Inhibition of Intrinsic Interferon-γ Function Prevents Neointima Formation After Balloon Injury
It is still controversial whether intrinsic interferon (IFN)-γ promotes or attenuates vascular remodeling in hyperproliferative vascular disorders, such as neointima formation after balloon injury. Thus, we investigated whether inhibition of intrinsic IFN-γ function prevents neointima formation. For this purpose, naked DNA plasmid encoding a soluble mutant of IFN-γ receptor α-subunit (sIFNγR; an IFN-γ inhibitory protein) or mock plasmid was injected into the thigh muscle of male Wistar rats 2 days before balloon injury (day −2). sIFNγR gene transfer significantly elevated serum levels of sIFNγR 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. sIFNγR 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-γ signal). Balloon injury also upregulated the key molecules of neointima formation, such as intercellular adhesion molecule-1 and platelet-derived growth factor β-receptor. These changes were suppressed by sIFNγR treatment. In conclusion, it is suggested that intrinsic IFN-γ 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-γ signaling may be a new therapeutic target for prevention of neointima formation of hyperproliferative vascular disorders.
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)-γ signaling.4 Thus, it is suggested that IFN-γ would play a role in SMC proliferation for neoinitma formation.
The pleiotropic cytokine IFN-γ 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 wall6,7 by activating signal transducer and activator of transcription-1 (STAT1) and IFN regulatory factor-1 (IRF-1), the major transcriptional activator mediating IFN-γ signal.5 IFN-γ is a known regulator of intercellular adhesion molecule (ICAM)-18,9 and platelet-derived growth factor (PDGF) β-receptor (PDGFR-β),4,10 molecules that mediate SMC proliferation. However, IFN-γ 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-γ would promote or prevent vascular lesion formation.
Accordingly, the aim of this study was to determine the role of intrinsic IFN-γ in neointima formation after balloon injury. It has been shown that a soluble mutant of IFN-γ receptor α-chain (sIFNγR) inhibits in vivo IFN-γ function and prevents lesion formation of the mouse model of systemic lupus erythematodes.12,13 In the present study, naked plasmid DNA encoding sIFNγR was injected into the thigh muscle to transfer the sIFNγR gene and to secrete the overexpressed sIFNγR protein into the systemic circulation as a means to block the binding of IFN-γ onto the receptors of the target cells and, subsequently, to inhibit the IFN-γ–induced signaling in the remote organs.
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).
The VICAL VR1255 plasmid vector encoding the extracellular portion of mouse IFN-γ receptor α-chain fused to mouse IgG1 Fc-fragment under control of the cytomegalovirus immediate–early enhancer/promoter (sIFNγR 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 sIFNγR protein to inhibit the actions of IFN-γ in vitro and in vivo was determined as described elsewhere.12,13
sIFNγR Gene Transfer
Gene transfer of sIFNγR 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, sIFNγR 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 sIFNγR gene transfer, we examined the expression of sIFNγR 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 sIFNγR 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-γ receptor α-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-γ receptor α-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 sIFNγR 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.
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 ×200 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.
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-β26 are shown in the Table. The PCR primer pairs for IFN-γ 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.
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.
Efficiency of sIFNγR Gene Transfer Into the Thigh Muscle
Immunofluorescence analysis was performed in the thigh muscle to determine sIFNγR protein expression at the injection site of the sIFNγR 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 sIFNγR gene transfer. Muscle degeneration, tissue necrosis, and inflammation were not observed at the injection site.
Next, we examined the temporal changes in serum sIFNγR protein levels to determine the secretion of the overexpressed sIFNγR protein into the systemic circulation. Immunoreactive sIFNγR was scarcely detected in intact rats. A single sIFNγR gene transfer increased serum sIFNγR levels with a peak 2 to 9 days after the injection, and sIFNγR was detected in the serum at least for 16 days (Figure 1B). Thus, sIFNγR 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-γ mRNA expression was insignificant in the intact artery. IFN-γ expression was upregulated by balloon injury at day 7 and declined thereafter (Figure 2B).
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-γ–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 sIFNγR Treatment on Neointima Formation
There were no significant differences in heart rate, blood pressure, or body weight between mock- and sIFNγR-treated rats during the observation period (data not shown). No rats showed apparent adverse effects of sIFNγR treatment.
sIFNγR treatment remarkably prevented neointima formation after balloon injury (Figure 3A). The minimum dose of injected sIFNγR plasmid to induce the maximum inhibition was 350 μg (Figure 3B). At day 14, 350 μg of sIFNγR 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 sIFNγR-treated rats (data not shown).
