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Cardiovascular Research 1997 36(3):408-428; doi:10.1016/S0008-6363(97)00184-3
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Selective {alpha}vβ3 integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury1:

Evidence for the functional importance of integrin {alpha}vβ3 and osteopontin expression during neointima formation

S.Sanjay Srivatsaa, Lorraine A Fitzpatrickb, Peter W Tsaoc,2, Thomas M Reillyc, David R Holmes, Jra, Robert S Schwartza and Shaker A Mousac,*

aDivision of Cardiology, Department of Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA
bDivision of Endocrinology and Metabolism, Department of Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA
cDuPont Merck Pharmaceutical Company, Route 141 & Henry Clay Road, Experimental Station, Wilmington, DE 19880-0400, USA

* Corresponding author. Tel: +1 (302) 695 8418; E-mail: mousasa@a1.lldmpc.umc.dupont.com

Received 12 May 1997; accepted 10 July 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Lumen loss from vascular restenosis remains a leading cause of chronic revascularization failure. Objective: We hypothesized that cell-matrix adhesion, migration, and differentiation events that underlie restenosis are mediated by {alpha}vβ3 integrin-ligand interactions. Methods: Using immunohistochemistry and in situ hybridization, we examined the spatial and temporal vessel wall expression of {alpha}vβ3 and osteopontin following deep coronary arterial injury. Cell migration and adhesion assays were performed to demonstrate the affinity and specificity of XJ735 for various vessel wall integrins. The effects of XJ 735 (a selective cyclic Arg-Gly-Asp (RGD) peptidomimetic {alpha}vβ3 antagonist) on neointimal hyperplasia and lumen stenosis were tested in a porcine coronary injury model. Normolipemic swine underwent oversized stent injury followed by XJ 735 administration (9 animals, 28 lesions; 1 mg/kg bolus+7 days 4 mg/kg/d infusion+21 days 2 mg/kg i.v. bolus 12 hourly) or placebo (10 animals, 30 arterial lesions). Results: Maximal {alpha}vβ3 immunoreactivity was observed between 7–14 days following injury in the neointima, media, and adventitia. Maximal osteopontin mRNA signal in the neointima, media, and adventitia was observed at 14, 7 and 28 days respectively. IC50 for XJ 735 {alpha}vβ3-mediated inhibition of human and porcine endothelial cell adhesion, and vascular smooth muscle cell migration, ranged from 0.6 to 4.4 µM. In contrast, IC50 for porcine or human {alpha}IIb/β3, {alpha}4β1, {alpha}vβ5, and {alpha}5β1 inhibition exceeded 100 µM. Steady state XJ 735 plasma levels exceeded 5 µM. Despite slightly higher injury scores in XJ 735 treated animals, significant reductions in mean neointima area (43% reduction; p = 0.0009), and mean percent lumen stenosis (~2.9 fold reduction; p = 0.04) were observed in XJ 735 treated animals. XJ 735 treatment did not significantly alter the relative size of the arterial injury and reference sites (geometric remodeling). Comparison of neontima area vs. injury score regression lines revealed significant reductions in slope (p = 0.0001) and intercept (p = 0.0001) for XJ 735. Conclusions: Selective {alpha}vβ3 blockade is an effective anti-restenosis strategy that potently limits neointimal growth and lumen stenosis following deep arterial injury. The co-ordinate spatial and temporal upregulation of {alpha}vβ3 expression following vessel wall injury, and the high affinity and specificity of XJ 735 for {alpha}vβ3, confirms the importance of this integrin in adhesive and migratory cell-matrix events underlying coronary restenosis.

KEYWORDS Alpha v beta 3; Porcine coronary; Stent; Adhesion; Migration; Restenosis; Remodeling; Integrins


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Integrins are cell surface heterodimeric glycoprotein receptors that integrate the cytoskeletal activities of a cell with that of its environment, via cell-to-cell and cell-to-extracellular matrix interactions [1]. Integrin-ligand interactions are involved in maintaining normal vascular structure, and in remodeling of the injured vessel wall associated with wound healing and angiogenesis [2]. Integrin {alpha}vβ3 is a promiscuous receptor that recognizes several extracellular matrix protein ligands including osteopontin, vitronectin, thrombospondin, and denatured collagen to which it binds via a specific ARG-GLY-ASP (RGD)-containing binding site [3, 4]. Prior studies have demonstrated a wide distribution of {alpha}vβ3 on endothelial cells (EC) [5], growth factor stimulated monocytes and T lymphocytes [6, 7], monocyte-derived macrophages [8], fibroblasts [9], vascular smooth muscle cells (VSMC) [4, 10–12]and platelets [13]. {alpha}vβ3 integrin-ligand interactions are known to mediate cell adhesion and migration [1], angiogenesis [14], osteoclast bone resorption [15], apoptosis [16], stimulation of extracellular matrix degrading proteinase expression [17], and tumor cell adhesion-invasion [17]. The {alpha}vβ3 integrin also participates in the migration of VSMC and EC towards osteopontin [12, 18], and the platelet derived growth factor (PDGF) stimulated migration of human VSMC over fibronectin, laminin, and collagens I and IV [19, 20]. We hypothesized that specific potent antagonism of {alpha}vβ3 mediated cell-matrix interactions in the vessel wall, would inhibit cell recruitment into the injury site, thereby limiting subsequent cell proliferation, migration and extracellular matrix protein synthesis. Recently, non-selective cyclic RGD peptide antagonists of the {alpha}vβ3 and {alpha}IIb/β3 integrins have been shown to limit neointimal hyperplasia in small animal models of restenosis, including the rat, rabbit, hamster and guinea-pig carotid angioplasty models [19, 21–24]. The ability of cyclic RGD peptides and specific neutralizing monoclonal {alpha}vβ3 antibodies to inhibit VSMC migration in vitro, suggests that this antirestenotic benefit derives from antagonism of {alpha}vβ3-mediated VSMC migration. However, platelet integrins ({alpha}IIb/β3) and several other SMC and vessel wall integrins (e.g {alpha}vβ1 and {alpha}vβ5), also depend on a functional RGD sequence [25, 26], suggesting that nonspecific RGD blockade may target a wide variety of integrins other than {alpha}vβ3 alone.

It is speculated but unproven that antagonism of {alpha}vβ3, {alpha}IIbβ3 or other integrins may have been responsible for the clinical benefit of the β3 neutralizing antibody c7E3 on repeat-target vessel revascularization in the EPIC trial [27]. We present evidence for the functional importance of {alpha}vβ3 by using a selective cyclic RGD peptidomimetic {alpha}vβ3 antagonist (Kd=40 nM), to potently limit neointimal formation and lumen stenosis following deep coronary arterial injury. Stent injury produces lesions which are histologically similar to human coronary restenosis [28, 29]. Stent deployment currently constitutes a large proportion of percutaneous revascularization world wide, since randomized trials demonstrated lower restenosis rates for stenting compared with balloon angioplasty alone [30, 31]. Stent restenosis represents a large and growing clinical problem with increased stent implantation [32]. As such, adjunctive pharmacological strategies that can effectively reduce neointimal growth and late lumen loss may have important relevance to all forms of percutaneous revascularization.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
XJ 735 a small molecule RGD peptidomimetic {alpha}vβ3 integrin antagonist was synthetized at The DuPont Merck Pharmaceutical Co. (Wilmington, DE). XJ 735 is a cyclic meta-amino benzoic acid derivative that contains both anionic and cationic binding sites that straddle the RGD binding domain of the {alpha}vβ3 integrin (Fig. 1). The chemical structure of XJ 735 is Cyclo[L-Alanyl-L-arginyl-glycyl-L-aspartyl)-3-aminomethyl benzoic acid].


Figure 1
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  		Fig. 1 Structure of XJ 735, a small molecule RGD peptidomimetic {alpha}vβ3 integrin antagonist. XJ 735 is a cyclic meta-amino benzoic acid derivative that contains both anionic and cationic binding sites that straddle the RGD binding domain of the {alpha}vβ3 integrin.

