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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 5, 2002 as doi:10.1096/fj.02-0094fje. |
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Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; and
* Discovery Research Laboratories, Tanabe Seiyaku Co., Tokyo, Japan
2Correspondence: Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyushu University, 3–1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: egashira{at}cardiol.med.kyushu-u.ac.jp
SPECIFIC AIMS
No prior study has addressed the definite role of monocyte chemoattractant protein-1 (MCP-1) in restenosis (neointimal hyperplasia) after coronary intervention. The aim of the present study is to test the hypothesis that MCP-1-mediated inflammation is essential in the development of restenotic changes after balloon injury in rats and monkeys.
PRINCIPAL FINDINGS
1. Development of neointimal formation associated with increased expression of MCP-1 and CCR2 after balloon injury in rats
Balloon injury of the right common carotid arteries of male Wistar-Kyoto rats and male adult cynomolgus monkeys was performed three times by passages of an infiltrated Fogarty balloon catheter. Significant fibromuscular neointimal hyperplasia was observed 28 days after balloon injury. We detected ED1-positive monocytes, CD3-positive T lymphocytes, and PCNA-positive cells in the intima and media (Fig. 1
). These inflammatory and proliferative changes persisted mainly in the neointima at 28 days.
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The MCP-1 and CCR2 mRNA levels were undetectably low in uninjured left carotid artery. Both MCP-1 and CCR2 mRNA levels significantly increased as early as day 3, but declined with time; however, gene expression of MCP-1 and CCR2 in the injured artery was significantly higher on days 3, 7, and 28 than that in the uninjured control artery. Immunostaining showed that MCP-1 was undetectable in the uninjured control artery, whereas intense MCP-1 immunoreactivity was evident mainly in the neointima and media 7 days and in the neointima 28 days after balloon injury.
2. Blockade of MCP-1 reduces neointimal hyperplasia in rats and monkeys
We have devised a strategy for anti-MCP-1 gene therapy by transfecting mutant MCP-1 gene into skeletal muscle. This mutant MCP-1 lacks the amino-terminal amino acid 2 to 8, called 7ND, and has been shown to work as a dominant-negative inhibitor of MCP-1. We have shown that the cells infected with 7ND secrete 7ND protein into the circulating blood and the 7ND protein binds to the MCP-1 receptor on monocytes or target cells in remote organs, thus blocking the signal of MCP-1. Such blockade of MCP-1 activity would suppress MCP-1-mediated inflammation and improve the function of the target organs. This method (intramuscular transfection of the gene) is useful because direct gene transfer into the injured arterial wall is not necessary and definitive roles of MCP-1 can be investigated under pathophysiological conditions in vivo.
In rats, both inflammation (ED1-positive cells) and proliferation (the PCNA index) were markedly less in 7ND-transfected rats than in empty plasmid-transfected rats at 7 and 28 days (Fig. 1)
. In contrast, the number of CD3-positive cells did not differ between the two groups. 7ND gene transfer significantly reduced the neointimal hyperplasia (increases in neointimal area and intima/media ratio) 28 days after balloon injury (Fig. 1)
. 7ND gene transfer markedly reduced the increases in MCP-1 and CCR2 gene expression and in MCP-1 immunoreactivity (Fig. 1)
. In monkeys, significant fibromuscular neointimal hyperplasia was noted 28 days after balloon injury, and 7ND gene transfer markedly reduced the neointimal formation (Fig. 2
).
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3. Tissue chemokine and cytokine concentrations
We measured carotid artery tissue concentrations of various cytokines and chemokines 7 and 28 days after balloon injury. Tissue concentrations of MCP-1, but not CINC-1 (the rat homologue of IL-8), were higher in the empty plasmid group than in the normal control group. Other cytokines (TNF-
, IL-1ß, IL-6, IL2, IL-4, IL-10, and INF-
) also were higher in the empty plasmid group than in the normal control group. 7ND gene transfer partly but significantly reduced the increase in tissue MCP-1 concentrations. 7ND gene transfer did not affect the increase in IL-2, IL-4, IL-6, and INF-, but did partly reduce the increases in TNF-, IL-1ß, and IL-10 levels.
4. Plasma concentrations of MCP-1 and 7ND plasma
MCP-1 concentrations did not change during the course of experiments, whereas 7ND was detected in plasma 3, 7, and 14 days after transfection.
CONCLUSIONS AND SIGNIFICANCE
Percutaneous coronary intervention, widely used in dilating flow-limiting coronary atherosclerotic stenosis, is hampered by restenosis in >40% of patients. Until now, only intravascular radiation and rapamycin-eluting stents have offered hope to patients with restenosis. Therefore, prevention of restenosis is a major challenge, which highlights the need of new therapeutic options. Neointimal hyperplasia is an essential stage in the development of restenosis after coronary intervention as well as atherosclerosis. Recent evidence implies a central role of chronic inflammation (recruitment and activation of monocytes) in atherogenesis. The common inflammation-driving factor after arterial injury is MCP-1, a member of the C-C chemokine family. However, no study had addressed the definite role of MCP-1 in the development of restenosis after balloon injury. Therefore, we hypothesized that inflammation mediated by MCP-1 might contribute to restenosis. We have demonstrated here for the first time the definite role of inflammatory responses mediated by MCP-1 in the development of restenotic changes after balloon injury. Our present data strongly suggest that the MCP-1-mediated vascular inflammatory response is strikingly important in neointimal formation after balloon injury.
A rapid and persistent increase in plasma MCP-1 within the first day of coronary angioplasty is reported in patients with restenosis. Persistent increase in MCP-1 after angioplasty was shown to be statistically significant and an independent predictor of restenosis. We have demonstrated persistent increases in gene expression of MCP-1 and CCR2 as well as in protein production of MCP-1 after balloon injury in rats. 7ND gene transfer markedly attenuated early inflammatory and proliferative changes, and later, neointimal hyperplasia, indicating the definite role of MCP-1 in neointimal hyperplasia after balloon injury (Fig. 1)
. Activated monocytes may serve as a source of growth factors and cytokines. Our present data, therefore, indicate that locally produced MCP-1 not only induced the recruitment of monocytes but also activated lesional monocytes and vascular smooth muscle cells to produce the inflammatory cytokines, which might in turn caused neointimal hyperplasia. Thus, MCP-1-mediated inflammation in the arterial wall is likely to create the positive feedback mechanism to enhance inflammation and proliferation of injured arterial wall (Fig. 3
). Although we did not determine the mechanism underlying the activation of the MCP-1 pathway after balloon injury, it is plausible to speculate that increased oxidative stress, redox-sensitive transcription factors such as nuclear factor-
B might have contributed to the increased MCP-1 expression.
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The outstanding effects (
70% inhibition) of 7ND gene transfer in rats and monkeys imply that this gene transfer strategy can be a novel therapy against human restenosis. Our finding in nonhuman primates seems meaningful, because many therapeutic strategies effective in reducing restenosis in nonprimate animal models have failed to demonstrate substantial effect on human restenosis. The present study is the first to demonstrate the suppression of restenotic changes after balloon injury in primates.
In conclusion, inflammatory changes mediated by MCP-1 are essential in the development of restenotic changes (neointimal formation) after balloon injury. This study may support the hypothesis that inflammation may play central part in the pathogenesis of restenosis. Finally, this strategy may be a useful form of gene therapy against human restenosis after coronary intervention. Future study requires careful observation over a long period of time to establish the true risk:benefit ratio.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0094fje; to cite this article, use FASEB J. (September 5, 2002) 10.1096/fj.02-0094fje 
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