A Novel v?3 Integrin Antagonist Suppresses Neointima Formation for More Than 4 Weeks After Balloon Injury in Rats
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动脉硬化血栓血管生物学 2005年第7期
From the Dainippon Pharmaceutical Co., Ltd., Suita, Osaka, Japan.
Correspondence to Yayoi Honda, Pharmacology and Microbiology Research Laboratories, Dainippon Pharmaceutical Co., Ltd, Enoki 33-94, Suita, Osaka 564-0053, Japan. E-mail yayoi-honda@dainippon-pharm.co.jp
Abstract
Objectives— We performed a detailed kinetic analysis in a rat balloon injury model to clarify the essential roles of v?3 integrin and endothelial cell (EC) regeneration in neointima formation. Using this model, we evaluated the antistenotic effect of Dainippon compound BS-1417, a novel v?3 integrin antagonist.
Methods and Results— Kinetic analysis using RT-PCR showed that v?3 integrin-related genes are upregulated before neointima formation. Morphological and functional analyses revealed that EC regeneration requires >4 weeks after injury, and that recovery of EC normal function coincides with the arrest of neointima formation. Subcutaneous infusion of BS-1417 for 2, 4, 7, or 12 weeks after injury potently inhibited neointima formation without affecting EC regeneration. Although withdrawal of treatment with BS-1417 after short-term administration after injury resulted in catch-up growth of neointima, a long-term study suggested that this catch-up growth can be prevented by continuous administration of BS-1417 until EC regeneration.
Conclusion— We clarified that v?3 integrin and EC regeneration play an essential role in neointima formation, and that continuous administration of BS-1417 potently and stably inhibits neointima formation without affecting EC regeneration. These findings suggest that BS-1417 might be useful as a novel systemic drug for the treatment of restenosis.
We clarified the essential roles ofv?3 integrin and endothelial cell (EC) regeneration in neointima formation using a rat balloon injury model. In this model, we demonstrated that systemic administration of BS-1417, a novelv?3 integrin antagonist, potently and stably inhibits neointima formation without affecting EC regeneration.
Key Words: v?3 integrin antagonist ? endothelial cell regeneration ? vascular smooth muscle ? gene expression ? rat balloon injury model
Introduction
Vascular smooth muscle cell (SMC) migration and proliferation are critical steps in neointima formation after vascular injury1,2 and are regulated by various molecules, including integrins and their extracellular matrix protein ligands, matrix metalloproteinases (MMPs), and growth factors and their receptors.3,4 It is also known that regeneration of endothelial cells (ECs) plays an important role in preventing SMC proliferation.5,6 Given systemically, numerous single molecule drugs for the prevention of neointima formation in animal models have been reported.7 However, these drugs have shown only short-term efficacy in animal models7 and no promising result in clinical trials.7,8
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v?3 integrin, also called vitronectin receptor, is expressed on a variety of cell types,9 including SMCs and ECs, and it is known to mediate migration or proliferation of certain cells in disease states, including SMCs in restenosis after vascular injury,10 and ECs and SMCs in angiogenesis.11 Although many cyclic peptide and nonpeptide v?3 integrin antagonists have been reported,12,13 only a few have been shown to potently inhibit neointima formation in rat models.
In preliminary in vitro studies aimed at finding selective inhibitors of SMC proliferation, we found a series of v?3 integrin antagonists that inhibit SMC proliferation without affecting EC proliferation. Based on this finding and the results of a previous study showing that small-molecule v?3 integrin antagonists effectively inhibit neointima formation in rats,14 we focused our efforts on v?3 integrin antagonists and recently found a new series of promising phenylpiperazine-based v?3 integrin antagonists.15 Extensive screening of these novel antagonists using a rat balloon injury model led us to the discovery of BS-1417 (Figure I, available online at http://atvb.ahajournals.org) as a suitable candidate for further development.15
In the present study, we first performed a detailed kinetic analysis of neointima formation in a rat balloon injury model to verify the concept that v?3 integrin plays an essential role in neointima formation. In the kinetic analysis, we also examined the time course of EC regeneration, especially recovery of EC function, to clarify the preventive role of EC regeneration in neointima formation. Then, using the rat balloon injury model, we evaluated the antistenotic effects of BS-1417, a novel v?3 integrin antagonist, for >2 weeks after injury. Finally, we examined the long-term effects of BS-1417 through a treatment withdrawal experiment.
Methods
Balloon Injury
Male Sprague-Dawley rats (Clea Japan, Inc.) weighing 340 to 360 g were used in this study. They were divided into groups based on body weight. Carotid arteries were injured essentially as described by Clowes et al.16 In brief, rats were anesthetized with pentobarbital (50 mg/kg IP), and the left femoral and the left carotid arteries were exposed. A 2F Fogarty balloon catheter (Baxter Healthcare Corporation) was introduced through the left femoral artery and advanced to the left carotid artery until it was recognized at the bifurcation of the common carotid artery. The balloon was inflated with 0.3 mL of air and pulled back to the aortic arch at an approximate speed of 0.3 cm/s. After 3x repetition of this procedure, the catheter was removed, the femoral artery was ligated, and the wounds were closed.
Kinetic Analysis of Rat Balloon Injury Model
Rats were first anesthetized with pentobarbital (50 mg/kg IP) and then exsanguinated at 6 hours or 1, 2, 3, 5, 7, or 10 days, or 2, 3, 4, 5, 8 or 12 weeks after balloon injury. Isolated arteries served for morphometric analysis, immunohistochemical analysis, or measurement of gene expression or cGMP production (see online supplement, available at http://atvb.ahajournals.org).
