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(Circulation Research. 1999;85:1040.)
© 1999 American Heart Association, Inc.

Integrative Physiology

Reduction in Atherosclerotic Lesion Size in Pigs by Vß3 Inhibitors Is Associated With Inhibition of Insulin-Like Growth Factor-I–Mediated Signaling

Timothy C. Nichols, Tracey du Laney, Bo Zheng, Dwight A. Bellinger, G. Allen Nickols, Wayne Engleman, David R. Clemmons

From the Department of Pathology and Laboratory Medicine (T.C.N., T.d.L.), the Division of Laboratory Animal Medicine (D.A.B.) and the Department of Medicine (T.C.N., B.Z., D.R.C.), University of North Carolina, Chapel Hill, NC; and Monsanto/Searle, Inc, Chesterfield, Mo (G.A.N., W.E.).

Correspondence to David R. Clemmons, Department of Medicine, University of North Carolina, CB No. 7170, Chapel Hill, NC 27599-7170. E-mail dpm{at}med.unc.edu

	Abstract

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Abstract—Insulin-like growth factor-I (IGF-I) is a potent stimulant of smooth muscle cell (SMC) migration and proliferation and has been implicated in the development of experimental atherosclerotic lesions. Because optimal stimulation of SMC in vitro by IGF-I requires ligand occupancy of Vß3, these studies were conducted to determine whether Vß3 antagonists would result in a change in lesion size and whether they could alter IGF-I-mediated actions. Clamps were placed on the carotid and femoral arteries of normal pigs that had been fed a high-cholesterol diet for 4 weeks. Vß3 inhibitors (SC-69000, SC-65811) (10^-6 mol/L) or saline were infused for 2 weeks into the peristenotic area. Lesion area, the number of SMC layers, and proliferating cell nuclear antigen positive cells were determined in a 1.2-mm segment of each artery. Lesion areas were 304 788±113 453 µ² (saline), compared with 149 799±35 456 µ² (SC-69000) (P<0.01). Lesion areas in arteries treated with SC-64258, a compound that does not bind to Vß3, were 310 284±160 467 µ², P=not significant. In a second experiment, lesion areas were 110 391±17 347 µ² (saline) and 59 533±17 568 µ² (SC-65811, P<0.001). Neointimal SMC layers were reduced by SC-65811 from 7.4±4.5 to 3.0±0.4 (P<0.001). To determine whether IGF-I action was altered, IGF binding protein-5, which is synthesized in response to IGF-I, was analyzed. IGF-I binding protein-5 mRNA abundance was reduced by 67±8% in the 6 lesions treated with SRL-69000 compared with saline controls (P<0.001). We conclude that Vß3 antagonists block the development of lesions in pigs that have been induced by a high-cholesterol diet and stenosis, and the effect of these compounds is associated with their ability to inhibit IGF-I–mediated signaling.

Key Words: disintegrin • arteriosclerosis • insulin • signal transduction

	Introduction

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Smooth muscle cell (SMC) migration and proliferation are critical steps in the development of atherosclerosis and in vascular wound repair.^{1 2} SMC migration from the media to the neointima occurs during lesion formation and has been linked to increased expression of integrins on the surface of SMC, especially the Vß3 integrin.^{3 4} This integrin has been shown to bind to several arg-gly-asp (RGD)-containing ligands, such as fibrinogen, von Willebrand factor, thrombospondin, and osteopontin, and Vß3 has been detected on multiple vascular cell types, including endothelial cells, SMC, and platelets.⁵ Increasing the concentrations of ligands, such as vitronectin and osteopontin, within the subendothelial matrix is believed to facilitate SMC migration and vascular remodeling.^{6 7} Antibodies to the Vß3 integrin and nonspecific Vß3 inhibitors have been shown to decrease SMC migration in vitro and to retard the development of intimal hyperplasia and restenosis after vascular injury.^{8 9 10} An antibody that specifically blocks ligand binding to both the IIBß3 platelet integrin and the Vß3 integrin on SMC has been shown to reduce target vessel reocclusion after balloon angioplasty or directional atherectomy in humans; a reduction in restenosis has not been documented angiographically.¹¹ These data suggest that small molecules that inhibit these integrins could retard atherogenesis.