The effects of sIFNγR 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%. sIFNγR treatment reduced intimal PCNA+ SMCs, resulting in 50% reduction in the PCNA labeling index. Moreover, sIFNγR treatment reduced the number of phosphorylated STAT1+ SMCs in the neointima at day 7. The phosphorylated STAT1 labeling index was significantly smaller in sIFNγR-treated rats than in mock-treated rats (Figure 4B).
We examined the effects of sIFNγR treatment on SMC apoptosis using the TUNEL method. There was no difference in the number of TUNEL+ SMCs in the neointima between mock- and sIFNγR-treated rats at day 7 (data not shown).
Effects of sIFNγR Treatment on Gene Expression
Sham rats did not show significant expressions of IRF-1, ICAM-1, and PDGFR-β in the carotid artery (Figure 5). In mock-treated rats, balloon injury elicited upregulations of IRF-1 ICAM-1, and PDGFR-β at day 7, which were prevented by sIFNγR treatment.
In the present study, it is likely that the interference with the IFN-γ receptor by increased circulating sIFNγR 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-β in mock-treated rats. sIFNγR treatment inhibited these changes. Thus, it is suggested that intrinsic IFN-γ promotes neointima formation after balloon injury probably through the IRF-1/ICAM-1–mediated and PDGFR-β–mediated mechanisms.
sIFNγR Gene Transfer
The IFN-γ receptor is composed of 2 ligand-binding α-chains associated with 2 signal-transducing β-chains.5 sIFNγR, containing the extracellular portion of the IFN-γ receptor α-chain, acts as a competitive inhibitor for the IFN-γ receptor on the target cell surface.12 Indeed, it has been shown that overexpression of sIFNγR is a safe and effective tool to investigate the roles of IFN-γ in the pathogenesis of experimental models of inflammatory diseases.12,13 Thus, we used sIFNγR as a means to interfere with the interaction of intrinsic IFN-γ with its receptor in this study. As shown in Figure 1, the sIFNγR gene was successfully incorporated into the transduced myocytes, and the overexpressed sIFNγR protein was secreted into systemic circulation for ≥16 days. Accordingly, in the present study, rats received a single sIFNγR gene transfer 2 days before balloon injury.
Role of Intrinsic IFN-γ in Balloon-Injured Artery
Earlier studies showed that neointima formation was reduced by systemic administration of recombinant IFN-γ27 or by local IFN-γ gene overexpression in the carotid artery,28 suggesting that IFN-γ would inhibit neointima formation. However, Zohlnhofer et al4 have demonstrated that the injury-induced neointima formation is inhibited in the IFN-γ α-receptor–deficient mice, indicating a crucial role of IFN-γ in neointima formation. Thus, there was discrepancy among the studies. Accordingly, in this study, we examined the effects of postnatal blocking of the IFN-γ receptor by sIFNγR to determine the causal relationship between intrinsic IFN-γ and neointima formation.
The present study demonstrated that IFN-γ 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-γ through STAT1 activation (Figure 5). These findings suggest that the IFN-γ–mediated signaling pathway is activated in the neointima. Moreover, sIFNγR treatment prevented neointima formation by inhibiting SMC proliferation (Figures 3 and 4⇑A). This result is consistent with the study by Zohlnhofer et al4 using mice in which IFN-γ pathway is genetically ablated. The present study has provided the notion that, in addition to the genetic ablation, postnatal blocking of the IFN-γ receptor can inhibit neointima formation by suppressing intrinsic IFN-γ function.
sIFNγR treatment suppressed both STAT1 phosphorylation and IRF-1 induction (Figures 4B and 5⇑), suggesting that sIFNγR interrupted the IFN-γ–mediated signaling in the injured artery. There are several possible mechanisms whereby sIFNγR 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-γ induces IRF-1 expression and, in turn, IRF-1 activates transcription of the target genes of IFN-γ, 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 sIFNγR treatment. Thus, it is suggested that IRF-1–mediated ICAM-1 induction would be one of the mechanisms whereby IFN-γ promotes neointima formation.
Another mechanism may be the attenuation of PDGFR-β 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-γ potentiates the PDGF-BB–induced SMC proliferation by upregulating PDGFR-β,10,30 although IFN-γ does not have a direct proliferative effect on cultured SMCs.11 As shown in Figure 5, PDGFR-β expression was induced in the injured artery and was suppressed by sIFNγR treatment. This finding may suggest that the IFN-γ–induced PDGFR-β upregulation would participate in neointima formation.
