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(Hypertension. 2007;49:909.)
© 2007 American Heart Association, Inc.

Original Articles

Inhibition of Intrinsic Interferon- Function Prevents Neointima Formation After Balloon Injury

Ken Kusaba; Hisashi Kai; Mitsuhisa Koga; Narimasa Takayama; Ayami Ikeda; Hideo Yasukawa; Yukihiko Seki; Kensuke Egashira; Tsutomu Imaizumi

From the Department of Internal Medicine (K.K., H.K., M.K., N.T., A.I., Y.S., T.I.), Division of Cardio-vascular Medicine, Kurume University School of Medicine, Kurume, Japan; the Department of Pharmaceutical Care and Health Sciences (M.K.), Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan; the Cardiovascular Research Institute (H.Y.), Kurume University, Kurume, Japan; and the Department of Cardiovascular Medicine (K.E.), Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.

Correspondence to Hisashi Kai, Department of Internal Medicine, Division of Cardio-vascular Medicine, Kurume University School of Medicine, 67 Asahimachi, Kurume 830-0011, Japan. E-mail naikai{at}med.kurume-u.ac.jp

	Abstract

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It is still controversial whether intrinsic interferon (IFN)- promotes or attenuates vascular remodeling in hyperproliferative vascular disorders, such as neointima formation after balloon injury. Thus, we investigated whether inhibition of intrinsic IFN- function prevents neointima formation. For this purpose, naked DNA plasmid encoding a soluble mutant of IFN- receptor -subunit (sIFNR; an IFN- inhibitory protein) or mock plasmid was injected into the thigh muscle of male Wistar rats 2 days before balloon injury (day –2). sIFNR gene transfer significantly elevated serum levels of sIFNR protein for 2 weeks. In mock-treated rats, balloon injury induced smooth muscle cell proliferation in the neointima with a peak at day 7 and produced thick neointima at day 14. sIFNR treatment reduced the number of proliferating intimal smooth muscle cells by 50% at day 7 and attenuated neointima formation with a 45% reduction of the intima/media area ratio at day 14. In mock-treated rats, at day 7, balloon injury induced phosphorylation of signal transducer and activator of transcription-1 and upregulations of IFN regulatory factor-1 (a transcription factor mediating IFN- signal). Balloon injury also upregulated the key molecules of neointima formation, such as intercellular adhesion molecule-1 and platelet-derived growth factor ß-receptor. These changes were suppressed by sIFNR treatment. In conclusion, it is suggested that intrinsic IFN- promotes neointima formation probably through IFN regulatory factor-1/intercellular adhesion molecule-1–mediated and platelet-derived growth factor–mediated mechanisms. Thus, inhibition of IFN- signaling may be a new therapeutic target for prevention of neointima formation of hyperproliferative vascular disorders.

Key Words: IFN- • neointima • gene therapy • inflammation • signaling

	Introduction

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Neointima formation, predominantly consisting of smooth muscle cell (SMC) proliferation, is a major pathological feature of hyperproliferative vascular disorders, such as atherosclerosis and restenosis after percutaneous coronary intervention.¹ The primary event in the development of hyperproliferative vascular disorders is the injury to the endothelium, which induces SMC migration from the media to the intima and subsequent proliferation.^2,3 However, the mechanism of neoinitma formation has not been fully understood. Recently, a transcriptome analysis has revealed that in atherectomy specimens obtained from restenotic coronary lesions, one third of 223 differentially expressed genes are related to the activation of interferon (IFN)- signaling.⁴ Thus, it is suggested that IFN- would play a role in SMC proliferation for neoinitma formation.