Naturally occurring peptides, termed disintegrins, have been shown to bind integrins and to block integrin-mediated functions. The disintegrin echistatin has been shown to be a potent inhibitor of Vß3 function in osteoclasts,¹² platelets,¹³ endothelial cells,¹⁴ and SMC.^{15 16} In in vitro studies, we have shown that the addition of echistatin to migrating SMC in culture markedly retards their movement over lateral surfaces.⁸ The molecular mechanism by which disintegrins alter SMC migration may not be solely dependent on blocking the ability of ligands to bind Vß3. Previous studies have demonstrated that growth factors work collaboratively with Vß3 to enhance several cellular functions, including cell migration, receptor clustering, and growth factor receptor tyrosine kinase activation.^{17 18 19}

Experiments done in our laboratory have shown that echistatin and synthetic Vß3 inhibitors block the SMC migration response to insulin-like growth factor-I (IGF-I) in vitro.^{8 20} These reagents also inhibit IGF-I stimulation of DNA synthesis as well as IGF-I receptor-mediated intracellular signaling.^{16 21} The present study was conducted to determine whether the synthetic Vß3 inhibitors would also reduce atheroma formation in vivo and whether this was related to their ability to inhibit a specific IGF-I–inducible effect. A porcine hypercholesterolemic-stenotic model of lesion formation was used, and the ability of these Vß3 antagonists to inhibit lesion formation was determined by measuring the number of SMC layers in the neointima, the lesion areas, proliferating cell nuclear antigen (PCNA) labeling, and the expression of IGF binding protein-5 (IGFBP-5), a protein whose synthesis is induced specifically by IGF-I.²¹

	Materials and Methods

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Animal Preparation
Male and female pigs, aged 6 months, were treated according to the Guide for Care and Use of Laboratory Animals (National Institutes of Health publication No. 85-23), using an approved protocol. Cholesterol values were determined by an enzymatic method.²² The pigs had 1 of 2 genotypes for apolipoprotein, Lpb-5/5 or Lpb-8/8.²³ The pigs were placed on a 1% cholesterol diet for 4 weeks, which raised serum cholesterol to 700 to 1000 mg/dL.

Under anesthesia, 0.5-cm Goldblatt steel clamps were applied to both carotid and both femoral arteries and were closed to produce a >80% stenosis.²⁴ Alzet pumps (volume=2.0 mL) (Alza Corp) and infusion catheters were placed within the vessel wall, as shown in Figure 1. They delivered the experimental or control compound at a rate of 5 µL/hr for 14 days. After injection of 500 µL (10^-6 mol/L) of each compound, the pump reservoir was filled and the infusion commenced. Both test compounds inhibited SMC migration in vitro.²⁰ SC-64258, a control compound that does not bind to Vß3, was also tested. Each vessel of 1 pair (eg, carotid and femoral arteries) was randomized to an individual treatment with the contralateral vessel receiving control. At the end of 6 weeks, the animals were killed.

View larger version (14K): [in this window] [in a new window]   Figure 1. Placement of the Alzet pump relative to vessel wall. The infusion port of the Alzet osmotic pump was sutured into place in a manner that delivered the compounds into the vessel wall. At the end of the experiment, the position of the infusion port was confirmed when the vessel was removed.

The arterial segments were fixed in 4% paraformaldehyde and paraffin embedded. Eight cross-sections per vessel were obtained 1.9 to 2.2 mm from the proximal edge of the clamps. When the plaque areas of the carotid and femoral lesions were compared, there was no statistically significant difference; therefore, the data from both vessels were pooled.