 
2.1 Purified {alpha}vβ3 receptor biotinylated vitronectin binding assay
The method is as previously described [33]. Briefly, purified receptor obtained from human placenta was diluted with coating buffer and coated on high capacity binding plates overnight at 4°C. Thirty microliters of biotinylated vitronectin plus 50 µL of either XJ 735 or buffer with 1.0% BSA were added to each well, and incubated for 25 min at room temperature. Plates were washed twice with buffer and incubated one hour at room temperature, with anti-biotin alkaline phosphatase. Finally, plates were washed twice with buffer followed by the addition of 100 µL of phosphatase substrate (1.5 mg/mL). The reaction was stopped by adding 2N NaOH and the developed color was read at 405 nm.

2.2 Endothelial cell-fibrinogen ({alpha}vβ3-mediated) adhesion assay
The method is as previously described [33]. In this cell based assay, the endothelial cell/fibrinogen-mediated adhesion is specifically blocked by a monoclonal antibody to {alpha}vβ3 but not by anti-{alpha}vβ5 or anti-β1 monoclonal antibodies. Briefly, a Costar 3590 plate was coated with 100 µL of fibrinogen (25 µg/well) overnight at 4°C. Resuspended human umbilical vein or porcine endothelial cells (~1x106 cells/mL, Clonetics Corp., San Diego, CA) in MCDB-131 standard media were used when confluent (passages 3–4). Cells were labeled with 2 µM Calcein-AM for 30 min at 37°C in a humidified incubator. Following calcein labeling, cells were washed twice with MCDB-131 medium, centrifuged at 1000 rpm and then resuspended; cell counts were adjusted to 1x106/mL. Cells were preincubated with 150 µL of XJ735 or medium for 15 min at room temperature, added to the assay plate and incubated for another 60 min at room temperature. Following washing, 100 µL of MCDB-131 medium was added to each well and fluorescence was read at 485 nm excitation, 530 nm emission.

2.3 Cell migration assays
Assays were performed using a 96 well chemotaxis chamber towards a vitronectin or osteopontin gradient. Cells were removed using EDTA/Trypsin (0.01%/0.025%). After removal, the cells were washed twice and resuspended (2x106/ml) in EBM (Endothelial cell basal media, Clonetics Inc.). 30 µl of either vitronectin or osteopontin (0.5–1.5 µg/well) was added to the lower wells of a disposable chemotaxis chamber. The cell suspension (45 µl) was added to a plate containing 5 µl of XJ735 at different concentrations and incubated for 5 minutes at 22°C. 25 µl of cell/XJ735 suspension was then added to the upper filter wells, and incubated for 22 h at 37°C. After overnight incubation, non migrated cells and excess media were removed. The filters were then washed twice in PBS (without Ca+2 or Mg+2) and fixed with 1% formaldehyde. Cell membranes were permeated using Triton X-100 and washed 2–3 times. Migrated cells were then stained with Rhodamine Phalloidin. Chemotaxis was quantitatively determined by fluorescence detection (530 nm excitation/590 nm emission).

2.4 Specificity studies
2.4.1 Antiplatelet ({alpha}IIb/β3) efficacy
Light Transmittance Aggregometry Assay: Venous blood was obtained from either healthy human donors or pigs maintained drug and aspirin-free for at least two weeks prior to collection as previously described [33]. Blood was collected in citrated tubes, centrifuged at 1000 rpm at room temperature, and platelet-rich plasma (PRP) removed. Samples were assayed on a PAP-4 Platelet Profiler, using platelet poor plasma as the blank (100% transmittance). Two hundred microliters of PRP (2–3x108 platelets/mL) were added to each micro test tube, and transmittance was set to 0%. Twenty microliters of the platelet agonist, ADP (10 µM final concentration) was added to each tube, and the aggregation profiles were plotted (% transmittance versus time). Twenty µL of XJ 735 were then added at different concentrations prior to ADP. Results were expressed as percent inhibition of agonist-induced platelet aggregation or IC50 (µM).

2.4.2 SKBR3 cell-vitronectin ({alpha}vβ5-mediated) adhesion assay
The method is as previously described [33]. In this cell based assay, the SKBR3/vitronectin-mediated adhesion is specifically blocked by a monoclonal antibody to {alpha}vβ5 but not by anti-{alpha}vβ3 or {alpha}4β1 monoclonal antibodies. The inhibitory effect of XJ 735 on SKBR3-vitronectin ({alpha}vβ5-mediated) adhesion was determined over a wide range of concentrations.

2.4.3 Jurkat-fibronectin ({alpha}4β1-mediated) adhesion assay
The method is as previously described [33]. In this cell based assay, jurkat/connecting segment 1 (CS-1)-mediated fibronectin adhesion is specifically blocked by a monoclonal antibody to {alpha}4β1 but not by anti-{alpha}v, anti-β3 or anti-{alpha}5 monoclonal antibodies. The inhibitory effects of XJ 735 on Jurkat-fibronectin ({alpha}4β1-mediated) adhesion was determined over a wide range of concentrations.

2.4.4 Purified {alpha}5β1 receptor-biotinylated fibronectin binding assay
The method is as previously described [33]. In this ELISA based assay, the purified {alpha}5β1/biotinylated fibronectin binding is specifically blocked by an anti-{alpha}5β1 monoclonal antibody but not by anti-{alpha}vβ3, {alpha}vβ5 or {alpha}4β1 monoclonal antibodies. The inhibitory effects of XJ 735 on fibronectin-{alpha}5β1-mediated binding was determined over a wide range of concentrations.

2.5 Experimental animal model
The porcine coronary restenosis model utilized has been extensively studied and validated [34–36]. This investigation conformed with "the guide for the care and use of laboratory animals" published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985. 25–30 kg juvenile normolipemic crossbred swine were fed a normal laboratory chow diet without lipid or cholesterol supplementation. Using an analgesia and anesthesia combination of ketamine (12 mg/kg) and xylazine (8 mg/kg) intramuscularly, animals underwent standard arterial sheath placement. A tunneled subcutaneous central venous Hickman catheter was placed in the the superior vena cava for intravenous drug delivery and blood removal. PTCA guide catheters were advanced under fluoroscopy and engaged in the left or right coronary artery ostia. Radio-opaque tantalum wire coronary stents were delivered in a standardized manner using 3.0–4.0 mm diameter angioplasty balloons inflated to exceed 20–30% of the vessel diameter at nominal balloon pressures, in each of the three main coronary arteries sequentially. All animals were given aspirin 650 mg (24 h pre-implant), a 10,000 U IV Heparin bolus (immediately after arterial sheath placement) and Verapamil SR 120mg (24 h pre-implant) to reduce peri-procedural mortality from spasm or acute thrombotic occlusion. Fluroscopy and selective contrast injection after stent implantation verified coil location, adequate coil expansion and vessel patency.

2.6 Drug delivery
XJ 735 a selective cyclic RGD peptidomimetic {alpha}vβ3 integrin antagonist (Kd=40 nM) was administered as a 1 mg/kg i.v. bolus followed by a 4 mg/kg/day intravenous infusion commencing 45 min prior to vascular injury. The continuous i.v. infusion at 4 mg/kg/day was maintained for 7 days, following which the drug was administered by intravenous 2 mg/kg i.v. bolus every 12 h for the next 21 days until sacrifice. Steady state plasma levels were measured in a separate group of 3 animals every 10 min up to 60 min following a 1 mg/kg iv bolus and then at 120 min, 180 min, 7 and 14 days following injury. Simultaneous samples were submitted for standard coagulation (PT, aPTT), liver function, creatinine, and platelet aggregation studies (ADP and collagen mediated platelet aggregation).

