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Human Molecular Genetics, 2003, Vol. 12, No. 2 177-187
© 2003 Oxford University Press

A central role of interferon regulatory factor-1 for the limitation of neointimal hyperplasia

Rainer Wessely1,*, Ludger Hengst2, Birgit Jaschke1, Franziska Wegener1, Thomas Richter3, Raffaella Lupetti2, Makarios Paschalidis1, Albert Schömig1, Richard Brandl4 and Franz-Josef Neumann1

1Deutsches Herzzentrum and 1. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität, Munich, Germany, 2Max Planck Institut für Biochemie, Martinsried, Germany, 3Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar, Technische Universität, Munich, Germany and 4Abteilung für Gefässchirurgie, Klinikum rechts der Isar, Technische Universität, Munich, Germany

Received September 30, 2002; Accepted November 15, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Neointima formation, the leading cause of restenosis after catheter angioplasty, is a paradigm for vascular proliferative responses. Neointima formation is self-limiting after a variable degree of tissue growth, causing significant renarrowing in a substantial number of patients. To investigate the mechanisms that limit neointima formation we studied the role of the transcription factor IRF-1, which is a regulator of interferons and a tumor suppressor. We demonstrate that IRF-1 is highly regulated in human vascular lesions and exhibits a growth inhibitory function in coronary artery smooth muscle cells (CASMC). IRF-1 deficient mice display a high grade of susceptibility towards neointima formation following vessel injury. IRF-1 leads to G1 cell cycle arrest in CASMC and induces the CDK inhibitor p21. In addition, IRF-1 induces NO production, which is known to attenuate endothelial dysfunction. Mitogen-mediated cellular migration is abrogated by IRF-1. In conclusion, IRF-1 displays pleiotropic anti-restenotic activities in vascular restenosis through transcriptional activation of several relevant mechanisms that limit neointima formation. These findings suggest an important role of this transcription factor as an endogenous inhibitor of neointimal growth following vessel injury and it is likely that IRF-1 regulation also plays a role in the pathophysiology of primary atherosclerosis. In addition, IRF-1 may be an interesting target for interventions to prevent neointimal hyperplasia.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Coronary artery disease is the most common cause of cardiac morbidity and mortality in industrial countries (1). The use of interventional cardiovascular procedures has expanded beyond all expectations due to considerable technical and procedural progress within the last decade. Currently, more than one million procedures are performed each year (2) and the use of coronary stents is widespread and increasing. A major limitation of invasive procedures is the development of restenosis in the dilated vessel, usually within 6 months after the procedure. Despite the fact that stenting may decrease restenosis following percutaneous coronary intervention (3), the majority of adverse outcomes after coronary interventions is due to in-stent restenosis.

In-stent restenosis is due to neointimal hyperplasia (4). More than 90% of the cellular compartment of the neointima consists of migrated and proliferated coronary artery smooth muscle cells (CASMC) originating from the tunica media (5).

In-stent restenosis develops within a complex pathophysiological background. Several independent and partially redundant pathways may lead to neointima formation (for review see 6). In the clinical setting, there are well-defined risk factors which increase the probability of restenosis. These include diabetes mellitus and arterial hypertension (7). However, the majority of patients develop restenosis despite reasonable therapy of treatable risk factors. Therefore, genetic risk factors may play the most important role in the disease development (7). To further elucidate the pathophysiology of restenosis, to predict the restenotic risk for an individual patient and to identify new therapeutic targets, it is important to identify and characterize genes critically involved in CASMC migration and proliferation which may be critical in the positive or negative regulation of vascular restenosis.

The transcription factor interferon regulatory factor 1 (IRF-1) was originally characterized as a regulator of the interferon beta gene family (8). During recent years there has been growing evidence that IRF-1 encompasses a broader biological spectrum and may act as a tumor suppressor, even in the absence of interferon (9). Recently, Horiuchi et al. (10) reported that IRF-1 plays a role in regulating cell proliferation of smooth muscle cells: the protein is downregulated in proliferating cells and upregulated in quiescence. In addition, IRF-1 can directly bind and activate the NOS2 promoter, thus potentially supporting restoration of endothelial function following vessel injury (11). Therefore, we aimed to investigate if this transcription factor is involved in neointima formation.