Morphometric Analysis
The arterial sections were stained, and the images were visualized on a Nikon microphot-FXA microscope with an Optronics TEC-470CCD video camera system. The images were captured by NIH Image and were analyzed for neointimal area by tracing the internal elastic lamina and the lumen of the vessel, then calculating the difference between these areas. The observer was blinded as to treatment group. The number of cell layers was quantified by counting the average number of neointimal cell layers between the internal elastic lamina and the lumen in at least 4 different areas per vessel. In general, the 4 or 5 highest quality cross-sections of each artery were used.

PCNA Staining
PCNA was used as an index of cell proliferation.^{24 25} The paraffin sections were fixed onto Probe-On Plus slides and then the paraffin was removed. The sections were exposed to a hydrogen peroxide/methanol solution. Nonspecific binding was blocked by exposure of the sections to 2% horse serum. The sections were then incubated with a 1:100 dilution of PC-10, a polyclonal antibody to PCNA (Dako Corp) for 1 hour at 22°C, then exposed to the ABC vectastain secondary antibody (horse anti-rabbit) and the chromagen DAB. Positively stained cells in both the neointima and the media were counted, and the results are expressed as a percentage of the total number of cells that stained.²⁴

Determination of IGFBP-5 mRNA Abundance
Liquid nitrogen frozen tissue samples were extracted with Tri-Reagent (Gibco BRL) and the RNA quantified by optical density readings at 260 nm. Samples were denatured in formaldehyde, processed by agarose gel electrophoresis (1% gel), transferred to nitrocellulose, and probed with a specific IGFBP-5 cDNA.²⁰ To control for loading artifacts, the blots were also probed for GAPDH. Twelve separate areas were analyzed: 6 saline treated and 6 exposed to SC-69000. The autoradiographic band intensities were quantified using a Phospho Imager and Image Quant software (Molecular Dynamics).

Assays of Integrin Inhibitor Specificity
The Vß3 and the IIbß3 integrins were purified from human placenta or platelets.²⁰ The ability of the integrin antagonists to compete for binding with biotinylated vitronectin to purified Vß3 or biotinylated fibrinogen to purified IIbß3 was determined.²⁶ To quantify the ability of the antagonists to inhibit vitronectin binding to Vß1, Vß3, and 5ß1, cell based attachment assays were used. For measuring their effects on 5ß1 mediated adhesion, K562 erythroleukemia cells were used with purified fibronectin.²⁷ The effect of these compounds on Vß1 mediated attachment was determined by assessing adherence of 293 cells that constitutively express this integrin to vitronectin.²⁸ A stably transfected line of 293 cells expressing Vß5 was used to determine the activity of the compounds in inhibiting attachment to vitronectin.²⁹

Statistical Analysis
Comparison of the lesion sizes and cell layer numbers were analyzed by 2-way ANOVA and Dunnett’s T test. The PCNA labeling and IGFBP-5 mRNA abundance were compared using Student’s t test.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Porcine arterial SMCs have been shown to contain the following integrins: 1ß1, Vß1, 2ß1, Vß3, 5ß1, and 3ß1.⁸ When the integrin antagonists that were used in this study (Figure 2) were tested for affinity for these integrins, it was found that the SC-69000 had an IC₅₀ for Vß3 of 0.56 nmol/L and a high affinity for Vß1, eg, 0.75 nmol/L. In contrast, its affinity for IIbß3 was reduced 30-fold, and for 5ß1 it was >900-fold lower (Table 1). When SC-65811 was tested, it had an IC₅₀ of 0.79 nmol/L for Vß3. It was more selective, because its affinity for IIbß3 was reduced >3000-fold, for Vß1>100-fold, and its affinity for 5ß1 was negligible. Both compounds competed effectively for binding to Vß5, but we have not detected this integrin on pSMC surfaces.⁸ Therefore, we conclude that the SC-65811 compound must be exerting its predominant effects through inhibiting binding to Vß3 and the SC-69000 compound through Vß3 and Vß1. Compound SC-64258 had almost no detectable inhibitory activity for any of the integrins at 10^-6 mol/L.