2.7 Pathology
Control and drug treated animals were sacrificed at 28±2 days. In a separate series of experiments 5 control animals were sacrificed at 24 hours, 7 days, 14 days and 28 days following both stent and angioplasty injury and arterial sections from these animals were used for immunohistochemistry and in situ hybridization studies as described below. Coronary arteries were perfusion fixed with 10% neutral buffered formalin and injured segments isolated together with uninvolved vessel at either end. Segments (roughly 2.5 cm in length) were sectioned transversely at 2 mm intervals and embedded in paraffin. 5 micron sections were cut and routinely stained with Hematoxylin-Eosin, Elastic van Gieson, and immunohistochemical stains to identify {alpha}-smooth muscle cell actin, macrophages, endothelial cells, and the {alpha}vβ3 integrin.

2.8 Immunohistochemistry
The following antibodies were used: (1) Purified mouse IgG1 monoclonal anti-human {alpha}vβ3 integrin (cross reactive to pig) clone LM609 (Chemicon International, Temecula, CA); (2) Mouse monoclonal IgG antibody (SWC3) against porcine monocyte-macrophage differentiation molecule (VMRC, Pullman, WA). (3) Mouse IgG2a monoclonal anti-{alpha}-smooth muscle cell actin (DAKO, Carpenteria, CA). (4) Rabbit polyclonal IgG anti-human osteopontin (LF-7) crossreactive with porcine tissue (courtesy of Dr. Larry Fisher, NIDR, NIH). Tissues were deparaffinized in xylene and rehydrated. Single label immunohistochemistry was performed using standard ABC-peroxidase methodology according to manufacturer's recommendations (Vector Laborotories, Burlingame, CA) with the following modifications. Sections were blocked using Tris buffered saline (TBS) containing 0.3% casein and 10% normal goat serum. All steps were carried out at room temperature followed by a 30 min wash with TBS containing 1% normal goat serum. Primary antibodies were applied at the indicated dilutions (determined by appropriate serial dilutions) and incubated either for one hour at room temperature or overnight at 400°C. Secondary antibody (biotinylated anti-primary antibody species) was applied in 1:400 dilution. Endogenous peroxidase was inhibited with 0.3% H2O2/methanol. Peroxidase conjugated avidin–biotin complex (ABC) was allowed to react with the secondary antibody for 30 min and visualized with 0.01% diamino-benzidine or 3-amino-9-ethylcarbazole chromagen and 0.01% H2O2 in Tris, pH 7.2 for 4–6 min. Normal human bone sections were used as positive control tissue for the {alpha}vβ3 immunohistochemistry. Negative control sections were obtained by eliminating the primary antibody, and by using isotype specific nonimmune mouse serum at the same concentrations as the primary antibody. No specific staining was observed in the negative controls confirming the specificity of the primary antibody staining reactions. Serial sections cut 2mm apart through the injury site were stained for {alpha}vβ3 integrin. At each injury site, the section displaying the maximum immunostaining in the 24 h, 7 day, 14 day, 21 day, control animals was semiquantified on a grading system from 0 to 3 in each of the neointima (NI), media (MED), and adventitia (ADV): grade 0=no immunostaining detectable; grade 1=positive immunolabeling of <25% of cells; grade 2=positive immunolabeling of 25–50% of cells; grade 3=positive immunolabeling of >50% of cells in either NI, MED, or ADV.

2.9 In situ hybridization
Target osteopontin mRNA sequence was selected from the known DNA sequence encoding this protein obtained via the GENBANK database. Synthetic DNA probes were constructed and purified using oligonucleotide purification cartridges (Applied Biosystems, Foster City, CA). Antisense osteopontin cDNA probe was synthesized as a 40 base pair synthetic oligomer complementary to the osteopontin mRNA sequence starting at position 1173: 5' GAA GCT TTT AGT TTA CAG GGA GTT TCC ATG AAG CCA CAA A 3'. A complementary sense probe to osteoponin was utilized as a control. 35S-[AMP]n labeled single stranded cDNA probe for messenger RNA was prepared using terminal deoxynucleotidyl transferase (TdT) and [{alpha}-35S] dATP provided as a NEP100 labeling kit (Dupont, Boston, MA). 35S-labeled probes were utilized at a specific activity of 1x106 cpm/µl. Tissue sections were deparaffinized in xylene, cleared in descending alcohols, and rehydrated. Permeabilization with 0.05% Triton X-100 was followed by deproteinization with 0.2N HCl and proteinase K digestion (10 mg/ml in 50 mM EDTA, 0.1 M Tris pH 8.0 for 30 min at 37°C). Acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine was used to reduce non-specific probe binding, followed by washing in 2xSSC (saline sodium citrate composed of 0.3M NaCl, 0.3M trisodium citrate, pH 7.0) and dehydration in ascending alcohols. Hybridization solution (50% deionized formamide, 10 mM DTT, 1 mM EDTA, 10 mM Tris-HCl pH 8.0, 0.3M NaCl, 1 mg/ml Herring sperm DNA, 10 mg/ml yeast tRNA, 2% Denhardt's solution, 10% dextran sulphate) containing approximately 5x104 cpm of 35S labeled probe was applied to pretreated sections and hybridized overnight twice, covered by acetylated coverslips at varying optimized hybridization temperatures based on the percentage of G+C content and formamide concentrations. Following hybridization, coverslips were removed and sections were desalted using decreasing concentrations of SSC (4x to 0.5x) containing 1 mM DTT as antioxidant. Slides were then uniformly coated with Kodak NTB-2 emulsion, dried and exposed at 4°C. Development in Kodak D-19 developer was followed by fixation and counterstaining (equal parts Toluidine blue 1:50 and Giemsa 1:50). Controls for in situ hybridization included: (i) sense DNA oligomers derived from the target mRNA sequence; (ii) pretreatment of tissue sections with RNAase prior to hybridization; (iii) positive pig bone sections processed in a manner identical to coronary artery sections; (iv) normal uninjured porcine coronary arteries. The total number of cells expressing in situ hybridization signal for osteopontin was counted for each arterial section. Contiguous sections across the injury site were analyzed. The total number of cells positive for osteopontin signal in each injury site (hybrid score) was determined for adventitia (ADV), media (MED), and neointima (NI) locations. A mean osteopontin signal was obtained for each vessel wall location by averaging across all lesions within each time point. Analysis of variance was used to compare mean osteopontin mRNA signal scores for each vessel wall location across and within time points. Where significant differences were observed using ANOVA, post hoc analysis was performed using two-tailed unpaired t-tests with Bonferroni/Dunn correction for comparison of groups.

2.10 Histology
Measurements were performed blinded to treatment strategy. Quantitative measurement of neointima area, lumen area, and arterial size was assessed at both injury and immediate proximal uninjured reference sites. Calibrated digital planimetry was used to obtain all morphometric measurements. The arterial injury at each wire site was scored ordinally as validated previously [35, 36]. An ordinal injury score from 0 to 3 was used where: 0=no injury noted; 1=internal elastic lamina violated and media compressed but not lacerated; 2=internal elastic lamina and media lacerated but external elastic lamina intact; 3=internal elastic lamina, media and external elastic lamina all violated. A mean injury score (INJ) was established for each injury site, by averaging the injury scores of all wire sites. The following measurements were directly measured: (1) Neointima area (NI, mm2); (2) Residual lumen area at injury site (LAL, mm2); (3) Lumen area at immediate proximal uninjured reference site (LAR, mm2); (4) Internal elastic lamina area at injury site (IELinj, mm2); (5) Internal elastic lamina area at immediate proximal uninjured reference site (IELref, mm2); (6) External elastic lamina area at injury site (EELinj, mm2); (7) External elastic lamina area at immediate proximal uninjured reference site (EELref, mm2).

The following were calculated using measured parameters:

1. Change in lumen area between injury and proximal reference sites=LAR-LAL.
2. Geometric arterial remodeling (dEELA) or change in external elastic lamina area between injured and proximal uninjured reference sites=EELinj-EEL ref.