The intention of this study was to analyze the significance of IRF-1 as a potential inhibitor of neointima formation in vivo in restenotic human lesions harvested during vessel surgery and in a murine model of neointimal hyperplasia. Furthermore, we sought to investigate the effect of IRF-1 on landmark characteristics of neointima formation in a cell culture model of low passage human coronary smooth muscle cells: influence on mitogen induced proliferation and migration, the induction of NO and interference with cell cycle and its G1 regulatory proteins. Some of these mechanisms have been characterized in different, mainly transformed cell lines but have not been proven to exist in coronary artery smooth muscle cells. Finally, we examined the effect of recombinant overexpression of IRF-1 to inhibit neointima formation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IRF-1 is consistently induced in human neointima and colocalizes with p21
To characterize IRF-1 expression in human neointima we collected 20 histological specimens harvested during vascular thromboendatherectomy due to symptomatic restenosis. The samples included carotid (n=16) and femoral lesions (n=4). Tissues were stained against IRF-1, p21 and the S-phase marker KI-67. In 85% of the samples (17/20), IRF-1 staining was detectable. This is in accordance with the number of human atherectomy samples derived from restenotic coronary arteries that were positive for IRF-1 mRNA expression examined by microarray analysis (12). IRF-1 was particularly upregulated in cell-rich areas with low extracellular matrix content (Fig. 1A). KI-67 staining was rare and could be detected occasionally in smooth muscle cells within cell rich lesions (Fig. 1C). The pattern of p21 expression was similar to IRF-1 (Fig. 1B). The CDK inhibitor colocalized with IRF-1 in cell-rich lesions. IRF-1 and p21 staining in matrix-rich regions with low cellular content was rare, KI-67 could not be detected in these lesion areas (data not shown). Neointimal cells staining positive for IRF-1 were predominantly smooth muscle cells as confirmed by immunostaining against smooth muscle actin (Fig. 1D). These data suggest a role for IRF-1 as an important factor involved in the endogenous limitation of smooth muscle cell proliferation in vivo.



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  		Figure 1. p21 expression in non-proliferating smooth muscle cells from human neointima samples is conferred to IRF-1 expressing cells. Immunohistochemistry was performed in cell-rich lesions (A–D, serial sections) and matrix rich lesions with only limited cellular content (not shown), respectively. The majority of cells with a smooth muscle cell phenotype stained positive for IRF-1 in lesions with high cellular content (A). Smooth muscle phenotype was confirmed by staining against smooth muscle actin (D). p21 expression was conferred to IRF-1 expressing cells (B). KI-67 as a proliferation marker stained only occasionally or not at all in these lesions, as displayed in (C). IRF-1 was not found to be coexpressed with KI-67. The inserted image in the upper right corner of (C) shows a positive control for KI-67 staining. Matrix rich lesions with low cellular compound stained mainly negative for both IRF-1 and p21 suggesting already abrogated proliferation. This was confirmed by complete lack of KI-67 staining in these areas. Staining with DAB appear brownish, Fast Red stained cells are stained in red colour. Magnification 100x. Number of positively staining cells with smooth muscle phenotype in cell rich lesion areas: IRF-1, 78.7±10.2%; p21, 55.3±9.2%, Ki67 1.0±1.1%.

 
IRF-1 is critical for the limitation of neointima formation in vivo: IRF-1 knockout mice display increased neointima formation following vessel injury
Since IRF-1 upregulation correlated with elevated p21 expression and decreased proliferation, we wished to determine if loss of the IRF-1 tumor suppressor gene enhances neointima formation. To elucidate the role of IRF-1 in restenosis following vascular injury, we analyzed IRF-1 knockout mice. Twenty-eight days after the induction of flow cessation in the mouse common carotid artery, histomorphometry was performed 600 µm proximal the ligation side. Quantitative morphometry revealed a significant increase of neointima formation in IRF-1 deficient mice compared with wild-type controls with identical genetic background (Fig. 2), supporting a key role of IRF-1 in limiting restenosis.