The pleiotropic cytokine IFN- is a key proinflammatory mediator.⁵ This cytokine is expressed at high levels in vascular lesions and regulates functions and properties of all of the cell types in vascular wall^6,7 by activating signal transducer and activator of transcription-1 (STAT1) and IFN regulatory factor-1 (IRF-1), the major transcriptional activator mediating IFN- signal.⁵ IFN- is a known regulator of intercellular adhesion molecule (ICAM)-1^8,9 and platelet-derived growth factor (PDGF) ß-receptor (PDGFR-ß),^4,10 molecules that mediate SMC proliferation. However, IFN- also has anti-inflammatory actions, such as interleukin-8 induction, in a range of cell types⁷ and antiproliferative action in cultured SMCs.¹¹ Thus, it has remained undetermined whether intrinsic IFN- would promote or prevent vascular lesion formation.

Accordingly, the aim of this study was to determine the role of intrinsic IFN- in neointima formation after balloon injury. It has been shown that a soluble mutant of IFN- receptor -chain (sIFNR) inhibits in vivo IFN- function and prevents lesion formation of the mouse model of systemic lupus erythematodes.^12,13 In the present study, naked plasmid DNA encoding sIFNR was injected into the thigh muscle to transfer the sIFNR gene and to secrete the overexpressed sIFNR protein into the systemic circulation as a means to block the binding of IFN- onto the receptors of the target cells and, subsequently, to inhibit the IFN-–induced signaling in the remote organs.

	Methods

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All of the procedures were approved by the Animal Care and Treatment Committee of Kurume University and were conducted in conformity with institutional guidelines. Male Wistar rats (350 to 400 g) were purchased from SLC Inc (Shizuoka, Japan).

sIFNR Plasmid
The VICAL VR1255 plasmid vector encoding the extracellular portion of mouse IFN- receptor -chain fused to mouse IgG1 Fc-fragment under control of the cytomegalovirus immediate–early enhancer/promoter (sIFNR plasmid)^12,13 was a generous gift of Dr Gérald J. Prud’homme (McGill University, Montreal, Canada). All of the sequences were confirmed by double-stranded DNA sequencing. The plasmid DNA was amplified, purified with an endotoxin-free purification kit (Quiagen), and stored at –20°C until use. The ability of the overexpressed sIFNR protein to inhibit the actions of IFN- in vitro and in vivo was determined as described elsewhere.^12,13

sIFNR Gene Transfer
Gene transfer of sIFNR using the naked DNA method was performed as described previously.^14,15 Briefly, under ether anesthesia, 12.5 µg/g of bupivacaine (AstraZeneca) was injected into the thigh muscle to improve the efficiency of gene introduction.¹⁶ Two days after bupivacaine injection, sIFNR plasmid solved in PBS (final concentration, 1 µg/µL) was injected at the same site of the muscle where bupivacaine had been injected.

To verify the efficiency of sIFNR gene transfer, we examined the expression of sIFNR protein in the thigh muscle 5 days after gene transfer. The cryosections were subjected to immunofluorescence using an FITC-labeled monoclonal antibody against mouse Fc-fragment (Santa Cruz Inc). Next, we determined the temporal changes in serum sIFNR levels. At denoted days, blood was drawn from the right atrium just before perfusion fixation under anesthesia with 30 mg/kg of IP pentobarbital. Aliquots of the serum (500 µL) were subjected to immunoprecipitation using a monoclonal antibody against the extracellular portion of the mouse IFN- receptor -chain (Santa Cruz), as described previously.¹⁷ The immunoprecipitants were separated by 7.5% SDS-PAGE and blotted onto polyvinyliodine difluoride membrane, as described previously.¹⁸ Blots were probed with the same anti–IFN- receptor -chain antibody used for immunoprecipitation. The signals were detected and analyzed by the chemiluminescence detection system (Pierce Biotechnology) according to the manufacturer’s instruction.

Balloon Injury Model and Study Protocol
Two days before balloon injury (day –2), denoted dose of sIFNR or mock plasmid (the empty VR1255 plasmid) was injected into the bupivacaine-pretreated thigh muscle, as described above. At day 0, rats were anesthetized with sodium pentobarbital (50 mg/kg), and then balloon injury of the left carotid artery was performed as described previously.^19,20 Sham rats underwent the same operation without balloon insertion. At denoted days, the rat was killed with an overdose of pentobarbital, and then the carotid artery was fixed by perfusion with 4% paraformaldehyde at 100 mm Hg, excised, and embedded in paraffin. In each rat, neointima formation was examined at 3 individual hematoxylin/eosin-stained sections (6 µm in thickness), each separated by 180 µm (n=5 per group at each time point).²¹ The medial and intimal areas were measured with a computerized digital image analysis system and averaged in 3 independent sections.