2.11 Statistical analysis
The sample size reflects the number of data points required for detection of differences in slope, intercepts, or both using the linear regression models described below. The minimum number of 14 data points per sample (2 data points per animal) allowed detection of a 26.9% change in the intercept at 80% power, and a 25.0% change in slope also at 80% power. These numbers are consistent with treatments that would give angiographically observable differences and thus likely to be of clinical benefit. Linear regression analysis compared control, and XJ 735 treated groups for the following:

1. Neointima area vs. injury score.
2. Neointima area vs. change in arterial size (dEELA).
3. Percent lumen stenosis (LAR-LAL/LAR) vs. Neointima area (NI).
4. Percent lumen stenosis (LAR-LAL/LAR) vs. change in arterial size (dEELA) between injury and proximal reference sites.
Two linear models were used to relate the dependent variable (e.g. mean neointima area) to the independent variable (e.g. injury score) in order to quantitatively compare the response to treatment of slope and intercept. A binary drug interaction variable (denoted 0 or 1) was added to the regression equations to evaluate the significance of XJ 735 treatment as compared to control. Differences in response to treatment are reflected by significant differences in slopes or intercepts for the two groups. The two linear regression models utilized test for differences in: (1) Intercept assuming equal slopes (Model I) and (2) Slope assuming equal intercepts (Model II).


Formula



Formula

µ and {alpha}1 are coefficients estimated by multiple regression. Statistical significance was established by evaluating the {alpha}1 for each model. A statistically significant {alpha}1 coefficient in each model can be interpreted as a significant difference between treated and control groups for either intercept or slope. Remodeling effects were assessed by comparing dEELA vs. NI for each group and by comparing mean dEELA between groups. All data are presented as mean±s.e.m. Comparison of mean NI area, INJ score, change in lumen area (LAR-LAL), percent lumen stenosis, and change in arterial size (dEELA) between XJ 735 and control treatment groups utilized unpaired two-tailed t-tests with significance accepted at p≤0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Immunohistochemistry
In the uninjured porcine coronary artery, minimal baseline {alpha}vβ3 immunoreactivity was observed along the luminal endothelium only (Fig. 2A). The expression of {alpha}vβ3 immunoreactivity along the luminal endothelium was similar in all uninjured coronary sections. Strong cellular {alpha}vβ3 immunoreactivity was observed along the resorption lines of porcine bone controls (data not shown). The marked temporal–spatial variation in {alpha}vβ3 immunoreactivity observed in injured coronary arteries is illustrated in Fig. 2A–2I and summarized in Table 1. At 24 h following injury, diffuse {alpha}vβ3 immunoreactivity was observed colocalized with the platelet thrombus accumulated at the arterial injury site (Fig. 2B). At 7 days following injury, strong {alpha}vβ3 immunoreactivity was observed within the neointima and regenerating endothelium (Fig. 2C). Strong immunoreactivity comparable to the neointima was observed in the adventitia at 7 days, with moderate {alpha}vβ3 immunoreactivity within the injured media (Fig. 2D). At 14 days following injury, peak immunoreactivity for {alpha}vβ3 was observed within the {alpha}-actin positive cells of the injured media and neointima, and the overlying regenerated endothelium (Figs. 2E, 2F). Strong {alpha}vβ3 immunoreactivity also localized with {alpha}-SMC actin and vimentin positive myofibroblast-like cells within the adjacent adventitia at 14 days (Figs. 2G, 2H). At 21 days following injury, {alpha}vβ3 immunoreactivity declined to moderate intensity within the media, neointima, and luminal endothelium. Adventitial immunoreactivity was confined to zones of ongoing cellular inflammation (macrophages), myofibroblasts, and the vasa vasora, where strong immunostaining of the endothelium and smooth muscle cells was observed (Fig. 2I). By 28 days following injury, {alpha}vβ3 immunoreactivity was quiescent in the media and luminal endothelium, with residual {alpha}vβ3 immunoreactivity around cellular infiltrates in the adventitia and neointima (data not shown).


Figure 2
Figure 2
Figure 2
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  		Fig. 2 233(A) High power photomicrograph showing baseline {alpha}vβ3 immunoreactivity (arrows) along the luminal endothelium of an uninjured porcine coronary artery. Arrows indicate positive staining of the luminal endothelium. M=media. Original magnification x200. Section was immunostained with a monoclonal antibody to integrin {alpha}vβ3. (B) Photomicrograph of porcine coronary artery within 24 h of angioplasty, showing diffuse {alpha}vβ3 immunoreactivity within platelet thrombus (P) accumulated at the injury site. Media (M) and adventitia (A) show baseline {alpha}vβ3 immunoreactivity using a monoclonal antibody to integrin {alpha}vβ3 (LM609). Arrows indicate lacerated ends of media. Original magnification x50. (C) High power photomicrograph showing strong {alpha}vβ3 immunoreactivity within the forming neointima (N) and regenerating endothelium (arrows) of a porcine coronary artery at 7 days following injury. Section was immunostained with a monoclonal antibody to integrin {alpha}vβ3 (LM609). Original magnification x250. (D) Low power view of artery illustrated in Fig. 2C, showing strong {alpha}vβ3 immunoreactivity around neointimal (N) and adventitial (A) cells with moderate diffuse immunoreactivity of the media (M) at 7 days following injury. Section was immunostained with a monoclonal antibody to integrin {alpha}vβ3 (LM609). Original magnification x100. (E) Negative control section stained with non-immune mouse IgG serum. Arterial section is taken 14 days following injury. Inset shows low power view of the same arterial section stained with a monoclonal antibody to {alpha}-SMC actin demonstrating {alpha}-smooth muscle cell actin positive cells within the media (m) and neointima (n). (a)=adventitia Original magnification x24.5. (F) Photomicrograph of arterial section shown in Fig. 2E taken 14 days following injury and immunostained with a monoclonal antibody to integrin {alpha}vβ3 (LM609). Diffuse prominent {alpha}vβ3 immunoreactivity is observed corresponding to locations containing {alpha}-SMC actin positive cells of the media (m) and neointima (n) seen in Fig. 2E. High power inset (x100 original magnification) shows strong {alpha}vβ3 immunoreactivity within the neointima (N) and endothelium (arrow). Section was immunostained with a monoclonal antibody to integrin {alpha}vβ3 (LM609). Original magnification x24.5. (G) Photomicrograph of arterial section shown in Figs. 2E, 2F taken 14 days following injury and immunostained with a monoclonal antibody to vimentin, a marker for both fibroblasts and smooth muscle cells. Prominent immunostaining is observed at the injury site (I) of cells within the adventitia (A), neointima (N), and to a lesser extent the uninjured media (M). Original magnification x24.5. (H) High power photomicrograph of arterial section shown in Fig. 2E inset taken 14 days following injury and immunostained with a monoclonal antibody to {alpha}-smooth muscle cell actin (original magnification x90). Prominent immunoreactivity for {alpha}-smooth muscle cell actin is observed within cells of not only the neointima (N) and injured media (M), but also within myofibroblast cells of the adjacent adventitia (A). These adventitial cells are shown in the inset at high power magnification (original x250) displaying strong immunoreactivity for {alpha}vβ3 using a monoclonal antibody to integrin {alpha}vβ3 (LM609). (I) Low and high power photomicrographs of porcine coronary artery taken at 21 days following injury, showing moderately strong {alpha}vβ3 immunoreactivity within the neointima (N), media (M), and luminal endothelium (straight arrows). Adventitial immunoreactivity (A) is confined to zones of ongoing cellular inflammation and remains strong around the vasa vasora (curved arrow). Section was immunostained with a monoclonal antibody to integrin {alpha}vβ3 (LM609). Original magnification x32; inset magnification x75.