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  		Figure 2. Increase of neointima formation in IRF-1 deficient mice following vessel injury. (A) Intima/media area ratio±standard error of the mean. *P<0,01, n=6 each group; (B) non-injured common carotid artery; (C) representative injured carotid artery of a wild-type mouse; (D) representative slide of an injured carotid artery derived from a carotid artery of an animal with identical genetic background lacking the IRF-1 gene. Animals were sacrificed 28 days post surgery.

 
IRF-1 induces cell cycle arrest in coronary artery smooth muscle cells
Having identified IRF-1 as an inhibitor of neointimal hyperplasia in vivo we sought to determine the mechanism how IRF-1 acts on human coronary artery smooth muscle cells (CASMC). CASMC were synchronized by serum withdrawal. Twenty-four hours prior to mitogen stimulation, cells were transduced by the appropriate adenovirus vector, encoding for either IRF-1 (Ad-IRF-1) or as a control lacZ (Ad-lacZ). There was a significant decrease of BrdU incorporation in IRF-1 overexpressing cells compared with Ad-lacZ or non-virally transduced cells (Fig. 3A). Cell cycle analysis by propidium iodine flow cytometry revealed cell cycle arrest by IRF-1 in the G0/G1 phase (Fig. 3B). Interestingly, at the time point of flow cytometry analysis, no significant upregulation of interferon ß in CASMC supernatant could be detected (data not shown), implying a direct mechanism of IRF-1-induced cell cycle inhibition.



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  		Figure 3. Molecular effects of ectopic expression of IRF-1 in cultured human coronary artery smooth muscle cells. (A) Significant decrease of BrdU incorporation of IRF-1 transduced CASMC 24 h and 48 h post mitogen stimulation (*P<0.01). There is no significant alteration in BrdU incorporation between lacZ transduced and control cells (P=n.s. denotes not significant). (B) Propidium iodine flow cytometry for cell cycle analysis of CASMC. Left peak denotes G0/1 phase, the blue intermedium S phase, right peak G2/M phase. The right panels display Ad-lacZ infected cells 6 h (top) and 36 h (bottom) after mitogen stimulation. The left panels show Ad-IRF-1 infected cells 6 h (top) and 36 h (bottom) after mitogen stimulation. (C) Cyclin A kinase assay. Lane 1, mitogen stimulated CASMC; lane 2, quiescent cells; lanes 3 and 4, Ad-IRF-1 MOI 10 and 100 infected cells; lanes 5 and 6, Ad-lacZ MOI 10 and 100 infected cells. There is a dose-dependent decrease of cyclin A-mediated histone H1 phosphorylation in IRF-1 transduced cells. (D) NO ELISA assay showing a significant increase of NO production in IRF-1 transduced CASMC (*P<0.05). The experiments were repeated twice with similar results.

 
IRF-1 leads to CDK inactivation and induces the CDK inhibitor p21
The cyclin A/cdk2 kinase complex is activated in late G1 at the G1/S transition (13) and can be inhibited by cycle dependent kinase inhibitors of the cip/kip family (14). Therefore, considering the cell cycle inhibitory effect of IRF-1, cyclin A/cdk2 activity may be repressed by IRF-1. To investigate this hypothesis, synchronized CASMC were infected 24 h prior to mitogen stimulation. Twenty-four hours after mitogen stimulation, cellular protein was isolated and kinase activities were determined. The activity of immunoprecipitated cyclin A/cdk2 was measured in vitro against the substrate histone H1. As shown in Fig. 3C, IRF-1 overexpression leads to a significant dose-dependent decrease of cyclin A-associated histone H1 kinase activity. IRF-1 overexpression may therefore lead to CDK inactivation, resulting in cell cycle arrest of CASMC.