Immunohistostaining
The paraffinized sections were subjected to immunohistostaining using the primary antibody against proliferating cell nuclear antigen (PCNA; Dako) or phosphorylated STAT1 (Assay BioTechnology) and a commercially available detection system (Dako), according to the manufacturer’s instructions. PCNA⁺ cells and phosphorylated STAT1⁺ cells were counted in 3 independent sections at x200 magnification in each rat (n=5 per group) by 3 observers in a blind manner.^21,22 The values obtained by the 3 observers were averaged in each section. The ratio of PCNA⁺ SMCs over total nucleated SMCs in the intima was expressed as the PCNA labeling index. The ratio of intimal SMCs with STAT1⁺ nucleus was evaluated in the same way. For the same specimen, apoptotic cells were detected by the TUNEL method using Apop Tag (Intergen).²² TUNEL⁺ intimal SMCs were counted, and the TUNEL index was calculated, being described as the PCNA labeling index.

RT-PCR Analysis
After the mouse was euthanized and perfused with ice-cold PBS, the carotid arteries (n=8 per group) were removed and snap frozen in dry ice/acetone and stored at –80°C until use. Two frozen samples of the carotid arteries were homogenized in TRIzol (Invitrogen Corp) using a FastPrep homogenizer (ThermoSavant). The total RNA was extracted and reverse transcribed by using first-strand reaction mix beads (Amersham Biosciences).²³ Aliquots of the reverse-transcription products were amplified using KOD-plus (Toyobo) and primer pairs, according to the manufacturer’s instructions. The primer nucleotide sequences and amplification conditions for rat IRF-1,²⁴ ICAM-1,²⁵ and PDGFR-ß²⁶ are shown in the Table. The PCR primer pairs for IFN- and GAPDH were purchased from Biosource International and Applied Biosystems, respectively. The RT-PCR products were electrophoresed on a 2% agarose gel stained with SYBR Gold (Invitrogen). The resulting bands were scanned and analyzed using a digital image analyzer. Expression level of the target gene was normalized by GAPDH level in each sample.

View this table: [in this window] [in a new window]   PCR Primer Sequences and Amplification Conditions

Statistical Analysis
Statistical analysis was performed by unpaired Student’s t test or ANOVA followed by Scheffé’s F test. A value of P<0.05 was considered significant. The interobserver or intraobserver variability was <5% in each experiment.

	Results

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Efficiency of sIFNR Gene Transfer Into the Thigh Muscle
Immunofluorescence analysis was performed in the thigh muscle to determine sIFNR protein expression at the injection site of the sIFNR plasmid. As shown in Figure 1A, the fluorescence signals (green color) were detected in 40% to 50% of striate muscle cells in the thigh muscle 5 days after sIFNR gene transfer. Muscle degeneration, tissue necrosis, and inflammation were not observed at the injection site.

View larger version (38K): [in this window] [in a new window]   Figure 1. Efficiency of sIFNR gene transfer. A, Representative microphotographs of light microscopy (left) and immunofluoroscopy against the mouse IgG Fc-fragment (right) of the thigh muscle 5 days after an injection with the sIFNR plasmid. Striate muscle cells expressing sIFNR protein are stained as green. B, Representative immunoblot showing the temporal changes in serum sIFNR protein levels after a single sIFNR gene transfer. Intact indicates intact rats without gene transfer. Three independent studies showed the similar results.