Evaluation of the Effects of BS-1417 on Neointima Formation
The effects of BS-1417 on neointima formation were evaluated first for a period of 2 weeks (short-term effects) after balloon injury, and then for a period of 4, 7, or 12 weeks (long-term effects) after balloon injury. BS-1417 was infused subcutaneously at doses of 0.1, 0.3, and 1.0 mg/rat per day (2-week experiment) or at a dose of 7.2 mg/rat per day (4-, 7-, and 12-week experiments) starting 1 day before injury using Alzet osmotic pumps (Alza Corporation). The difference in dosage between the 2-week experiment and the 4-, 7-, and 12-week experiments was attributable to the fact that treatment with BS-1417 at 1.0 mg/rat per day for 4 weeks resulted in catch-up growth of neointima during the latter 2 weeks (data not shown). To confirm the effects of BS-1417 on neointima formation, a treatment withdrawal experiment in which BS-1417 (7.2 mg/rat per day) was first infused subcutaneously for a period of 2, 4, or 12 weeks then withdrawn for a period of 2, 3, or 4 weeks, respectively, was carried out. In all experiments, BS-1417 was dissolved in 0.3 mol/L HCl, and the pH was adjusted to 4 with NaOH. Dosing formulations were prepared by appropriate dilution with water or saline. Control rats in all experiments were either untreated rats or rats that underwent the same procedure as those treated with BS-1417, except that saline was substituted for BS-1417.
Evaluation of the Effects of BS-1417 on Relevant Gene Expression
The effects of BS-1417, given subcutaneously at a dose of 7.2 mg/rat per day, on relevant gene expression were evaluated for a period of 6 hours, 1 or 3 days, or 2 or 8 weeks after balloon injury. Genes that were evaluated are listed in supplemental Table I (available online at http://atvb.ahajournals.org).
Evaluation of the Effects of BS-1417 on EC Regeneration
The effects of BS-1417, given subcutaneously at a dose of 7.2 mg/rat per day, on EC regeneration were evaluated for a period of 4 or 12 weeks after balloon injury.
Statistical Analysis
Data are expressed as mean±SEM. Comparison between drug-treated and control groups was performed using the 2-tailed Student’s t test. The short-term effects of BS-1417 were compared in 4 groups using parametric William’s test. A P value <0.05 denoted the presence of a statistically significant difference.
Results
Time Course of Neointima Formation
Neointima started to form within 5 days after balloon injury and continued to thicken for 8 weeks, with a maximum intima media (I/M) ratio of 2.2 (Figure 1A). From 8 to 12 weeks after injury, neointima formation subsided gradually, with an I/M ratio of 1.5 at 12 weeks after injury. Proliferating SMCs, identified as proliferating cell nuclear antigen (PCNA)–positive cells, first appeared in the adventitia 3 days after injury (Figure 1B). Along with neointima formation, PCNA-positive cells appeared in the neointima and continued to be present until 4 weeks after injury.
Figure 1. Kinetic analysis in rat balloon injury model. A, Time course of neointima formation after injury. B, Representative photomicrographs of PCNA-stained carotid arteries. PCNA-positive cells are shown in brown (bar=100 μm). C, Changes in gene expression after injury. D, EC regeneration after injury as indicated by cGMP production in response to acetylcholine stimulation. Photomicrographs in D show tissue sections stained with hematoxylin-eosin (bar=50 μm).
Changes in Gene Expression after Balloon Injury
Examination of relevant gene expression after balloon injury revealed that along with neointima formation, ?3 integrin gene expression was upregulated 6 hours after injury, followed by an increase in MMP-9 and fibronectin gene expression (Figure 1C). On the other hand, platelet-derived growth factor (PDGF) ? receptor gene expression increased 2 weeks after injury, and collagen type I and MMP-2 gene expression was downregulated during the first 3 days after injury. Endothelial NO synthase (eNOS) gene expression was suppressed just after injury but recovered to normal levels 8 weeks after injury. Previous studies have indicated that fibronectin serves as a ligand for v?3 integrin, whereas MMP-9 cleaves native type IV collagen to yield proteolysed collagen that is known to serve as a ligand for v?3 integrin.17 Together, these findings indicate that the genes that are upregulated before neointima formation constitute ?3 integrin, a potential endogenous ligand–fibronectin and a potential ligand-producing proteinase–MMP-9, supporting the concept that v?3 integrin plays an essential role in neointima formation.
EC Regeneration After Balloon Injury
As shown in Figure 1D (left graph), cGMP production in normal uninjured arteries increased 3- to 9-fold after stimulation with acetylcholine (10–6 mol/L). However, in injured arteries, no significant change in cGMP production was observed during the first 5 weeks after injury, although after the fifth week and up to the 12th week after injury, cGMP production increased 4- to 6-fold (Figure 1D, right graph). Morphological analysis of EC after injury showed that along with neointima formation, ECs were clearly regenerated 4 weeks after injury, although these regenerated ECs were morphologically different (round-shaped) from the normal flat EC (Figure 1D, photomicrographs). The regenerated round-shaped ECs were replaced by flat ECs at 12 weeks after injury. These findings suggest that after balloon injury, ECs require >4 weeks to recover normal function despite their presence, and that recovery of EC normal function coincides with the arrest of neointima formation.
Our results also show that recovery of response to acetylcholine stimulation correlated well with eNOS gene expression in injured arteries, suggesting that eNOS plays a central role in EC function.
Effects of BS-1417 on Neointima Formation in Rats
As shown in Figure 2A, subcutaneous infusion of BS-1417 (0.1, 0.3, and 1 mg/rat per day) for 2 weeks after injury dose-dependently reduced I/M ratio. At the highest dose of 1 mg/rat per day, BS-1417 remarkably reduced I/M ratio by 59% (P<0.001). Further analyses showed that this 59% reduction in I/M ratio was on the plateau level of inhibition of neointima formation (data not shown). In addition, BS-1417, even at the lowest dose of 0.1 mg/rat per day, significantly increased lumen area (Figure 2B) and almost completely inhibited luminal narrowing at 1 mg/rat per day (Figure 2C). Extended analysis revealed that the 59% reduction in I/M ratio caused by treatment with BS-1417 corresponded to a 43% increase in lumen area or 88% inhibition of luminal narrowing. These findings indicate that BS-1417, given subcutaneously for a period of 2 weeks after balloon injury, potently inhibits neointima formation in rats.
Figure 2. Dose-dependent effects of BS-1417 on neointima formation. I/M ratio (A), lumen area (B), and luminal narrowing (C) were measured in the arteries of rats treated subcutaneously with 0.1, 0.3, or 1 mg/rat per day of BS-1417 from 1 day before to 2 weeks after injury. Data represent mean±SEM (A and B) or mean (C). **P<0.01; ***P<0.001 vs saline control.