CDK inactivation and subsequent growth arrest can be accomplished by a number of mechanisms. In G1 control, regulation by CDK inhibitory proteins (CKIs) plays a prominent role. Cip/Kip proteins like p21 and p27 can bind to and inhibit a broad spectrum of CDK complexes. A single molecule of p21 inactivates cyclin A/Cdk2 kinase (14). p21 is a transcriptional target of the tumor suppressor p53. Since p53 and IRF-1 share common DNA-binding motives (15), we speculated that p21 might also be directly induced by IRF-1 in CASMC. It was demonstrated recently that IRF-1 can induce p21 in Friend leukemia cells (16). Further, p21 overexpression has been proven to be effective in limiting neointima formation in the rat carotid injury model (17). Tanaka et al. (18) were able to show that p21 is regulated in response to DNA damage by both p53 and IRF-1 in mouse embryonic fibroblasts. Therefore we wished to determine whether IRF-1 was able to directly induce the p21 promoter in differentiated, non-transformed coronary artery smooth muscle cells. A construct containing a firefly luciferase encoding gene under the control of the p21 promoter sequence was cotransfected with either a plasmid vector encoding for IRF-1 or lacZ as a control into CASMC. As shown in Fig. 4A, there was a significant induction of luciferase expression in IRF-1 transfected CASMC as compared with the lacZ control. To confirm these findings, western blotting was performed following recombinant IRF-1 expression in CASMC. Again, compared with the controls, there was an elevated p21 expression level detectable when IRF-1 was overexpressed in CASMC (Fig. 4B). Therefore, p21 induction may be responsible for CDK inactivation as observed in IRF-1 transduced cells (Fig. 3C).



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  		Figure 4. p21 is induced by IRF-1 both in vitro and in vivo. (A) Induction of luciferase under the control of a p21 reporter gene by an IRF-1 encoding plasmid vector. *P<0.01 by ANOVA two way analysis. (B) Western blotting of a CASMC lysate using a p21 antibody (top) or an IRF-1 antibody (bottom) 48 h after infection with either Ad-IRF-1 or Ad-lacZ, respectively. Lane 1, non-virally transduced cells; lane 2, Ad-lacZ infection; lane 3, Ad-IRF-1 infection. IRF-1 overexpressing cells reveal elevated levels of p21. (C) Induction of p21 by IRF-1 following recombinant gene transfer of IRF-1 into the vessel wall. The right panel displays an immunohistological staining of an IRF-1 transduced rat carotid artery 3 days after recombinant gene transfer of IRF-1 (brown DAB staining) into the vessel wall. The left panel shows colocalization with p21 (Fast Red red staining). In control tissues, either sham or Ad-lacZ transduced, there was neither staining of IRF-1 nor p21 detectable at this time point (data not shown).

 
IRF-1 induces NO synthesis in CASMCs
Nitric oxide (NO) was identified in numerous studies as an important molecule in vascular biology which may improve endothelial function (19) and lead to cell cycle arrest in smooth muscle cells (20). Overexpression of inducible NO-synthase (NOS2) significantly limits neointima formation following vascular injury (21). It has been shown previously that IRF-1 transcriptionally binds to and activates the NOS2 promoter (11), however, it is not known whether IRF-1 may induce NO synthesis in coronary artery smooth muscle cells. Compared with non-virally transduced and Ad-lacZ infected cells, we could detect a significant increase in NO production following IRF-1 overexpression (Fig. 3D).

Inhibition of mitogen induced smooth muscle cell migration by IRF-1
Cell migration is a key pathophysiological event in restenosis. To investigate whether enhanced IRF-1 expression has an effect on mitogen-induced CASMC migration we used a Boyden chamber assay. This assay measures haptotaxis induced by a fibronectin gradient. Twenty-four hours after plating Ad-IRF-1 or as a control Ad-lacZ-transduced cells into the Boyden chamber, cells on the bottom side of the membrane were stained and 10 random 100x power fields counted on an inverted microscope. There was a highly significant decrease of migration when cells were transduced by IRF-1 (IRF1, 17±3.4 cells, lacZ 67.8±8.8; P<0.01). The experiment was repeated twice with similar results.