Enhanced SMC apoptosis is a possible mechanism to prevent neointimal formation.22 However, in our study, sIFNγR 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 sIFNγR treatment prevented neointima formation in this model. Finally, sIFNγR 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.
The present study has provided the first evidence suggesting that inhibition of intrinsic IFN-γ represses STAT1 activation and inductions of IRF-1, ICAM-1, and PDGFR-β in the balloon-injured artery, which results in the prevention of neointima formation. Thus, it is suggested that inhibition of the IFN-γ–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-γ, a local delivery system of the recombinant sIFNγR protein using a drug-eluting stent might be desirable.
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.).
- Received December 22, 2006.
- Revision received January 12, 2007.
- Accepted January 24, 2007.
Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004; 75: 163–189.
Harvey EJ, Ramji DP. Interferon-γ and atherosclerosis: pro- or anti-atherogenic? Cardiovasc Res. 2005; 67: 11–20.
Jaruga B, Hong F, Kim WH, Gao B. IFN-gamma/STAT1 acts as a proinflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines and adhesion molecules: A critical role of IRF-1. Am J Physiol Gastrointest Liver Physiol. 2004; 287: G1044–G1052.
Yokota T, Shimokado K, Kosaka C, Sasaguri T, Masuda J, Ogata J. Mitogenic activity of interferon gamma on growth-arrested human vascular smooth muscle cells. Arterioscler Thromb. 1992; 12: 1393–1401.
Hansson GK, Jonasson L, Holm J, Clowes MM, Clowes AW. Gamma-interferon regulates vascular smooth muscle proliferation and Ia antigen expression in vivo and in vitro. Circ Res. 1988; 63: 712–719.
Tahara N, Kai H, Niiyama H, Mori T, Sugi Y, Takayama N, Yasukawa H, Numaguchi Y, Matsui H, Okamura K, Imaizumi T. Repeated gene transfers of naked prostacyclin synthase plasmid into skeletal muscles attenuate monocrotaline-induced pulmonary hypertension and prolong survival in rats. Hum Gene Ther. 2004; 15: 1270–1278.
Kai H, Griendling KK, Lassegue B, Ollerenshaw JD, Runge MS, Alexander RW. Agonist-induced phosphorylation of the vascular type 1 angiotensin II receptors. Hypertension. 1994; 24: 523–527.
Kai H, Griendling KK, Lassègue B, Fukui T, Minieri CA, Alexander RW. Prolonged exposure to agonist results in a reduction in the levels of the Gq/G11 α-subunit in cultured vascular smooth muscle cells. Mol Pharmacol. 1996; 49: 96–104.
Yasukawa H, Imaizumi T, Matsuoka H, Nakashima A, Morimatsu M. Inhibition of intimal hyperplasia after balloon injury by antibodies to ICAM-1. Circulation. 1997; 98: 1515–1522.
Seki Y, Kai H, Shibata R, Nagata T, Yasukawa H, Yoshimura A, Imaizumi T. Roles of JAK/STAT pathway in rat carotid artery remodeling after vascular injury. Circ Res. 2000; 87: 12–18.
Shibata R, Kai H, Seki Y, Kato S, Morimatsu M, Kaibuchi K, Imaizumi T. Role of Rho-associated kinase in neointima formation after vascular injury. Circulation. 2001; 103: 284–289.
Kuwahara F, Kai H, Tokuda K, Kai M, Takeshita A, Egashira K, Imaizumi T. Transforming growth factor-β function blocking prevents myocardial fibrosis and diastolic dysfunction in pressure-overloaded rats. Circulation. 2002; 106: 130–135.
Egwuagu CE, Sztein J, Mahdi RM, Li W, Chao-Chan C, Smith JA, Charukamnoetkanok P, Chepelinsky AB. IFN-γ increases the severity and accelerates the onset of experimental autoimmune uveitis in transgenic rats. J Immunol. 1999; 162: 510–517.
Hansson GK, Holm J. Interferon-gamma inhibits arterial stenosis after injury. Circulation. 1991; 84: 1266–1272.
Ribault S, Neuville P, Mechine-Neuville A, Auge F, Parlakian A, Gabbiani G, Paulin D, Calenda V. Chimeric smooth muscle–specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy. Circ Res. 2001; 88: 468–475.
Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev. 1999; 79: 1283–1316.
Sata M. Role of circulating vascular progenitors in angiogenesis, vascular healing, and pulmonary hypertension: Lessons from animal models. Arterioscler Thromb Vasc Biol. 2006; 26: 1008–1014.