View larger version (11K): [in this window] [in a new window]   Figure 2. Chemical structures of the three test compounds. These structures have been published previously. (Reproduced with permission. © The Endocrine Society. Clemmons DR, Horvitz G, Engleman W, Nichols T, Moralez A, Nickols GA. Synthetic Vß3 antagonists inhibit insulin-like growth factor-I–stimulated smooth muscle cell migration and replication. Endocrinology. 1999;140(10):4616–4621.)

View this table: [in this window] [in a new window]   Table 1. IC50 Values for Test Compounds

In the first experiment, 4 pigs (14 vessels) were used. The active compound, SC-69000, was infused into 7 vessels and the results compared with the 3 vessels that received the control compound, SC-64258, and 3 that received a saline solution. The average weight of the pigs at the start of the protocol was 129±15 kg, and at the end of the protocol, it was 150±0.0 kg. Total cholesterol was 116±4 mg/dL at the beginning of the protocol and 642±93 mg/dL at the end. Morphometric analysis of the lesions (Figure 3) showed that SMC layers and neointimal area were significantly reduced by SC-69000 compared with saline or SC-64258 (Tables 2 and 3). The media areas in the regions of the lesions showed no significant difference between controls and compound SC-69000. In contrast, PCNA labeling in the neointima or media showed no significant change in any of the treatment groups, including the SC-69000 exposed animals (Table 4). This suggests that the proliferative rate of SMC was not affected by the SC-69000 infusion. In contrast, the marked reduction of lesion volume suggests that either synthesis of extracellular matrix proteins or cell migration or both had been inhibited. The control compound, SC-64258, showed an increased number of SMC layers. However, because of the large standard error and the small number of lesions tested, the difference was not statistically significant. Conversely, the effect of SC-69000 on both the number of SMC layers and lesion area was significant when compared with saline control–treated lesions.

View larger version (62K): [in this window] [in a new window]   Figure 3. Integrin Vß3 blockade inhibits arterial neointimal lesions induced by shear stress and an atherogenic diet in porcine carotid arteries. Vessels exposed to compounds SC-69000 and SC-65811 are shown in panels A and B, respectively. There is reduced neointimal formation when compared with saline-treated control vessel (panel C) or an artery treated with compound SC-64258 (panel D) (inactive compound). All four cross-sections were stained with hematoxylin and eosin and photographed at an original magnification of x10.

View this table: [in this window] [in a new window]   Table 2. Changes in Neointimal Area

View this table: [in this window] [in a new window]   Table 3. Changes in Smooth Muscle Cell Layers

View this table: [in this window] [in a new window]   Table 4. PCNA Labeling

To confirm that the effect of the SC-69000 compound was specific for Vß3 and that it was reproducible, a second Vß3 antagonist, SC-65811, was infused using a similar experimental design. In the second experiment, 7 lesions were treated with the SC-65811 compound for 2 weeks, and the results were compared with 7 lesions that were exposed to saline alone. SC-65811 is also a more specific Vß3 inhibitor, because SC-69000 also inhibits ligand occupancy of Vß1 and IIbß3 at the concentrations that were used (Table 1). These animals weighed 95±37 kg at the beginning of the study and 115±41 kg at the end, and their cholesterol rose from 128±7 to 1460±396 mg/dL. There was a significant reduction in the lesion area and the number of SMC layers in the SC-65811–treated lesions compared with saline controls (Figure 3; Tables 2 and 3). The level of decrease was similar to that observed with SC-69000. The number of SMC layers decreased from 7.4±4.5 in the saline-treated animals to 3.0±0.4 (P<0.05) in the SC-65811–treated animals. The neointimal area was significantly less in the SC-65811 group: 110 391±17 374 µ² compared with 59 533±17 568 µ² (P<0.001). Comparison of the media areas showed no significant difference. The number of PCNA-positive cells in the neointima was reduced significantly, but there was no significant change in PCNA labeling in the media (Table 4). The adventitia areas were not analyzed because it was partially removed during clamp placement. No inflammation was noted in the adventitia that remained.