Long-Term Effects of BS-1417 on Neointima Formation in Rats
As shown in Figure 3A through 3C and Figure II (available online at http://atvb.ahajournals.org), subcutaneous administration of BS-1417 at 7.2 mg/rat per day for 4 weeks after balloon injury potently inhibited neointima formation in rats. This long-term effect of BS-1417 was confirmed for 7 weeks and up to 12 weeks after injury. These results demonstrate that BS-1417, infused continuously at a dose of 7.2 mg/rat per day, potently and stably inhibits neointima formation for up to 12 weeks after balloon injury in rats.
Figure 3. Long-term effects of BS-1417 on neointima formation after injury. I/M ratio (A), lumen area (B), and luminal narrowing (C) were measured in the arteries of rats treated subcutaneously with 7.2 mg/rat per day of BS-1417 from 1 day before to 4, 7, or 12 weeks after injury. Data represent mean±SEM (A and B) or mean (C). **P<0.01; ***P<0.001 vs untreated or saline-treated control. Representative photomicrographs of PCNA-stained carotid arteries (D) from rats treated subcutaneously with 7.2 mg/rat per day of BS-1417 from 1 day before to 4 or 7 weeks after injury. PCNA-positive cells are shown in brown (bar=100 μm).
Next, we compared the number of PCNA-positive cells in BS-1417–treated groups with that in untreated groups at 4 and 7 weeks after injury. At 4 weeks after injury, the number of PCNA-positive cells in BS-1417–treated group was much lower than that in the untreated group, although some PCNA-positive cells were still present (Figure 3D). However, at 7 weeks after injury, there were almost no proliferating PCNA-positive cells in BS-1417–treated and untreated groups, as was shown at 8 weeks after injury in kinetic analysis.
Effects of BS-1417 on Relevant Gene Expression in Injured Arteries
As shown in supplemental Figure III (available online at http://atvb.ahajournals.org), BS-1417 upregulated MMP-9 gene expression at 2 weeks after injury. As for other relevant genes, BS-1417 had minimum or no effect on the expression of v and ?3 integrins, fibronectin, collagen type I, PDGF? receptor, vascular endothelial growth factor receptor, MMP-2, and eNOS genes at any time after injury.
Effects of BS-1417 on EC Regeneration in Injured Arteries
The effects of BS-1417 on EC regeneration were evaluated on the basis of recovery of cGMP production after acetylcholine stimulation in injured arteries. In the 4-week and 12-week experiments, response to acetylcholine stimulation in injured arteries of saline-treated rats was recovered with a 3- to 5-fold increase that brought it to the same level as that in uninjured arteries (Figure 4). BS-1417, given subcutaneously at a dose of 7.2 mg/rat per day, did not inhibit response to acetylcholine stimulation in uninjured arteries but enhanced it in injured arteries. However, this enhancement may not be attributable to enhancement of EC regeneration because as indicated above, BS-1417 had no effect on eNOS gene expression. Next, the effects of BS-1417 on EC regeneration were evaluated morphologically. There was no marked difference in the morphology of regenerated ECs between BS-1417–treated and untreated groups (Figure 3D). These results indicate that BS-1417 has no inhibitory effect on either EC regeneration or EC normal function.
Figure 4. Effect of BS-1417 on acetylcholine-stimulated cGMP production after injury. Acetylcholine-stimulated cGMP production was measured in the arteries of rats treated subcutaneously with 7.2 mg/rat per day of BS-1417 from 1 day before to 4 or 12 weeks after injury.
Withdrawal Study
When treatment with BS-1417 at 7.2 mg/rat per day was withdrawn for 2 weeks after treatment for 2 weeks, catch-up growth of neointima occurred. This catch-up growth resulted in an increase in I/M ratio, indicating larger neointima formation after BS-1417 withdrawal (Figure 5A). However, this larger neointima formation neither decreased lumen area nor increased luminal narrowing (Figure 5B and 5C). Catch-up growth of neointima was also observed when treatment with BS-1417 was withdrawn for 3 weeks after 4 weeks of treatment, whereas it was prevented to a considerable degree when treatment with BS-1417 was withdrawn for 4 weeks after 12 weeks of treatment (Figure 5).
Figure 5. Effects of BS-1417 withdrawal on neointima formation after injury. I/M ratio (A), lumen area (B), and luminal narrowing (C) were measured in the arteries of rats in which treatment with BS-1417 was withdrawn for a period of 2, 3, or 4 weeks after continuous treatment for 2, 4, or 12 weeks after injury, respectively. During treatment, BS-1417 was given subcutaneously at a dose of 7.2 mg/rat per day starting 1 day before injury. Data represent mean±SEM (A and B) or mean (C). **P<0.01 vs untreated or saline-treated control.
Discussion
Previous studies have suggested that v?3 integrin plays an important role in SMC proliferation,9,10 with some reports showing that v?3 integrin protein level increases in the early stage of neointima formation.18,19,20 In the present study, we first performed a detailed kinetic analysis in a rat balloon injury model to clarify the essential roles of v?3 integrin and EC regeneration in neointima formation. Next, we evaluated using in this model the antistenotic effect of BS-1417, a novel v?3 integrin antagonist. In the kinetic analysis, gene expression of v and ?3 integrins was compared with that of various key genes relevant to SMC proliferation. Our results clearly show that after balloon injury, the genes that are upregulated before neointima formation constitute ?3 integrin, a potential endogenous ligand–fibronectin and a potential ligand-producing proteinase–MMP-9, supporting the concept that v?3 integrin plays an essential role in neointima formation. In addition, the present kinetic analysis clearly demonstrates the relationship between neointima formation and EC regeneration. Specifically, our results show that EC regeneration requires >4 weeks to recover normal function despite EC presence, and that recovery of EC normal function coincides with the arrest of neointima formation in the rat balloon injury model. These findings suggest that EC regeneration, especially recovery of EC normal function, plays an important role in preventing neointima formation. In one of our experiments, no EC regeneration was observed even 5 weeks after injury (Figure 1D, right graph), whereas in another experiment, it was observed 4 weeks after injury (Figure 4). From these observations, it is likely that the time required for EC regeneration after balloon injury depends on the degree of damage inflicted by the injury. Thus, in the rat balloon injury model, although the beginning of neointima formation after injury may be reproducible, the end of its progression may vary depending on the time required for ECs to recover normal function.