 

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  		Table 1 Time course and location of {alpha}vβ3 expression following vessel wall injury

 
3.2 In situ hybridization
In the first 24 h following injury in situ hybridization signal for osteopontin was detectable overlying a few trapped mononuclear cells within the platelet thrombus overlying the injury site (Fig. 3A). At 4 days following injury, osteopontin mRNA signal was detectable overlying scattered cellular infiltrates within the organizing neointima and adventitia overlying the stent wire injury site (Fig. 3B). At 7 days following injury, with increased cellular inflammation of the adventitia, increased osteopontin mRNA signal was detectable in the adventitia (Fig. 3C). Less intense signal was detectable over the lacerated media and developing neointima. Medial osteopontin signal peaked at 7 days following injury declining without further change between 14 and 28 days (ANOVA p = 0.009; 7 d vs. 14 d vs. 28 d, Fig. 4). At 14 days following injury, osteopontin signal increased compared with earlier time points and was greatest in the adventitia and developing neointima (Fig. 3D). Osteopontin signal scores were equivalent for neointima and adventitia, and significantly greater than the signal detected in the media (ANOVA p = 0.01, NI vs. MED vs. ADV, Fig. 4). Osteopontin mRNA signal in the neointima increased between 7 and 14 days (ANOVA p = 0.004; 7 d vs. 14 d vs. 28 d), to peak at 14 days following injury, with no change between 14 and 28 days. At 28 days following injury, maximal osteopontin mRNA signal was detected overlying dense cellular infiltrates in the adventitia and around the sites of stent wire implantation (Figs. 3E, 3F, 3G). This signal localized with monocyte-macrophage cell infiltrates and to a lesser extent with a-actin positive cells in these zones (Fig. 3F inset, 3H). Similar results were obtained for either angioplasty or stent-induced injury, and hybridization experiments utilizing corresponding sense controls were negative (Fig. 3E). Adventitial osteopontin signal scores were significantly higher than either media or neointimal signal scores (ANOVA p<0.0001, NI vs. MED vs. ADV, Fig. 4). Highest scores for adventitial osteopontin signal were observed at 28 days (ANOVA p = <0.0001; 7 d vs. 14 d vs. 28 d). Within the adventitia, osteopontin signal was observed overlying cellular infiltrates (smooth muscle cells/myofibroblasts and mononuclear cells) surrounding the vasa vasora (Fig. 3G inset, 3H). Sections demonstrating positive osteopontin mRNA signal underwent immunostaining with LF-7 anti-osteopontin antibody, to confirm osteopontin protein expression in these zones (Fig. 3H).


Figure 3
Figure 3
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  		Fig. 3 (A) In situ hybridization signal for osteopontin overlyimg mononuclear cells trapped within platelet thrombus (P) formed at the injury site (I) within 24 h following injury. Hybridization signal is seen as black signal against a blue (Toluidine blue/Giemsa) counterstained tissue background. (M)=media. Original magnification x24.5. (B) 4 days following injury moderate osteopontin mRNA signal is observed within the organizing neointima (N) and adventitia (A) overlying the stent wire injury sites (asterisk). Minimal osteopontin mRNA signal is observed overlying the media (M). Original magnification x50. (C) 7 days following injury, strong osteopontin mRNA signal is observed overlying cellular infiltrates within the adventitia (A) over the stent wire injury site (asterisk). Less intense osteopontin signal is detectable overlying the media (M) and developing neointima (N). Original magnification x50. (D) 14 days following injury, osteopontin signal has increased compared with earlier time points, and is observed to be greatest overlying cells within the neointima (N) and adventitia (A). Original magnification x32. High power inset (original magnification x75) shows the dense osteopontin signal overlying neointimal and adventitial cellular infilltrates, with minimal signal overlying the lacerated ends of the media (M) at the stent wire injury site (asterisk). (E) 28 days following angioplasty injury, maximal osteopontin mRNA signal is observed overlying dense cellular infiltrates in the adventitia (A). Less intense signal was also observed overlying the mature neointima (N). Minimal signal is present within the media (M). Original magnification x48.5. Inset (magnification x48.5) shows contiguous negative control section hybridized with sense probe, to show absence of specific hybridization signal. (F) 28 days following stent injury, maximal osteopontin mRNA signal is evident overlying dense cellular infiltrates in the adventitia (A) and around the sites of stent wire implantation (asterisk). Less intense signal is observed overlying the mature neointima (N), and the media (M) exhibits minimal in situ hybridization signal. Areas demonstrating strong osteopontin mRNA expression around the stent wire sites correspond to zones infiltrated with mononuclear cells, identified by immunostaining as porcine monocyte- macrophages (inset). High power inset (original magnification x75) shows immunostaining of monocyte-macrophage cells around the stent wire implantation site (asterisk). L=Lumen. (G) Demonstration of strong osteopontin mRNA signal expression around stent wire implantation sites (asterisk) and adjacent adventitia (A) 28 days following injury (original magnification x75). Inset shows high power view (original magnification x125) of the adventitia with extensive osteopontin signal overlying cellular infiltrates around the vasa vasora (vv). (H) High power views of stent wire injury sites (asterisk) expressing osteopontin mRNA signal shown in Figs. 3F, 3G, illustrating corresponding strong osteopontin protein expression identified with antibody LF-7 (left panel) to osteopontin (OP). {alpha}-smooth muscle cell actin positive cells are also identified around the stent wire sites (arrows, right panel). Original magnification x75 for osteopontin staining and x100 for actin staining.

 

Figure 4
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  		Fig. 4 Mean in situ hybridization scores for osteopontin mRNA signal in neointima, media, and adventitia at 7 days, 14 days and 28 days following injury. Osteopontin mRNA signal within the neointima increased between 7 d and 14 d to peak at 14 d following injury and no further change between 14 d and 28 d (ANOVA p = 0.004; 7 d vs. 14 d. vs. 28 d). Medial osteopontin signal peaked at 7 days following injury, declining without further change between days 14 and 28 (ANOVA p = 0.009 7 d vs. 14 d vs. 28 d). Adventitial osteopontin signal exceeded that found in either media or adventitia, with highest adventitial signal observed at 28 days post injury (ANOVA p≤0.0001; 7 d vs. 14 d vs. 28 d).

 
3.2.1 Plasma XJ 735 levels
Plasma XJ 735 levels rose rapidly following bolus administration to a mean of ~12.5 µM and declined to basal levels (<2.5 mM) within the next hour (Fig. 5). Following commencement of the infusion steady state plasma levels of 7.8±3.1 µM were obtained at 180 min, and remained ≥5 µM between 7 and 14 days. Steady state plasma XJ 735 levels were approximately ten-fold and two-fold higher respectively than the IC50 for XJ 735 inhibition of human and porcine VSMC migration towards osteopontin or vitronectin. No platelet aggregation, hemostatic, or biochemical disorders were observed in XJ 735-treated animals.


Figure 5
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  		Fig. 5 Mean plasma XJ 735 plasma levels in three animals with sampling every 12 min up to 60 min following a 1 mg/kg intravenous bolus, and then at 30 min and 60 min following commencement of a 1 mg/kg/6 h intravenous infusion of XJ 735. Steady state levels achieved were in excess of 5 µM.

 
3.2.2 Integrin specificity of XJ 735
The IC50 values for XJ 735 inhibition of {alpha}vβ5, {alpha}IIbβIIIa, {alpha}4β1, and {alpha}5β1 are summarized in Table 2. XJ 735 IC50 for inhibition of porcine {alpha}IIbβIIIa-mediated platelet aggregation was 500 µM. Similarly, XJ 735 IC50 for inhibition of porcine/human {alpha}5β1-mediated fibronectin adhesion, porcine/human {alpha}4β1-mediated fibronectin adhesion, and porcine/human {alpha}vβ5- mediated vitronectin adhesion all exceeded 100 µM. XJ 735 IC50 for inhibition of human {alpha}IIbβIIIa mediated platelet aggregation was 20 µM. In contrast, XJ 735 IC50 for inhibition of purified human {alpha}vβ3 receptor vitronectin binding was 40 nM (Table 3).