Next, we examined the temporal changes in serum sIFNR protein levels to determine the secretion of the overexpressed sIFNR protein into the systemic circulation. Immunoreactive sIFNR was scarcely detected in intact rats. A single sIFNR gene transfer increased serum sIFNR levels with a peak 2 to 9 days after the injection, and sIFNR was detected in the serum at least for 16 days (Figure 1B). Thus, sIFNR gene transfer was performed only once 2 days before balloon injury (day –2) throughout the following experiments.

Neointima Formation After Balloon Injury
In mock-treated rats, balloon injury induced neointima formation and marked neointimal thickening developed at day 14 (Figure 2A). IFN- mRNA expression was insignificant in the intact artery. IFN- expression was upregulated by balloon injury at day 7 and declined thereafter (Figure 2B).

View larger version (69K): [in this window] [in a new window]   Figure 2. Neointima formation and IFN- induction. A, Representative microphotographs of hematoxylin/eosin-stained cross-sections of the carotid arteries 7 and 14 days after balloon injury. The similar results were obtained from 5 independent experiments. B, Representative photographs of electrophoresis of RT-PCR products for IFN- (a) and pooled data showing the temporal changes in IFN- mRNA levels (b) in the injured carotid arteries. IFN- expression level was normalized by GAPDH level in each sample. Bar=1xSD (n=4). *P<0.05 and P<0.05 vs sham and day 7, respectively. C, Representative microphotographs of immunohistostaining against PCNA and phosphorylated STAT1 (pSTAT1) in the injured arteries at day 7 (n=5). The nuclei of the PCNA+ proliferating cells are stained as brown. pSTAT1 typically shows the nuclear localization (arrowheads). Sham indicates sham-operated rats.

As we reported previously,^21,22 PCNA labeling revealed that neointimal SMC proliferation peaked at day 7 (Figure 2C). STAT1 phosphorylation was investigated for the activation of IFN-–mediated signaling at day 7. STAT1 phosphorylation was not found in the carotid artery of sham rats (Figure 2C). In mock-treated rats, phosphorylated STAT1⁺ SMCs were increased mainly in the neointima.

Effects of sIFNR Treatment on Neointima Formation
There were no significant differences in heart rate, blood pressure, or body weight between mock- and sIFNR-treated rats during the observation period (data not shown). No rats showed apparent adverse effects of sIFNR treatment.

sIFNR treatment remarkably prevented neointima formation after balloon injury (Figure 3A). The minimum dose of injected sIFNR plasmid to induce the maximum inhibition was 350 µg (Figure 3B). At day 14, 350 µg of sIFNR plasmid significantly reduced the intima/media area ratio by 45% (Figure 3C). The intima and media remained intact in the right intact carotid artery of sIFNR-treated rats (data not shown).

View larger version (50K): [in this window] [in a new window]   Figure 3. Effects of sIFNR treatment on neointima formation. A, Representative microphotographs of hematoxylin/eosin-stained cross-sections of the carotid arteries 14 days after balloon injury (n=5). sIFNR or mock plasmid (350 µg) was injected in the thigh muscle 2 days before balloon injury. B, The minimum dose of sIFNR plasmid to induce the maximum reduction in intima/media area ratio at day 14 was 350 µg. C, Pooled data of the effects of sIFNR () or mock () treatment (350 µg) on neointima formation at day 14. Bar=1xSD (n=5). *P<0.05 and **P<0.01 vs mock treatment, respectively.

The effects of sIFNR treatment on intimal SMC proliferation were evaluated at day 7 (Figure 4A). In mock-treated rats, the PCNA labeling index in the neointima was 60%. sIFNR treatment reduced intimal PCNA⁺ SMCs, resulting in 50% reduction in the PCNA labeling index. Moreover, sIFNR treatment reduced the number of phosphorylated STAT1⁺ SMCs in the neointima at day 7. The phosphorylated STAT1 labeling index was significantly smaller in sIFNR-treated rats than in mock-treated rats (Figure 4B).

View larger version (22K): [in this window] [in a new window]   Figure 4. Effects of sIFNR treatment on neointimal cell proliferation and STAT1 phosphorylation. Pooled data show the effects of sIFNR treatment on PCNA labeling index (A) and pSTAT1 labeling index (B) of the intima. Sham indicates sham-operated rats. Bar=1xSD (n=5). *P<0.05 and P<0.05 vs sham and mock-treated rats, respectively.