In a rat balloon injury model, continuous administration of BS-1417 (1 mg/rat per day) for 2 weeks after injury potently inhibited neointima formation with 59% reduction in I/M ratio. Detailed morphometric analysis revealed that this 59% reduction in I/M ratio represented 88% inhibition of luminal narrowing. These results suggest that BS-1417 has a potent antistenotic effect that almost completely prevents neointima formation for 2 weeks after injury. Using a rat balloon injury model, previous studies have commonly examined the antistenotic effects of investigational drugs for a period of 2 weeks after injury with, to our knowledge, only 1 exception, in which the antistenotic effects of a cyclic Arg-Gly-Asp (RGD) peptide v?3 antagonist were investigated for 75 days.21 In the present study, the potent in vivo effects of BS-1417 prompted us to examine its efficacy for >2 weeks after injury. Continuous administration of BS-1417 (7.2 mg/rat per day) for a period of 4, 7, or 12 weeks after injury potently and stably inhibited neointima formation, suggesting that BS-1417 can sustain its potent antistenotic effect for a long period after injury. In addition to our results in the kinetic analysis, the long-term effect of BS-1417 on neointima formation strongly supports the concept that v?3 integrin plays an essential role in neointima formation.
Unexpectedly, continuous administration of BS-1417 at 1.0 mg/rat per day for 4 weeks resulted in catch-up growth of neointima during the latter 2 weeks (data not shown). However, neointima formation and catch-up growth could be prevented when BS-1417 was administrated continuously at 7.2 mg/rat per day for 4 weeks. The reason for this dose discrepancy is still unclear but might be associated with the fact that SMCs were actively proliferating in the media 3 to 5 days after injury, whereas they were active in the intima from 7 days up to 4 weeks after injury (Figure 1B). Thus, it seems that for the first 2 weeks after injury, BS-1417 (1.0 mg/rat per day) inhibits SMC proliferation in the media, whereas for the subsequent weeks, a higher dose of BS-1417 is required (7.2 mg/rat per day) to inhibit SMC proliferation in the intima. The reason for BS-1417 different inhibitory potency of SMC proliferation in the media and intima is still unclear. However, given that ligands for v?3 integrin are more abundant in the intima than in the media, it is plausible that a higher dose of BS-1417 is required to inhibit SMC proliferation in intima than in media.
We also found that withdrawal of treatment with BS-1417 (7.2 mg/rat per day) for 2 weeks after 2 weeks of continuous administration after injury resulted in catch-up growth of neointima. This phenomenon was also observed when BS-1417 was withdrawn after 4 weeks of treatment. In that case, although neointima formation was prevented at 4 weeks after injury, PCNA-positive cells were still present in neointima, and ECs were not completely regenerated. Therefore, it is likely that withdrawal of BS-1417, when PCNA-positive cells are still present in the neointima and ECs are only partially regenerated, results in catch-up growth of neointima. On the other hand, neointima catch-up growth was prevented to a considerable degree when treatment with BS-1417 was withdrawn after 12 weeks of administration after injury, when PCNA-positive cells were scarcely present in neointima and ECs were fully regenerated. Recent experiments with BS-1417 using a mild injury model in which EC regeneration may occur earlier than in the severe injury model used in this study revealed that catch-up growth of neointima is prevented when treatment with BS-1417 is withdrawn after 4 weeks of administration (data not shown). These results suggest that BS-1417 inhibition of neointima formation is associated with regeneration of ECs and recovery of their normal function. In other words, continuous administration of BS-1417, until EC regeneration, can prevent not only neointima formation but also neointima catch-up growth after treatment withdrawal.
In BS-1417 withdrawal experiments, catch-up growth of neointima resulted in an increase in I/M ratio, indicating a larger neointima development after treatment withdrawal. However, this larger neointima development neither decreased lumen area nor increased luminal narrowing. These findings suggest that the larger neointima development, after BS-1417 withdrawal, is associated with expansive remodeling of vessels. Actually, morphological analyses including that performed in this study indicated that BS-1417 substantially enlarges vessel size or internal elastic lamina length, although this enlargement was not always statistically significant (data not shown). Margolin et al21 also reported an expansive remodeling by a cyclic RGD peptide v?3 antagonist in a rat balloon injury model. On the other hand, monoclonal antibody to v?3 in a rabbit balloon injury model showed a dose-dependent constrictive remodeling.22 These inconsistent results may be caused by a difference in species, inhibitors, or treatment regimen. Further studies are needed to clarify the effects of BS-1417 on vascular remodeling. In the present study, we found that treatment with BS-1417 for 2 weeks after injury upregulates MMP-9 gene expression, although we have no direct evidence that BS-1417 increases MMP-9 activity. Recent studies have shown that MMP-9 is implicated in expansive arterial remodeling.23,24 Therefore, given that BS-1417 can cause expansive remodeling, it would be of interest to examine the possible involvement of MMP-9 in expansive remodeling by BS-1417.
In conclusion, the results of our kinetic analysis of neointima formation in the rat balloon injury model support the concept that v?3 integrin plays an essential role in neointima formation and elucidate the preventive role of EC regeneration, especially recovery of EC function, in neointima formation. Using this model, we demonstrated that continuous administration of BS-1417, a novel v?3 integrin antagonist, potently and stably inhibits neointima formation for >4 weeks after injury without affecting EC regeneration. Although withdrawal of treatment with BS-1417 after short-term administration after injury resulted in catch-up growth of neointima, a long-term study suggested that this catch-up growth can be prevented by administration of BS-1417 until EC regeneration. We conclude that BS-1417 has great potential as a systemic drug for the treatment of restenosis after angioplasty.