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  		Table 2 Integrin specificity of XJ 735

 

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  		Table 3 Anti-integrin {alpha}vβ3 affinity and efficacy of XJ 735

 
3.2.3 Anti-integrin {alpha}vβ3 specificity and efficacy of XJ 735
The IC50 values for XJ 735 inhibition of human and porcine endothelial cell {alpha}vβ3-mediated fibrinogen adhesion were 4.4 µM and 0.6 µM, respectively (Fig. 6, left panel, Table 3). The IC50 for XJ 735 inhibition of avb3 mediated human and porcine vascular SMC migration towards osteopontin and vitronectin ranged from 0.84 to 4.0 µM (Fig. 6, right panel, Table 3). For comparison similar inhibition experiments were conducted using the chimeric antibody c7E3. The IC50 for c7E3 inhibition of human vascular SMC migration towards vitronectin or osteopontin ranged between 0.35–0.42 µM.


Figure 6
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  		Fig. 6 (Left panel) Porcine endothelial cell-fibrinogen ({alpha}vβ3-mediated) adhesion assay. Dose response curve for XJ735 inhibition of porcine endothelial cell adhesion to fibrinogen. IC50 for inhibition of endothelial adhesion is 0.6 µM, which compares with demonstrated in vivo steady state levels of >5 µM in XJ 735 treated animals (Fig. 5). Wells were precoated with fibrinogen, and the binding of calcein labeled endothelial cells preincubated with increasing concentrations of XJ 735, assessed by assaying fluorescence after 60 min. Data are % maximal inhibiton of adhesion. (Right panel) Porcine vascular smooth muscle cell/vitronectin directed ({alpha}vβ3-mediated) migration assay. Dose response curve for XJ 735 inhibition of porcine vascular smooth muscle cell migration toward osteopontin. IC50 for inhibition of VSMC migration is 1.9 µM, which compares with demonstrated in vivo steady state levels of >5 µM in XJ 735 treated animals (Fig. 5). Cell suspensions containing varying concentrations of XJ 735 were plated in the upper wells of a chemotaxis chamber and tested for chemotaxis toward osteopontin in the lower chamber. After overnight incubation, cells migrated to the bottom of the filter were quantitated using fluorescence detection. Data are % maximal inhibiton of migration.

 
3.2.4 In vivo anti-restenotic efficacy of XJ 735
Thirty arterial lesions from untreated controls and twenty-eight arterial lesions from XJ 735 treated animals were available for histologic analysis.

3.3 Mean comparisons
Mean injury score (INJ) was significantly (17%) higher in the XJ 735-treated animals (2.30±0.05) as compared with untreated controls (1.93±0.12, p = 0.01, Fig. 7). Mean injury site neointimal area (NI) was significantly reduced (43%) in the XJ 735 treated animals (1.26±0.14 mm2) as compared with untreated controls (2.28±0.25 mm2, p = 0.0009, Fig. 7). Mean injury site neointima+media area (NI) was also significantly reduced in the XJ 735-treated animals (2.06±0.25 mm2) as compared with untreated controls (2.95±0.33 mm2, p = 0.04). No effects of XJ 735 were observed on the mean change in arterial size between the injured and uninjured proximal reference sites (dEELA), which was used as an index of geometric arterial remodeling. dEELA was similar for XJ 735 (2.44±0.30 mm2) and untreated controls (2.71±0.31 mm2, p=NS, Fig. 7). Mean percent lumen stenosis at the injury site was significantly reduced (~2.9 fold) in XJ 735 treated animals (–13.7±6.2%) as compared with untreated controls (7.3±7.9%, p = 0.04, Fig. 7). Mean change in lumen area between injured and reference sites was reduced (~2.5 fold) in the XJ 735-treated animals (increase in lumen area of 0.38±0.21 mm2) as compared with untreated controls (reduction in lumen area of 0.25±0.19 mm2, p = 0.03).


Figure 7
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  		Fig. 7 Comparison of mean neointimal area (left upper), injury score (right upper panel), change in arterial size (left lower panel), and percent injury site lumen stenosis (right lower panel). A 43% reduction in mean neointimal area (p = 0.0009) despite slightly higher (17% greater) injury score (p = 0.01), was observed in the XJ 735 treated group as compared with controls. There was no difference in the change in arterial size between injury and reference sites, comparing XJ 735 treated and control groups. Mean lumen stenosis of the injury site compared with the proximal reference site was reduced ~2.9 fold (p = 0.04) in the XJ 735 group as compared with controls. All data displayed are mean±s.e.m. The antirestenotic effect of XJ 735 is seen to be derived from inhibition of neointimal growth and not by promoting an overall increase in arterial size (geometric remodeling).

 
3.4 Regression comparisons
Regression data for the NI vs. INJ score relationship are shown in Fig. 8. Significant NI vs. INJ score correlations were obtained for both control (R = 0.72, p<0.0001) and XJ 735-treated animals (R = 0.4, p = 0.04). Linear regression Model II comparing slopes assuming equal intercepts showed a significant 45% reduction in slope of the NI vs. INJ score relationship by XJ 735 (p = 0.0001, Fig. 8). Linear regression Model I comparing intercepts assuming equal slopes showed a significant 76% reduction in intercept of the NI vs. INJ score relationship by XJ 735 (p = 0.0001, Fig. 8). These findings indicate a significant attenuation in neointimal growth with incremental arterial injury, over a wide range of injury scores. Regression data for the NI vs. dEELA relationship are shown in Fig. 8. Significant NI vs. dEELA correlations were observed for both control (R = 0.73, p<0.0001) and XJ 735 treated animals (R = 0.5, p = 0.007) confirming a linear increase in arterial size with increasing neointimal growth (geometric arterial remodeling) in both treatment groups. No significant change in either the slope or intercept of the NI vs. dEELA relationship was observed comparing XJ 735 and control groups (Fig. 8). Thus, the primary anti-restenotic benefit of XJ 735 treatment is seen to be a reduction in NI growth rather than an alteration in geometric arterial remodeling.


Figure 8
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  		Fig. 8 Regression data comparing neointimal area vs. injury score relationship (left panel) and change in arterial size between inury and reference sites ({Delta} EELA) vs. neointima area (right panel). Significant reductions in both slope and intercept of the neointima area vs. injury score relationship (p = 0.0001) are demonstrated for the XJ 735 group compared with controls. No significant change in either slope or intercept of the {Delta} EELA vs. neointima area relationship was observed comparing treated and control groups.

 
Fig. 9 examines the relationship of NI and dEELA to percent lumen stenosis at the injury site for both XJ 735 and control groups. Fig. 9 illustrates the strong correlation between neointimal area and percent lumen stenosis (%LS) for the control group (R = 0.62, p = 0.003). No relationship between NI and %LS is evident for XJ 735-treated animals. In contrast a strong negative correlation is observed between dEELA and %LS in XJ 735-treated animals (R = 0.52, p = 0.005), whereas no such relationship is evident for control animals (Fig. 9). These results illustrate the dependence of %LS on NI growth in control animals, as compared with the XJ 735-treated group where NI is significantly attenuated such that the final %LS is now determined predominantly by changes in arterial size rather than neointimal growth.


Figure 9
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  		Fig. 9 Relationship between percent lumen stenosis at the injury site and neointima area (NI) is shown in the left panel. Relationship between percent lumen stenosis at the injury site and {Delta} EELA (the change in arterial size between injury and reference sites), is shown in the right panel. A strong correlation is observed between neointimal growth and lumen stenosis for control animals, whereas neointimal growth is not significantly correlated with injury site percent lumen stenosis in XJ 735 treated animals (left panel). In contrast, NI is attenuated significantly in the XJ 735 treated group, such that percent lumen stenosis is now inversely correlated with {Delta} EELA, a parameter of geometric arterial remodeling (right panel). {Delta} EELA does not correlate significantly with percent lumen stenosis for the control group, as this remains predominantly determined by neointimal growth.