Overexpression of IRF-1 in vivo leads to significant limitation of neointima formation
Since IRF-1 displays potent anti-proliferative and anti-migratory effects in human coronary artery smooth muscle cells, recombinant overexpression of IRF-1 may have therapeutic relevance by limiting the formation of restenotic lesions. To determine whether overexpression of IRF-1 is sufficient to limit neointima formation in vivo, recombinant adenovirus was delivered into the lumen of previously injured rat common carotid arteries. Gene transfer efficacy was monitored by ß-galactosidase staining of Ad-lacZ transduced arteries. Compared with Ad-lacZ, there was a significant decrease in neointima formation in IRF-1 treated arteries (Fig. 5). p21 expression was examined at day 3 following intraluminal gene transfer by immunohistochemistry. Consistently, IRF-1 overexpressing cells showed enhanced p21 levels (Fig. 4c), whereas non-IRF-1 expressing cells lacked p21 expression. Therefore, overexpression of IRF-1 is able to attenuate neointima formation following vascular injury probably through induction of p21. This supports the hypothesis that endogenous expression of IRF-1 is important to limit smooth muscle cell proliferation and thus neointima formation in vivo.



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  		Figure 5. Overexpression of IRF-1 via recombinant gene transfer into the vessel wall following balloon injury leads to significant decrease of neointima formation 14 days after surgery. (A) Mean intima to media ratio of injured vessels as measured by histomorphometry, *P<0.05. (B) An uninjured vessel. (C) A representative histological slide of a lacZ transduced carotid artery. (D) An IRF-1 transduced vessel. Media areas were not significantly different in both groups.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Atherosclerosis and restenosis following percutaneous vascular intervention are common diseases, especially in industrialized countries. To fully understand the pathology and to appreciate why only a subgroup of patients are affected by these diseases, it is essential to identify protective genes which are endogenously activated to limit neointima formation. These genes may also serve as potential therapeutic targets to prevent neointima forma-tion and may account for genetic disposition for restenosis.

IRF-1 was initially characterized as a transcription factor involved in interferon beta regulation. Recently, IRF-1 was found to regulate proliferation and differentiation of various cell types and the gene was characterized as a tumor suppressor (for review see 9). IRF-1 is a critical determinant of oncogene-induced cell transformation or apoptosis (22) and has been demonstrated to play a role in the regulation of DNA repair (23). Loss of the IRF-1 gene, which maps to chromosome 5q31.1, can be frequently found in human leukemia and preleukemic myelodysplastic syndromes (24). A recent report suggests that there may be a loss of IRF-1 expression in human breast cancer (25). Loss of IRF-1 increases tumor susceptibility in mice carrying the Ha-ras oncogene or in animals deficient of the tumor suppressor gene p53 (26). On the other hand, IRF-1 is able to suppress growth of hepatocellular carcinoma cells and is regarded as a potential candidate for gene therapy of hepatocellular carcinoma (27).

The importance of IRF-1 as a tumor suppressor in eukaryotic cells is underscored by the finding that IRF-1 and p53 share a common transcriptional regulatory element (15), indicating that they may regulate some common effectors. Overexpression of p53 in VSMC leads to cell cycle arrest in G1 phase and inhibits mitogen induced cell proliferation (28). A key target of p53 induced G1 arrest is p21. Since we also observed growth arrest after IRF-1 overexpression we sought to determine whether the antiproliferative effect of IRF-1 includes the induction of p21. Accordingly, human atherectomy samples were immunostained to characterize the expression pattern of IRF-1, p21 and the S-phase marker KI-67. We found IRF-1 upregulation in 85% of the specimens. IRF-1 stained predominantly in cell rich lesion areas and colocalized frequently with p21. KI-67 was rarely detected in these regions suggesting inhibited cell proliferation. There was no colocalization of IRF-1 and KI-67. IRF-1 level, frequency and colocalization with p21 implies a substantial anti-proliferative role for IRF-1 in vascular smooth muscle cells. Therefore, the molecular and cellular effects of recombinant IRF-1 overexpression were examined in this particular cell type.