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Srivatsa SS, Fitzpatrick LA, Tsao PW, Reilly TM, Holmes DR Jr. Schwartz RS, Mousa SA. Selective v?3 integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury: evidence for the functional importance of integrin v?3 and osteopontin expression during neointima formation. Cardiovasc Res. 1997; 36: 408–428.
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Correspondence to Yayoi Honda, Pharmacology and Microbiology Research Laboratories, Dainippon Pharmaceutical Co., Ltd, Enoki 33-94, Suita, Osaka 564-0053, Japan. E-mail yayoi-honda@dainippon-pharm.co.jp
Abstract
Objectives— We performed a detailed kinetic analysis in a rat balloon injury model to clarify the essential roles of v?3 integrin and endothelial cell (EC) regeneration in neointima formation. Using this model, we evaluated the antistenotic effect of Dainippon compound BS-1417, a novel v?3 integrin antagonist.
Methods and Results— Kinetic analysis using RT-PCR showed that v?3 integrin-related genes are upregulated before neointima formation. Morphological and functional analyses revealed that EC regeneration requires >4 weeks after injury, and that recovery of EC normal function coincides with the arrest of neointima formation. Subcutaneous infusion of BS-1417 for 2, 4, 7, or 12 weeks after injury potently inhibited neointima formation without affecting EC regeneration. Although withdrawal of treatment with BS-1417 after short-term administration after injury resulted in catch-up growth of neointima, a long-term study suggested that this catch-up growth can be prevented by continuous administration of BS-1417 until EC regeneration.
Conclusion— We clarified that v?3 integrin and EC regeneration play an essential role in neointima formation, and that continuous administration of BS-1417 potently and stably inhibits neointima formation without affecting EC regeneration. These findings suggest that BS-1417 might be useful as a novel systemic drug for the treatment of restenosis.
We clarified the essential roles ofv?3 integrin and endothelial cell (EC) regeneration in neointima formation using a rat balloon injury model. In this model, we demonstrated that systemic administration of BS-1417, a novelv?3 integrin antagonist, potently and stably inhibits neointima formation without affecting EC regeneration.
Key Words: v?3 integrin antagonist ? endothelial cell regeneration ? vascular smooth muscle ? gene expression ? rat balloon injury model
Introduction
Vascular smooth muscle cell (SMC) migration and proliferation are critical steps in neointima formation after vascular injury1,2 and are regulated by various molecules, including integrins and their extracellular matrix protein ligands, matrix metalloproteinases (MMPs), and growth factors and their receptors.3,4 It is also known that regeneration of endothelial cells (ECs) plays an important role in preventing SMC proliferation.5,6 Given systemically, numerous single molecule drugs for the prevention of neointima formation in animal models have been reported.7 However, these drugs have shown only short-term efficacy in animal models7 and no promising result in clinical trials.7,8
See page 1309
v?3 integrin, also called vitronectin receptor, is expressed on a variety of cell types,9 including SMCs and ECs, and it is known to mediate migration or proliferation of certain cells in disease states, including SMCs in restenosis after vascular injury,10 and ECs and SMCs in angiogenesis.11 Although many cyclic peptide and nonpeptide v?3 integrin antagonists have been reported,12,13 only a few have been shown to potently inhibit neointima formation in rat models.
In preliminary in vitro studies aimed at finding selective inhibitors of SMC proliferation, we found a series of v?3 integrin antagonists that inhibit SMC proliferation without affecting EC proliferation. Based on this finding and the results of a previous study showing that small-molecule v?3 integrin antagonists effectively inhibit neointima formation in rats,14 we focused our efforts on v?3 integrin antagonists and recently found a new series of promising phenylpiperazine-based v?3 integrin antagonists.15 Extensive screening of these novel antagonists using a rat balloon injury model led us to the discovery of BS-1417 (Figure I, available online at http://atvb.ahajournals.org) as a suitable candidate for further development.15
In the present study, we first performed a detailed kinetic analysis of neointima formation in a rat balloon injury model to verify the concept that v?3 integrin plays an essential role in neointima formation. In the kinetic analysis, we also examined the time course of EC regeneration, especially recovery of EC function, to clarify the preventive role of EC regeneration in neointima formation. Then, using the rat balloon injury model, we evaluated the antistenotic effects of BS-1417, a novel v?3 integrin antagonist, for >2 weeks after injury. Finally, we examined the long-term effects of BS-1417 through a treatment withdrawal experiment.
Methods
Balloon Injury
Male Sprague-Dawley rats (Clea Japan, Inc.) weighing 340 to 360 g were used in this study. They were divided into groups based on body weight. Carotid arteries were injured essentially as described by Clowes et al.16 In brief, rats were anesthetized with pentobarbital (50 mg/kg IP), and the left femoral and the left carotid arteries were exposed. A 2F Fogarty balloon catheter (Baxter Healthcare Corporation) was introduced through the left femoral artery and advanced to the left carotid artery until it was recognized at the bifurcation of the common carotid artery. The balloon was inflated with 0.3 mL of air and pulled back to the aortic arch at an approximate speed of 0.3 cm/s. After 3x repetition of this procedure, the catheter was removed, the femoral artery was ligated, and the wounds were closed.
Kinetic Analysis of Rat Balloon Injury Model
Rats were first anesthetized with pentobarbital (50 mg/kg IP) and then exsanguinated at 6 hours or 1, 2, 3, 5, 7, or 10 days, or 2, 3, 4, 5, 8 or 12 weeks after balloon injury. Isolated arteries served for morphometric analysis, immunohistochemical analysis, or measurement of gene expression or cGMP production (see online supplement, available at http://atvb.ahajournals.org).