 
3.5 Histology
Representative Elastic van Gieson stained coronary arterial sections retrieved at 28 days post injury from equivalently injured control and XJ735-treated animals are shown in Fig. 10A. A marked reduction in neointimal growth is observed with XJ735 treatment. The neointima was histologically identical in both groups. However, arteries from XJ735-treated animals displayed a marked reduction in the density of cellular infiltrates and neovascular channels formed within the adventitia surrounding the stent wire implantation sites, as compared with untreated controls (Figs. 10B, 10C).


Figure 10
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  		Fig. 10 (A) Representative photomicrographs of Elastic van Gieson stained coronary arterial sections taken from control (left panel) and XJ 735 treated animals (right panel) at 28 days following equivalent stent injury. Mean injury scores for the coronary sections taken from control and XJ 735 groups were 1.8 and 2 respectively. A marked reduction in neointimal hyperplasia is evident in the XJ 735 treated artery. Original magnification x24.5. (B) High power photomicrographs of the stent wire injury site (asterisk) and cellular infiltrates (arrows) within the adjacent adventitia (A), in Elastic van Gieson stained coronary arterial sections taken from control (right panel) and XJ 735 treated animals (left panel). A marked reduction in both neointima (N) and the adventitial cellular infiltrates (A) surrounding the stent wire implantation site (asterisk) is observed for XJ 735 treated animals at 28 days following injury. M=media, Original magnification x75. (C) Corresponding high power photomicrographs of the stent wire injury site (asterisk) and adjacent adventitia (ADV) in Elastic van Gieson stained coronary arterial sections taken from control (left panel) and XJ 735 treated animals (right panel). The marked adventitial neovascularization observed surrounding the stent wire injury site in the control section is markedly attenuated in the XJ 735 treated section studied at 28 days following stent injury. High power view of adventitial neovascular channels is illustrated in the inset. M=media, NC=neovascular channel, NI=neointima. Original magnification x75 left panel, x100 inset, x50 right panel.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
{alpha}vβ3-ligand interactions mediate cell-matrix adhesion, migration, and differentiation events that underlie neointimal growth [22], angiogenesis [14], apoptosis [10, 37], and tissue remodeling [4], which are fundamental to the reparative response to arterial injury. The present findings indicate that selective potent antagonism of integrin {alpha}vβ3 using a small molecule cyclic RGD peptidomimetic can limit neointimal hyperplasia and lumen stenosis at sites of deep coronary arterial injury. These results confirm the hypothesis that {alpha}vβ3 mediated cell-matrix interactions play a key modulatory role in coronary restenosis. The widespread cellular distribution of {alpha}vβ3 within the arterial wall, and the spatial–temporal co-induction of {alpha}vβ3 and osteopontin in the vessel wall following injury, support a physiologic role for {alpha}vβ3-ligand interactions in restenosis. Following arterial injury, there is early upregulation of integrin {alpha}vβ3 at sites of cell accumulation within the neointima and adventitia at 7 days, followed by persistent high levels of {alpha}vβ3 expression within the media and neointima up to 21 days, decreasing towards baseline by 28 days (Table 1). The extensive osteopontin mRNA expression in the neointima and adventitia between 14 and 28 days (Figs. 3D, 3E, 4) and the continued {alpha}vβ3 expression in the neointima and adventitia up to 21 days (Figs. 2H, 2I, Table 1), suggests that sustained pharmacologic blockade of {alpha}vβ3 beyond 14 days post injury, may be required to achieve maximal anti-restenotic efficacy.

We hypothesize that the coordinate spatial and temporal induction of osteopontin and {alpha}vβ3 expression observed in this study may serve to recruit EC, VSMC, monocyte-macrophage, and myofibroblast cells into the neointima. Integrin {alpha}vβ3 mediates the osteopontin-directed migration of VSMC and EC [11, 18, 38]; and the adhesion of VSMC, EC and fibroblasts to osteopontin [12]. {alpha}vβ3 supports the RGD-dependent binding of extracellular matrix ligands present at the injury site including fibronectin, fibrinogen, osteopontin, vitronectin, denatured collagen, von Willebrand factor and thrombospondin [25, 26]. Morever, platelet derived growth factor (PDGF) and transforming growth factor-β (TGF-β) which are enriched in restenotic neointima, potently induce VSMC migration by inducing β3 expression in these cells [20].

Non-specific cyclic RGD peptides capable of blocking RGD- recognizing integrins such as {alpha}vβ3 and {alpha}IIbβIIIa, can inhibit VSMC migration in vitro and decrease neointimal formation in rat, rabbit, hamster and guinea-pig carotid injury models [19, 21–24]. The antirestenotic benefit of peptides such as PenRGD (which targets all {alpha}v-containing integrins and {alpha}IIbβIIIa) and G4120 (which inhibits both {alpha}vβ3 and {alpha}IIbβIIIa) implicates at least platelet and smooth muscle cell integrin events in restenosis [19, 22]. The EPIC trial utilized a chimeric monoclonal antibody Fab fragment (c7E3), directed against β3-containing integrins, and demonstrated a marked reduction in repeat target vessel revascularization at 6 months [27, 39]. c7E3 potently inhibits the platelet {alpha}IIbβIIIa integrin but avidly cross reacts with {alpha}vβ3 and the leucocyte integrin Mac-1 [40]. Since specific short acting platelet {alpha}IIbβIIIa integrin anatgonists such as Integrelin have not demonstrated similar anti-restenotic efficacy [41], it remains uncertain whether {alpha}vβ3, {alpha}IIbβIIIa, or other integrins such as Mac-1, underlie this benefit of c7E3. The high affinity and specificity of XJ 735 for {alpha}vβ3 clarifies the functional importance of this integrin in coronary restenosis.

While {alpha}IIbβIIIa remains the most abundant platelet surface protein (~50,000 heterodimers/platelet), normal platelets also express 100–500 {alpha}vβ3 receptors/platelet which may mediate platelet adhesion to exposed extracellular matrix ligands at the injury site [13]. Antagonism of {alpha}vβ3-mediated platelet adhesion at the injury site may retard neointimal growth. Further, Reverter et al. have shown that chimeric c7E3 Fab can inhibit thrombin generation initiated by tissue factor in the presence of platelets, through both {alpha}IIbβIIIa and {alpha}vβ3 blockade [42]. Thus, inhibition of thrombin generation via {alpha}vβ3 mechanisms may also attenuate neointimal growth by decreasing thrombin driven chemotaxis and mitogenesis of VSMC. Inhibition of thrombin generation and platelet adhesion by {alpha}vβ3 blockade may be more imporatnt in retarding early neointimal growth than inhibition of osteopontin-{alpha}vβ3 depenedent cell migration, which may assume greater relevance later between 7 and 21 days following injury (Figs. 2, 4, Table 1).

A porcine coronary injury model was used in the present study to allow greater clinical applicability of results [36]. Small animal models of peripheral arterial injury such as the rat, hamster or rabbit emphasize medial VSMC migration and proliferation as key restenosis determinants [43]. In contrast, VSMC proliferation may not be predominant in large animals, where neointimal volume may be largely derived by cellular migration, with subsequent synthesis of extracellular matrix [27, 43, 44]. Our data show prominent {alpha}vβ3 immunoreactivity and osteopontin expression surrounding {alpha}-SMC actin and vimentin positive adventitial cells (Fig. 2G–I, 3E). Evidence from this porcine restenosis model suggests that migration and functional modulation of adventitial mesenchymal cells with myofibroblast properties may contribute to both restenotic neointimal formation and tissue remodeling [45, 46]. Since migration of VSMC is known to require {alpha}vβ3, and given the comparable efficacy of c7E3 and XJ 735 in inhibiting VSMC migration (Table 3); we suggest that XJ 735 may retard neointimal growth by inhibiting migration of VSMC and other {alpha}vβ3 bearing cells including EC, monocyte-macrophages, and myofibroblasts. This is corroborated by steady state plasma XJ 735 levels greater than the IC50 required to achieve potent ex vivo inhibition of EC adhesion and VSMC migration.