We examined the effects of sIFNR treatment on SMC apoptosis using the TUNEL method. There was no difference in the number of TUNEL⁺ SMCs in the neointima between mock- and sIFNR-treated rats at day 7 (data not shown).

Effects of sIFNR Treatment on Gene Expression
Sham rats did not show significant expressions of IRF-1, ICAM-1, and PDGFR-ß in the carotid artery (Figure 5). In mock-treated rats, balloon injury elicited upregulations of IRF-1 ICAM-1, and PDGFR-ß at day 7, which were prevented by sIFNR treatment.

View larger version (37K): [in this window] [in a new window]   Figure 5. Gene expression analysis. Representative photographs of electrophoresis of RT-PCR products for IRF-1, ICAM-1, and PDGFR-ß in the injured arteries at day 7 (A). Pooled data showing the effects of sIFNR treatment on IRF-1 (B), ICAM-1 (C), and PDGFR-ß (D) mRNA levels in the injured arteries. Expression level of the target gene was normalized by GAPDH level in each sample. Bar=1xSD (n=4). *P<0.05 and P<0.05 vs sham and mock, respectively.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, it is likely that the interference with the IFN- receptor by increased circulating sIFNR level was responsible for the reduction in neointimal SMC proliferation and prevention of neoinitma formation after balloon injury. Balloon injury induced phosphorylation of STAT1 and upregulations of IRF-1, ICAM-1, and PDGFR-ß in mock-treated rats. sIFNR treatment inhibited these changes. Thus, it is suggested that intrinsic IFN- promotes neointima formation after balloon injury probably through the IRF-1/ICAM-1–mediated and PDGFR-ß–mediated mechanisms.

sIFNR Gene Transfer
The IFN- receptor is composed of 2 ligand-binding -chains associated with 2 signal-transducing ß-chains.⁵ sIFNR, containing the extracellular portion of the IFN- receptor -chain, acts as a competitive inhibitor for the IFN- receptor on the target cell surface.¹² Indeed, it has been shown that overexpression of sIFNR is a safe and effective tool to investigate the roles of IFN- in the pathogenesis of experimental models of inflammatory diseases.^12,13 Thus, we used sIFNR as a means to interfere with the interaction of intrinsic IFN- with its receptor in this study. As shown in Figure 1, the sIFNR gene was successfully incorporated into the transduced myocytes, and the overexpressed sIFNR protein was secreted into systemic circulation for 16 days. Accordingly, in the present study, rats received a single sIFNR gene transfer 2 days before balloon injury.

Role of Intrinsic IFN- in Balloon-Injured Artery
Earlier studies showed that neointima formation was reduced by systemic administration of recombinant IFN-²⁷ or by local IFN- gene overexpression in the carotid artery,²⁸ suggesting that IFN- would inhibit neointima formation. However, Zohlnhofer et al⁴ have demonstrated that the injury-induced neointima formation is inhibited in the IFN- -receptor–deficient mice, indicating a crucial role of IFN- in neointima formation. Thus, there was discrepancy among the studies. Accordingly, in this study, we examined the effects of postnatal blocking of the IFN- receptor by sIFNR to determine the causal relationship between intrinsic IFN- and neointima formation.

The present study demonstrated that IFN- was upregulated in the balloon-injured artery at day 7 (Figure 2B) when neointimal SMC proliferation is peaked in this model.^21,22 Concurrently, the nuclear localization of phosphorylated STAT1 was observed in neointimal SMCs (Figure 2C). In addition, balloon injury induced IRF-1 expression, which is known to be upregulated by IFN- through STAT1 activation (Figure 5). These findings suggest that the IFN-–mediated signaling pathway is activated in the neointima. Moreover, sIFNR treatment prevented neointima formation by inhibiting SMC proliferation (Figures 3 and 4A). This result is consistent with the study by Zohlnhofer et al⁴ using mice in which IFN- pathway is genetically ablated. The present study has provided the notion that, in addition to the genetic ablation, postnatal blocking of the IFN- receptor can inhibit neointima formation by suppressing intrinsic IFN- function.