Evaluation of the Effects of BS-1417 on Neointima Formation
The effects of BS-1417 on neointima formation were evaluated first for a period of 2 weeks (short-term effects) after balloon injury, and then for a period of 4, 7, or 12 weeks (long-term effects) after balloon injury. BS-1417 was infused subcutaneously at doses of 0.1, 0.3, and 1.0 mg/rat per day (2-week experiment) or at a dose of 7.2 mg/rat per day (4-, 7-, and 12-week experiments) starting 1 day before injury using Alzet osmotic pumps (Alza Corporation). The difference in dosage between the 2-week experiment and the 4-, 7-, and 12-week experiments was attributable to the fact that treatment with BS-1417 at 1.0 mg/rat per day for 4 weeks resulted in catch-up growth of neointima during the latter 2 weeks (data not shown). To confirm the effects of BS-1417 on neointima formation, a treatment withdrawal experiment in which BS-1417 (7.2 mg/rat per day) was first infused subcutaneously for a period of 2, 4, or 12 weeks then withdrawn for a period of 2, 3, or 4 weeks, respectively, was carried out. In all experiments, BS-1417 was dissolved in 0.3 mol/L HCl, and the pH was adjusted to 4 with NaOH. Dosing formulations were prepared by appropriate dilution with water or saline. Control rats in all experiments were either untreated rats or rats that underwent the same procedure as those treated with BS-1417, except that saline was substituted for BS-1417.
Evaluation of the Effects of BS-1417 on Relevant Gene Expression
The effects of BS-1417, given subcutaneously at a dose of 7.2 mg/rat per day, on relevant gene expression were evaluated for a period of 6 hours, 1 or 3 days, or 2 or 8 weeks after balloon injury. Genes that were evaluated are listed in supplemental Table I (available online at http://atvb.ahajournals.org).
Evaluation of the Effects of BS-1417 on EC Regeneration
The effects of BS-1417, given subcutaneously at a dose of 7.2 mg/rat per day, on EC regeneration were evaluated for a period of 4 or 12 weeks after balloon injury.
Statistical Analysis
Data are expressed as mean±SEM. Comparison between drug-treated and control groups was performed using the 2-tailed Student’s t test. The short-term effects of BS-1417 were compared in 4 groups using parametric William’s test. A P value <0.05 denoted the presence of a statistically significant difference.
Results
Time Course of Neointima Formation
Neointima started to form within 5 days after balloon injury and continued to thicken for 8 weeks, with a maximum intima media (I/M) ratio of 2.2 (Figure 1A). From 8 to 12 weeks after injury, neointima formation subsided gradually, with an I/M ratio of 1.5 at 12 weeks after injury. Proliferating SMCs, identified as proliferating cell nuclear antigen (PCNA)–positive cells, first appeared in the adventitia 3 days after injury (Figure 1B). Along with neointima formation, PCNA-positive cells appeared in the neointima and continued to be present until 4 weeks after injury.
Figure 1. Kinetic analysis in rat balloon injury model. A, Time course of neointima formation after injury. B, Representative photomicrographs of PCNA-stained carotid arteries. PCNA-positive cells are shown in brown (bar=100 μm). C, Changes in gene expression after injury. D, EC regeneration after injury as indicated by cGMP production in response to acetylcholine stimulation. Photomicrographs in D show tissue sections stained with hematoxylin-eosin (bar=50 μm).
Changes in Gene Expression after Balloon Injury
Examination of relevant gene expression after balloon injury revealed that along with neointima formation, ?3 integrin gene expression was upregulated 6 hours after injury, followed by an increase in MMP-9 and fibronectin gene expression (Figure 1C). On the other hand, platelet-derived growth factor (PDGF) ? receptor gene expression increased 2 weeks after injury, and collagen type I and MMP-2 gene expression was downregulated during the first 3 days after injury. Endothelial NO synthase (eNOS) gene expression was suppressed just after injury but recovered to normal levels 8 weeks after injury. Previous studies have indicated that fibronectin serves as a ligand for v?3 integrin, whereas MMP-9 cleaves native type IV collagen to yield proteolysed collagen that is known to serve as a ligand for v?3 integrin.17 Together, these findings indicate that the genes that are upregulated before neointima formation constitute ?3 integrin, a potential endogenous ligand–fibronectin and a potential ligand-producing proteinase–MMP-9, supporting the concept that v?3 integrin plays an essential role in neointima formation.
EC Regeneration After Balloon Injury
As shown in Figure 1D (left graph), cGMP production in normal uninjured arteries increased 3- to 9-fold after stimulation with acetylcholine (10–6 mol/L). However, in injured arteries, no significant change in cGMP production was observed during the first 5 weeks after injury, although after the fifth week and up to the 12th week after injury, cGMP production increased 4- to 6-fold (Figure 1D, right graph). Morphological analysis of EC after injury showed that along with neointima formation, ECs were clearly regenerated 4 weeks after injury, although these regenerated ECs were morphologically different (round-shaped) from the normal flat EC (Figure 1D, photomicrographs). The regenerated round-shaped ECs were replaced by flat ECs at 12 weeks after injury. These findings suggest that after balloon injury, ECs require >4 weeks to recover normal function despite their presence, and that recovery of EC normal function coincides with the arrest of neointima formation.
Our results also show that recovery of response to acetylcholine stimulation correlated well with eNOS gene expression in injured arteries, suggesting that eNOS plays a central role in EC function.
Effects of BS-1417 on Neointima Formation in Rats
As shown in Figure 2A, subcutaneous infusion of BS-1417 (0.1, 0.3, and 1 mg/rat per day) for 2 weeks after injury dose-dependently reduced I/M ratio. At the highest dose of 1 mg/rat per day, BS-1417 remarkably reduced I/M ratio by 59% (P<0.001). Further analyses showed that this 59% reduction in I/M ratio was on the plateau level of inhibition of neointima formation (data not shown). In addition, BS-1417, even at the lowest dose of 0.1 mg/rat per day, significantly increased lumen area (Figure 2B) and almost completely inhibited luminal narrowing at 1 mg/rat per day (Figure 2C). Extended analysis revealed that the 59% reduction in I/M ratio caused by treatment with BS-1417 corresponded to a 43% increase in lumen area or 88% inhibition of luminal narrowing. These findings indicate that BS-1417, given subcutaneously for a period of 2 weeks after balloon injury, potently inhibits neointima formation in rats.