Functional co-induction of {alpha}vβ3 and osteopontin expression in the injured vessel wall may be a specific example of a general wound repair mechanism. Induction of {alpha}vβ3 expression has been demonstrated during porcine cutaneous wound repair [47]. In both cases, the wound matrix contains osteopontin, fibrin, fibronectin, and vitronectin which can all interact with {alpha}vβ3 on EC, VSMC and fibroblasts to promote angiogenesis and granulation tissue formation. The stimulation of EC migration by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) during angiogenesis, has similarly been shown to involve a cooperative mechanism between {alpha}vβ3 and osteopontin [48].

Osteopontin is expressed by numerous vascular cells including EC, VSMC and macrophages in both atherosclerotic and restenotic neointima [49]. Spatial–temporal induction of osteopontin expression is observed in both porcine and rodent models of balloon injury, coincident with VSMC migration into the neointima [50]. Osteopontin is chemotactic to both EC and VSMC in vitro, and to macrophages in vivo, with even low concentrations of osteopontin (~30 nM) being sufficient to promote cell migration [49]. Hence, osteopontin production within the adventitia and neointima of injured vessels (Fig. 3) may serve to perpetuate cell recruitment via osteopontin-{alpha}vβ3 dependent mechanisms. Since {alpha}vβ3 expression in the neointima and adventitia is largely quiescent by 28 days, we speculate that {alpha}vβ3 dependent cell recruitment may be largely complete between 7 and 21 days. The prominent osteopontin expression in the adventitia beyond 21 days may facilitate interaction with other integrins e.g. {alpha}vβ1, or {alpha}vβ5, which may underlie effects occuring at this time e.g. geometric vascular remodeling.

Large amounts of thrombin are liberated during deep arterial injury and augment osteopontin effects by molecular cleavage [51]. Morever, thrombin activates vascular endothelium to support monocyte adhesion via integrin {alpha}vβ3 [7]. In pigs, monocyte-macrophage derived interleukin-1β promotes intimal hyperplasia via a PDGF mediated mechanism [52]. Thus, {alpha}vβ3 antagonism may retard neointimal growth both indirectly by attenuating monocyte recruitment and interleukin-1β release, and directly by antagonizing PDGF-stimulated VSMC migration. The marked attenuation of adventitial mononuclear cell infiltrates by XJ 735 observed in the present study is consistent with this hypothesis (Fig. 10B).

Liaw et al. have described {alpha}vβ1, {alpha}vβ3 and {alpha}vβ5 as VSMC osteopontin recetors, with migration toward osteopontin being solely dependent on {alpha}vβ3, and osteopontin being supported by {alpha}vβ1, {alpha}vβ3 and {alpha}vβ5 integrins [12]. Given the redundancy in extracellular matrix protein receptors mediating cell adhesion and migration, the antirestenotic benefit of selective {alpha}vβ3 blockade, emphasizes the functional importance of this integrin. Prior small animal studies of balloon injury have utilized nonselective cyclic RGD peptides to achieve a reduction in neointimal growth [19, 22]. These nonselective cyclic RGD peptide antagonists occupy both {alpha}v-containing and platelet {alpha}IIbβ3a integrins; or as in the case of PenRGD occupy all {alpha}v-containing integrins [25]. In fact, Suchiro et al. have demonstrated crossreactivity between {alpha}vβ3 and {alpha}IIbβ3a integrins for linear RGD, cyclic RGD, vitronectin, fibrinogen, and {gamma}-chain peptides, dependent on divalent cation conditions [53]. This lack of selectivity makes it unclear whether reductions in platelet aggregation and platelet derived growth factor release via {alpha}IIbβ3a antagonism, or alterations in VSMC adhesion ({alpha}vβ1, {alpha}vβ3, and {alpha}vβ5-mediated) and/or migration ({alpha}vβ3 mediated), underlie the neointimal reduction produced by these peptides. The present findings confirm that selective {alpha}vβ3 antagonism is sufficient to inhibit neointimal growth and lumen stenosis (Figs. 8 and 9).

Another potential anti-restenotic benefit of {alpha}vβ3 antagonism relates to suppression of growth factor stimulated angiogenesis required to support neointimal growth [14]. Co-induction of {alpha}vβ3 and osteopontin promotes EC migration during VPF/VEGF stimulated angiogenesis [48]. Enhanced expression of {alpha}vβ3 occurs in angiogenic areas of human granulation tissue, in cytokine stimulated chick embryonic vessels, and in retinal neovascularization [14, 54]. Since angiogenesis requires {alpha}vβ3-dependent EC migration, it can be blocked using either monoclonal antibody to {alpha}vβ3 (LM609) or XJ 735 [54, 55]. In the present study, prominent {alpha}vβ3 expression was associated with adventitial neovascular channels of injured coronary arteries (Fig. 2I). Since injury-induced intimal hyperplasia can be linked to adventitial angiogenesis [56], XJ 735 blockade of {alpha}vβ3 mediated angiogenesis may have decreased neointimal growth. The marked reduction in the density of adventitial neovascular channels observed for XJ 735 treated animals compared with controls is concordant with this postulate (Fig. 10C).

Recent studies have debated the differing contributions of neointimal growth and geometric remodeling to final lumen outcome following differing types of arterial injury [57]. XJ 735 treatment resulted in a marked reduction in neointimal growth and lumen stenosis without significant effect on artery size, implying a dominant contribution of neointimal hyperplasia to stent restenosis (Figs. 7–9). This is supported by human intravascular ultrasound studies of stent restenosis [32]. Stent deployment aims to maximize vessel size by eliminating acute recoil and minimizing chronic vascular constriction, in order to overcome the lumen loss incurred by intimal hyperplasia [57]. The ‘Achilles heel’ of this "bigger is better" strategy is the increasing vascular counterreaction elicited by deeper levels of injury [35]. Avoidance of stent restenosis will require a combination of the best mechanical strategy to resists recoil and constrictive remodeling, and a pharmacologic strategy such as {alpha}vβ3 blockade to limits neointimal accretion.

In summary, we have demonstrated co-induction of integrin {alpha}vβ3 and osteopontin expression in the vessel wall following deep arterial injury. In this large animal coronary stent restenosis model, use of a selective high affinity {alpha}vβ3 antagonist resulted in a marked reduction in neointimal hyperplasia and lumen stenosis. These findings underline the functional importance of {alpha}vβ3-mediated cell-matrix interactions, and establish selective {alpha}vβ3 blockade as an effective anti-restenotic strategy. Unlike atherosclerosis or transplant arteriopathy, coronary restenosis is complete over a short time period, obviating the need for long term exposure to integrin antagonists [58]. By restricting selective {alpha}vβ3 blockade to a limited time period following injury and by using local delivery systems, systemic side effects such as impaired angiogenesis and wound healing can be avoided. Further studies are needed to determine the minimum dose and period of {alpha}vβ3 blockade required for anti-restenotic efficacy, and to ascertain the relative importance of distinct {alpha}vβ3-mediated cellular events in coronary restenosis.

Time for primary review 29 days.


   Acknowledgements
 
We thank LaDonna Camrud, Alan Camrud, Jodi Johnson, Michael Jorgenson, Avery Lafleur and Peggy Button for excellent technical assistance. This work was supported in part by PHS NHLB 51736 (L.A.F.), American Heart Association Minnesota Affiliate grant in aid (S.S.S.), and by an American College of Cardiology–Merck research fellowship (S.S.S.). We are also indebted to Dr. Larry Fisher, NIDR, NIH for the generous gift of LF-7 antibody.


   Notes
 
1 This work was presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, Louisiana, November 1996. Back

2 Present address: Tanabe Seiyaku Co., Saitama, Japan. Back


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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