Possible Mechanisms
sIFNR treatment suppressed both STAT1 phosphorylation and IRF-1 induction (Figures 4B and 5), suggesting that sIFNR interrupted the IFN-–mediated signaling in the injured artery. There are several possible mechanisms whereby sIFNR treatment prevents neointima formation. One of the possible mechanisms is the suppression of ICAM-1 induction in the injured artery (Figure 5). Our previous study demonstrated that ICAM-1 was upregulated in neointimal and medial SMCs after vascular injury and that an anti-ICAM-1 neutralizing antibody prevented neointima formation.¹⁹ These findings suggest an important role of ICAM-1 in neointima formation after vascular injury. To examine the upstream of ICAM-1, we analyzed the upregulation of IRF-1. IFN- induces IRF-1 expression and, in turn, IRF-1 activates transcription of the target genes of IFN-, including ICAM-1.^8,9 As shown in Figure 5, IRF-1 and ICAM-1 were concurrently upregulated in the injured artery, which was inhibited by sIFNR treatment. Thus, it is suggested that IRF-1–mediated ICAM-1 induction would be one of the mechanisms whereby IFN- promotes neointima formation.

Another mechanism may be the attenuation of PDGFR-ß induction. The importance of PDGF-BB in neointima formation has been established in the balloon injury model. PDGF-BB plays roles in SMC migration, proliferation, and synthesis of the extracellular matrix.²⁹ There is increasing evidence that IFN- potentiates the PDGF-BB–induced SMC proliferation by upregulating PDGFR-ß,^10,30 although IFN- does not have a direct proliferative effect on cultured SMCs.¹¹ As shown in Figure 5, PDGFR-ß expression was induced in the injured artery and was suppressed by sIFNR treatment. This finding may suggest that the IFN-–induced PDGFR-ß upregulation would participate in neointima formation.

Enhanced SMC apoptosis is a possible mechanism to prevent neointimal formation.²² However, in our study, sIFNR treatment did not affect SMC apoptosis in the injured artery (data not shown). Thus, it was unlikely that the enhancement of SMC apoptosis contributed to the mechanism whereby sIFNR treatment prevented neointima formation in this model. Finally, sIFNR protein was secreted into the systemic circulation for long time. Thus, we were not able to exclude the possibility of the involvement of indirect systemic effects, such as the reduction in mobilization of vascular smooth muscle progenitor cells.³¹ Future study should address this issue.

Perspectives
The present study has provided the first evidence suggesting that inhibition of intrinsic IFN- represses STAT1 activation and inductions of IRF-1, ICAM-1, and PDGFR-ß in the balloon-injured artery, which results in the prevention of neointima formation. Thus, it is suggested that inhibition of the IFN-–mediated pathway may be a new target for prevention and treatment of hyperproliferative vascular disorders, such as neointima formation after balloon injury. Although no apparent adverse effects were observed during the period of this study, a careful observation is necessary over longer periods before the consideration for clinical application. To avoid possible adverse effects related to immunosuppression induced by systemic inhibition of IFN-, a local delivery system of the recombinant sIFNR protein using a drug-eluting stent might be desirable.

	Acknowledgments

 
We appreciate Miyuki Ouchida, Kaoru Moriyama, Yayoi Yoshida, Miho Kogure, and Kimiko Kimura for their skillful technical assistance.

Sources of Funding

This study was supported in part by a grant for Science Frontier Research Promotion Centers and Grants-in-Aid for Scientific Research (H.K., Y.S., T.I.) from the Ministry of Education, Science, Sports and Culture, Japan, and by a research grants from Kimura Memorial Heart Foundation (T.I.).

Disclosures

None.

Received December 22, 2006; first decision January 12, 2007; accepted January 24, 2007.

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