Figure 2. Dose-dependent effects of BS-1417 on neointima formation. I/M ratio (A), lumen area (B), and luminal narrowing (C) were measured in the arteries of rats treated subcutaneously with 0.1, 0.3, or 1 mg/rat per day of BS-1417 from 1 day before to 2 weeks after injury. Data represent mean±SEM (A and B) or mean (C). **P<0.01; ***P<0.001 vs saline control.
Long-Term Effects of BS-1417 on Neointima Formation in Rats
As shown in Figure 3A through 3C and Figure II (available online at http://atvb.ahajournals.org), subcutaneous administration of BS-1417 at 7.2 mg/rat per day for 4 weeks after balloon injury potently inhibited neointima formation in rats. This long-term effect of BS-1417 was confirmed for 7 weeks and up to 12 weeks after injury. These results demonstrate that BS-1417, infused continuously at a dose of 7.2 mg/rat per day, potently and stably inhibits neointima formation for up to 12 weeks after balloon injury in rats.
Figure 3. Long-term effects of BS-1417 on neointima formation after injury. I/M ratio (A), lumen area (B), and luminal narrowing (C) were measured in the arteries of rats treated subcutaneously with 7.2 mg/rat per day of BS-1417 from 1 day before to 4, 7, or 12 weeks after injury. Data represent mean±SEM (A and B) or mean (C). **P<0.01; ***P<0.001 vs untreated or saline-treated control. Representative photomicrographs of PCNA-stained carotid arteries (D) from rats treated subcutaneously with 7.2 mg/rat per day of BS-1417 from 1 day before to 4 or 7 weeks after injury. PCNA-positive cells are shown in brown (bar=100 μm).
Next, we compared the number of PCNA-positive cells in BS-1417–treated groups with that in untreated groups at 4 and 7 weeks after injury. At 4 weeks after injury, the number of PCNA-positive cells in BS-1417–treated group was much lower than that in the untreated group, although some PCNA-positive cells were still present (Figure 3D). However, at 7 weeks after injury, there were almost no proliferating PCNA-positive cells in BS-1417–treated and untreated groups, as was shown at 8 weeks after injury in kinetic analysis.
Effects of BS-1417 on Relevant Gene Expression in Injured Arteries
As shown in supplemental Figure III (available online at http://atvb.ahajournals.org), BS-1417 upregulated MMP-9 gene expression at 2 weeks after injury. As for other relevant genes, BS-1417 had minimum or no effect on the expression of v and ?3 integrins, fibronectin, collagen type I, PDGF? receptor, vascular endothelial growth factor receptor, MMP-2, and eNOS genes at any time after injury.
Effects of BS-1417 on EC Regeneration in Injured Arteries
The effects of BS-1417 on EC regeneration were evaluated on the basis of recovery of cGMP production after acetylcholine stimulation in injured arteries. In the 4-week and 12-week experiments, response to acetylcholine stimulation in injured arteries of saline-treated rats was recovered with a 3- to 5-fold increase that brought it to the same level as that in uninjured arteries (Figure 4). BS-1417, given subcutaneously at a dose of 7.2 mg/rat per day, did not inhibit response to acetylcholine stimulation in uninjured arteries but enhanced it in injured arteries. However, this enhancement may not be attributable to enhancement of EC regeneration because as indicated above, BS-1417 had no effect on eNOS gene expression. Next, the effects of BS-1417 on EC regeneration were evaluated morphologically. There was no marked difference in the morphology of regenerated ECs between BS-1417–treated and untreated groups (Figure 3D). These results indicate that BS-1417 has no inhibitory effect on either EC regeneration or EC normal function.
Figure 4. Effect of BS-1417 on acetylcholine-stimulated cGMP production after injury. Acetylcholine-stimulated cGMP production was measured in the arteries of rats treated subcutaneously with 7.2 mg/rat per day of BS-1417 from 1 day before to 4 or 12 weeks after injury.
Withdrawal Study
When treatment with BS-1417 at 7.2 mg/rat per day was withdrawn for 2 weeks after treatment for 2 weeks, catch-up growth of neointima occurred. This catch-up growth resulted in an increase in I/M ratio, indicating larger neointima formation after BS-1417 withdrawal (Figure 5A). However, this larger neointima formation neither decreased lumen area nor increased luminal narrowing (Figure 5B and 5C). Catch-up growth of neointima was also observed when treatment with BS-1417 was withdrawn for 3 weeks after 4 weeks of treatment, whereas it was prevented to a considerable degree when treatment with BS-1417 was withdrawn for 4 weeks after 12 weeks of treatment (Figure 5).
Figure 5. Effects of BS-1417 withdrawal on neointima formation after injury. I/M ratio (A), lumen area (B), and luminal narrowing (C) were measured in the arteries of rats in which treatment with BS-1417 was withdrawn for a period of 2, 3, or 4 weeks after continuous treatment for 2, 4, or 12 weeks after injury, respectively. During treatment, BS-1417 was given subcutaneously at a dose of 7.2 mg/rat per day starting 1 day before injury. Data represent mean±SEM (A and B) or mean (C). **P<0.01 vs untreated or saline-treated control.
Discussion
Previous studies have suggested that v?3 integrin plays an important role in SMC proliferation,9,10 with some reports showing that v?3 integrin protein level increases in the early stage of neointima formation.18,19,20 In the present study, we first performed a detailed kinetic analysis in a rat balloon injury model to clarify the essential roles of v?3 integrin and EC regeneration in neointima formation. Next, we evaluated using in this model the antistenotic effect of BS-1417, a novel v?3 integrin antagonist. In the kinetic analysis, gene expression of v and ?3 integrins was compared with that of various key genes relevant to SMC proliferation. Our results clearly show that after balloon injury, the genes that are upregulated before neointima formation constitute ?3 integrin, a potential endogenous ligand–fibronectin and a potential ligand-producing proteinase–MMP-9, supporting the concept that v?3 integrin plays an essential role in neointima formation. In addition, the present kinetic analysis clearly demonstrates the relationship between neointima formation and EC regeneration. Specifically, our results show that EC regeneration requires >4 weeks to recover normal function despite EC presence, and that recovery of EC normal function coincides with the arrest of neointima formation in the rat balloon injury model. These findings suggest that EC regeneration, especially recovery of EC normal function, plays an important role in preventing neointima formation. In one of our experiments, no EC regeneration was observed even 5 weeks after injury (Figure 1D, right graph), whereas in another experiment, it was observed 4 weeks after injury (Figure 4). From these observations, it is likely that the time required for EC regeneration after balloon injury depends on the degree of damage inflicted by the injury. Thus, in the rat balloon injury model, although the beginning of neointima formation after injury may be reproducible, the end of its progression may vary depending on the time required for ECs to recover normal function.
