in early thrombus with each agent. The lack of measurable effects of lamifiban on processes other than platelet aggregation implies that a reduction in thrombus leads, directly or indirectly, to a diminished neointimal response.

Selective _vß₃ inhibition had no measurable effect on platelet aggregation, whereas it potently inhibited VSMC adhesion to vitronectin, a function mediated by _vß₃ and other integrins sharing the _v subunit. A role for _vß₃ in platelet adhesion to the damaged vessel wall has been suggested,²³ although this is usually mediated by other mechanisms, such as glycoprotein Ib/IX.²⁴ We cannot exclude an effect of _vß₃ on platelet adherence, but our data do suggest that this is unimportant to overall thrombus formation.

At 28 days, _vß₃ inhibition reduced neointima formation by 57.1%, suggesting that the demonstrated upregulation of _vß₃ in the neointima after PTCA is functionally important in the arterial response to injury; this is consistent with other studies.^{13 16 25} The mechanism for this is unclear, possibly reflecting inhibition of some aspect of VSMC function.

Inhibition of both _IIbß₃ and _vß₃ with a combined inhibitor reduced neointima at 28 days compared with control, but there was no evidence of an additive effect over inhibition of either integrin alone. This may be due to a number of reasons. Retrospective plasma level data indicated that _vß₃ inhibition is unlikely to have been equivalent between G3580 and VO514. This could explain the absence of a synergistic effect with G3580, which may have been acting as a pure _IIbß₃ inhibitor. Alternatively, if inhibiting thrombus formation leads directly to less neointima, then any subsequent anti-_vß₃ effect on neointima formation would be reduced, perhaps to a level that could not be detectable with the methods and numbers used in this study.

Our data support a role for both thrombus and _vß₃ in the development of neointima. Thrombus may act as a scaffold into which VSMCs migrate via _vß₃ mechanisms. Equally, volume of thrombus may be a surrogate for other biological signals arising from platelet aggregation, including cooperation of the platelet in the coagulation cascade or release of growth factors. The weak inhibitory effect of _IIbß₃ antagonists on platelet activation by agonists released at the site of injury,²⁶ however, suggests that _IIbß₃ inhibition exerts its influence by a reduction in the actual amount of thrombus.

_vß₃ antagonism has been shown to reduce neointima in other studies,^{13 14 15 17} although agent specificity has varied, and effects on _IIbß₃ are often discounted.¹⁶ _vß₃ inhibition may have effects not related solely to the recruitment of VSMCs to the thrombus in the lesion. _vß₃ also has effects on proliferation²⁵ and apoptosis.^{17 27} Inhibition of these processes may affect the amount of neointima formed independently of thrombus or may affect the reorganization of thrombus by VSMCs.

Some of our data correspond with clinical experience. Most obvious is the correlation between a reduction in ischemic complications and a reduction in thrombus after PTCA. Several clinical trials of _IIbß₃ antagonists with PTCA have shown a reduction in ischemic end points during the active-treatment infusions²⁸ with the same monitoring and platelet inhibition targets as our study. Trials using small-molecule agents or a bolus of abciximab without an infusion, however, showed that treatment postponed but did not abolish ischemic complications, with a "catching up" of events after the treatment was discontinued.²⁵ A bolus plus infusion of abciximab is able to permanently reduce ischemic events and possibly restenosis. A definite treatment duration seems to be necessary to permanently reduce ischemic complications (and presumably thrombus formation) after PTCA. It is possible that the prolonged duration of treatment in our study exceeded this duration and thus translated into meaningful reduction of neointima.

Conclusions
We conclude that the neointimal response to injury engendered by angioplasty is inhibited by antagonists of either integrin _IIbß₃ or _vß₃. The reduction in neointima with _IIbß₃ antagonists appears to be related to a reduction in thrombus formation at the site of injury, whereas the reduction seen with _vß₃ inhibition is independent of any effects on platelet aggregation or thrombus formation. These findings are both supportive of and supported by clinical trials and suggest novel approaches to the problem of restenosis.

	Acknowledgments

 
This study was funded by Genentech Inc. Professor Horton is supported by the Wellcome trust.

Received July 10, 2000; revision received August 23, 2000; accepted September 11, 2000.

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