To determine whether these compounds were having any effect on IGF-I–stimulated actions, IGFBP-5 mRNA abundance was assessed. IGF-I has been shown to be a potent stimulant of IGFBP-5 synthesis, and ligand occupancy of Vß3 has been shown to be necessary for IGF-I stimulation to be detected in vitro.^{16 24} In the absence of IGF-I, echistatin has no effect on the basal expression of IGFBP-5 mRNA.²⁰ The abundance of IGFBP-5 mRNA was reduced significantly in the lesions that had been exposed to SC-69000 compared with the saline controls (Figure 4). When the changes in scanning units were adjusted for differences in RNA loading (based on GAPDH abundance), there was a 3.8-fold reduction (P<0.01) in the IGFBP-5 mRNA from the 6 SC-69000 exposed lesions compared with the 6 saline control lesions. Because IGF-I is the only known stimulant of IGFBP-5 synthesis by SMC and Vß3 occupancy augments the IGFBP-5 response to IGF-I,²⁰ this change represents an attenuation of IGF-I action.

View larger version (86K): [in this window] [in a new window]   Figure 4. Northern blot of IGFBP-5 mRNA abundance. Lesion and nonlesion tissue was obtained from the vessel walls of animals that either received the SC-69000 compound or saline infusion. The tissues were processed and total RNA extracted. RNA was separated by agarose gel electrophoresis, transferred to nylon membranes, and the amount of IGFBP-5 determined by Northern blotting using a specific IGFBP-5 cDNA probe, as described in Methods. The results show that, although the amount of total RNA loaded was similar in each lane, the SC-69000 lesions showed markedly diminished IGFBP-5 mRNA, which is represented by the 3.6-KB band. The results shown are from 2 representative lesions. When 6 SC-69000 exposed lesions and 6 saline control lesions were analyzed and the results quantified by Phosphor Image analysis, the IGFBP-5 mRNA band intensities were 12 732±1416 (SC-69000) and 47 111±4609 (saline) scanning units.

	Discussion

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Genotypically-selected animals showed significant increases in total cholesterol levels and proliferative lesions after 6 weeks on an atherogenic diet in the distal aortas and in the carotid and femoral arteries with the Goldblatt clamps. This model involves not only diet-induced atherosclerotic lesions, as evidenced by the accumulation of macrophages, but also a clamp-induced stenosis, which results in changes in shear stress that result in vascular remodeling and partial vessel occlusion. Shear stress has been reported to play an important role in both vascular remodeling and wound repair that is typical of restenosis and intimal hyperplasia, as well as atherosclerosis (reviewed in References 24 and 30^{24 30} ). Partial clamping of the carotid artery in rabbits has been shown to induce neointimal lesion formation both with and without atherosclerotic diets.³⁰ Therefore, this model represents the combined effects of diet and stenosis.

Morphometrically, the animals responded in the same manner to both Vß3 inhibitors, with >45% reductions in neointimal area compared with saline controls irrespective of the compound used. There were fewer SMC layers in both groups of animals, with the degree of reduction being 54% for SC-69000 and 60% for SC-65811. These changes were not significant when compared with each other, but they were highly significant when compared with the saline controls. This indicates that both Vß3 inhibitors blocked SMC migration and/or hypertrophy in vivo.

Disintegrin blockade of Vß3 in SMC has been shown to block several cellular processes that are determinants of lesion volume and are stimulated by IGF-I. These include cell migration and division, protein synthesis, and accumulation of extracellular matrix.^{16 20} An inhibition of one or more of these processes could lead to decreased lesion volume; however, the decreased cell layer number is specific for either migration or proliferation and suggests that one or both of these processes is specifically involved. The PCNA results suggest that only SC-65811 was altering the cell proliferation response. There were no differences in the neointima or media in PCNA positivity when SC-69000–treated and saline-treated vessels were compared. This suggests, but does not prove, that the compound with more Vß3 selectivity had a greater effect on SMC proliferation. However, because the overall rate of proliferation was lower in experiment 2, in which compound SC-65811 was tested, this difference could also be due to experimental differences in sensitivity to Vß3 blockade. In both experiments, there were high rates of proliferative activity by PCNA staining, indicating that this is a reliable method for assessing proliferation and that actively growing young pigs with stenotic lesions have a high proliferative index. We conclude from this data that the Vß3 inhibitors appear to reduce lesion size at least in part by inhibiting SMC migration, although an effect on apoptosis cannot be excluded.