In a rat balloon injury model, continuous administration of BS-1417 (1 mg/rat per day) for 2 weeks after injury potently inhibited neointima formation with 59% reduction in I/M ratio. Detailed morphometric analysis revealed that this 59% reduction in I/M ratio represented 88% inhibition of luminal narrowing. These results suggest that BS-1417 has a potent antistenotic effect that almost completely prevents neointima formation for 2 weeks after injury. Using a rat balloon injury model, previous studies have commonly examined the antistenotic effects of investigational drugs for a period of 2 weeks after injury with, to our knowledge, only 1 exception, in which the antistenotic effects of a cyclic Arg-Gly-Asp (RGD) peptide v?3 antagonist were investigated for 75 days.21 In the present study, the potent in vivo effects of BS-1417 prompted us to examine its efficacy for >2 weeks after injury. Continuous administration of BS-1417 (7.2 mg/rat per day) for a period of 4, 7, or 12 weeks after injury potently and stably inhibited neointima formation, suggesting that BS-1417 can sustain its potent antistenotic effect for a long period after injury. In addition to our results in the kinetic analysis, the long-term effect of BS-1417 on neointima formation strongly supports the concept that v?3 integrin plays an essential role in neointima formation.
Unexpectedly, continuous administration of BS-1417 at 1.0 mg/rat per day for 4 weeks resulted in catch-up growth of neointima during the latter 2 weeks (data not shown). However, neointima formation and catch-up growth could be prevented when BS-1417 was administrated continuously at 7.2 mg/rat per day for 4 weeks. The reason for this dose discrepancy is still unclear but might be associated with the fact that SMCs were actively proliferating in the media 3 to 5 days after injury, whereas they were active in the intima from 7 days up to 4 weeks after injury (Figure 1B). Thus, it seems that for the first 2 weeks after injury, BS-1417 (1.0 mg/rat per day) inhibits SMC proliferation in the media, whereas for the subsequent weeks, a higher dose of BS-1417 is required (7.2 mg/rat per day) to inhibit SMC proliferation in the intima. The reason for BS-1417 different inhibitory potency of SMC proliferation in the media and intima is still unclear. However, given that ligands for v?3 integrin are more abundant in the intima than in the media, it is plausible that a higher dose of BS-1417 is required to inhibit SMC proliferation in intima than in media.
We also found that withdrawal of treatment with BS-1417 (7.2 mg/rat per day) for 2 weeks after 2 weeks of continuous administration after injury resulted in catch-up growth of neointima. This phenomenon was also observed when BS-1417 was withdrawn after 4 weeks of treatment. In that case, although neointima formation was prevented at 4 weeks after injury, PCNA-positive cells were still present in neointima, and ECs were not completely regenerated. Therefore, it is likely that withdrawal of BS-1417, when PCNA-positive cells are still present in the neointima and ECs are only partially regenerated, results in catch-up growth of neointima. On the other hand, neointima catch-up growth was prevented to a considerable degree when treatment with BS-1417 was withdrawn after 12 weeks of administration after injury, when PCNA-positive cells were scarcely present in neointima and ECs were fully regenerated. Recent experiments with BS-1417 using a mild injury model in which EC regeneration may occur earlier than in the severe injury model used in this study revealed that catch-up growth of neointima is prevented when treatment with BS-1417 is withdrawn after 4 weeks of administration (data not shown). These results suggest that BS-1417 inhibition of neointima formation is associated with regeneration of ECs and recovery of their normal function. In other words, continuous administration of BS-1417, until EC regeneration, can prevent not only neointima formation but also neointima catch-up growth after treatment withdrawal.
In BS-1417 withdrawal experiments, catch-up growth of neointima resulted in an increase in I/M ratio, indicating a larger neointima development after treatment withdrawal. However, this larger neointima development neither decreased lumen area nor increased luminal narrowing. These findings suggest that the larger neointima development, after BS-1417 withdrawal, is associated with expansive remodeling of vessels. Actually, morphological analyses including that performed in this study indicated that BS-1417 substantially enlarges vessel size or internal elastic lamina length, although this enlargement was not always statistically significant (data not shown). Margolin et al21 also reported an expansive remodeling by a cyclic RGD peptide v?3 antagonist in a rat balloon injury model. On the other hand, monoclonal antibody to v?3 in a rabbit balloon injury model showed a dose-dependent constrictive remodeling.22 These inconsistent results may be caused by a difference in species, inhibitors, or treatment regimen. Further studies are needed to clarify the effects of BS-1417 on vascular remodeling. In the present study, we found that treatment with BS-1417 for 2 weeks after injury upregulates MMP-9 gene expression, although we have no direct evidence that BS-1417 increases MMP-9 activity. Recent studies have shown that MMP-9 is implicated in expansive arterial remodeling.23,24 Therefore, given that BS-1417 can cause expansive remodeling, it would be of interest to examine the possible involvement of MMP-9 in expansive remodeling by BS-1417.
In conclusion, the results of our kinetic analysis of neointima formation in the rat balloon injury model support the concept that v?3 integrin plays an essential role in neointima formation and elucidate the preventive role of EC regeneration, especially recovery of EC function, in neointima formation. Using this model, we demonstrated that continuous administration of BS-1417, a novel v?3 integrin antagonist, potently and stably inhibits neointima formation for >4 weeks after injury without affecting EC regeneration. Although withdrawal of treatment with BS-1417 after short-term administration after injury resulted in catch-up growth of neointima, a long-term study suggested that this catch-up growth can be prevented by administration of BS-1417 until EC regeneration. We conclude that BS-1417 has great potential as a systemic drug for the treatment of restenosis after angioplasty.
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