We have shown previously in in vitro studies that these compounds inhibit the effects of IGF-I on SMC function.²⁰ Specifically, they are potent inhibitors of IGF-I–stimulated migration, DNA synthesis, induction of IGFBP-5 synthesis, and IGF-I–stimulated receptor phosphorylation. IGFBP-5 synthesis increases 6-fold in response to IGF-I, and unlike the other biologic effects listed, this effect is specific for IGF-I. It is not stimulated by other mitogens.²⁰ SC-65811 and SC-69000 have been shown to attenuate the IGFBP-5 synthesis response to IGF-I in pSMC in vitro. Based on that fact, we analyzed IGFBP-5 mRNA abundance in the lesions. Because IGF-I receptor antagonists lower the synthesis rate of IGFBP-5 by these cells in vitro,²⁰ the results suggest that the inhibition of IGFBP-5 mRNA abundance that was noted after exposure to SC-69000 is secondary to a decrease in IGF-I action. Studies concomitantly infusing IGF-I and Vß3 inhibitors would be required to confirm this observation.

IGF-I has been shown to be an important modulator of atherosclerotic lesion formation. After balloon denudation, there is an increase in IGF-I mRNA synthesis that peaks at 7 days after injury.³¹ In addition, human atherosclerotic lesions have been shown to contain abundant IGF-I.³² IGF-I appears to be a growth factor for normal SMC in vivo because over-expression of an IGF binding protein, IGFBP-4, that is known to inhibit IGF-I actions in vitro in SMC-containing tissues (bladder and aorta) results in SMC hypoplasia within these tissues.³³ This suggests that IGF-I is a required trophic factor for normal SMC and that inhibition of its ability to bind to receptors results in attenuation of its actions. This has been further confirmed using a protease-resistant form of IGFBP-5 that was shown to markedly inhibit IGF-I actions in SMC.³⁴ Although our findings do not address the mechanism by which blocking Vß3 occupancy inhibits IGF-I stimulation of lesion formation in vivo, they suggest that because targeting to the IGF receptor has not been possible, it may be possible to target IGF-I–stimulated lesion development with specific Vß3 inhibitors.

Previous reports have shown that specific anti-ß3 antibodies and a specific RGD-containing peptide inhibit lesions that are induced in rodent models of vascular injury.^{9 10} These results further support the conclusion that stimulation of Vß3 may be an important component of lesion development. A recent report analyzed the effect of a more selective Vß3 inhibitor on arterial lesions induced by stents in pigs. That study reported a 42% reduction in lesion volume after a 3-week infusion of active compound.³⁵ The investigators reported an increase in Vß3 expression and of its ligand, osteopontin,³⁵ in the lesions that developed after stent placement. This increase is consistent with this type of mechanical injury and led them to postulate that this change led to increased sensitivity to the inhibitory effect of Vß3 antagonists. Our findings are consistent with the degree of improvement seen by those investigators and with the proposed role of Vß3 changes that are seen after increases in sheer forces.³⁶ Future studies will be necessary to determine the degree to which altering ligand occupancy of Vß3 is effective in reducing responsiveness to IGF-I and the other variables that are known to regulate this process.

	Acknowledgments

 
These studies were supported by a grant from the National Institutes of Health, HL-56850 and a grant from Monsanto, Inc. The authors wish to thank George Mosley for his help in preparing the manuscript.

Received May 17, 1999; accepted September 13, 1999.

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