IRF-1 was found to be consistently upregulated in quiescent smooth muscle cells of human neointima. To elucidate the significance of IRF-1 in limiting neointima formation, we analyzed a model of IRF-1 deficiency. Accordingly, we utilized IRF-1-/- mice and applied the carotid flow cessation model to induce neointima formation. This model represents an established vascular injury model in mice (29,30). As in humans, the development of neointima in mice following vessel injury is highly dependent on the genetic background (31). For example, FVB mice display a high degree of neointima formation following carotid ligation. However, wild-type C57BL/6J mice as they were utilized in our studies do not necessarily develop a high degree of neointima after vascular injury (32). Since we expected an increase in neointima formation in case of a deficiency in IRF-1 expression, we decided to use mice with the C57BL/6J background that display a low degree of neointima formation in the wild-type scenario. Consistent with our hypothesis, IRF-1-/- mice revealed a pronounced increase in neointima formation compared with wild-type C57BL/6J mice. These data demonstrate that endogenous IRF-1 has potent growth inhibitory effects in vivo following vessel injury. This finding may not only be crucial for the pathophysiology and therapy of restenosis, but may also be important for clinical risk stratification for restenosis. Since there are known polymorphisms in the IRF-1 gene (33), a polymorphism in the IRF-1 gene may be one of the candidates responsible for genetic susceptibility to restenosis. Therefore, it would be challenging to identify various polymorphisms and their prognostic value in order to identify patients with increased risk for coronary restenosis.

IRF-1 is a pluripotent transcription factor. Its anti-tumor effects are present even in the absence of interferons (34). In fact, we were never able to demonstrate significant interferon beta levels in CASMC overexpressing IRF-1 by ELISA. Inhibition of mitogen-induced coronary vascular smooth muscle cell proliferation by IRF-1 as demonstrated here is based on several partially independent mechanisms. Our data demonstrate an IRF-1 induced growth arrest of CASMC in G0/G1. The activity of a major G1/S-Kinase, cyclin A/cdk2 was inhibited by IRF-1 in a dose-dependent manner. It was shown previously that p21 efficiently inactivates cyclin A/cdk2 kinase (14). In transformed leukaemia cells, it has been demonstrated that IRF-1 may induce p21 (16). In primary cultured coronary artery smooth muscle cells, we could confirm that p21 is induced by IRF-1. Immunohistochemical analysis of p21 expression in IRF-1 transduced rat carotid arteries revealed enhanced p21 expression in IRF-1 overexpressing cells. In human atherectomy samples we also found a consistent pattern of p21 expressing cells in IRF-1 expression areas. Accordingly, in regions with low or no detectable IRF-1 expression, p21 expression was also rare or non detectable. Thus, it could be demonstrated that induction of p21 by IRF-1 is an important mechanism in the IRF-1 induced G1 arrest.

An additional mechanism by which IRF-1 may induce cell cycle arrest in coronary artery smooth muscle cells is the induction of NO. It is known that IRF-1 can bind to the NOS2 (inducible nitric oxide synthase) promoter (11), thus promoting NO release. In the heart, IRF-1 has been found to be a major regulator of NO release following inflammatory heart disease (35). Nitric oxide is capable of inducing cell cycle arrest in various cell types (20) and is known to improve endothelial function (36). We could show that IRF-1 overexpression induces NO in CASMC. Since NO may limit neointima formation following vascular injury (21), IRF-1 mediated NO release may contribute to the inhibition of restenosis.

Interestingly, IRF-1 was not only able to inhibit CASMC proliferation but was sufficient to inhibit CASMC migration towards a fibronectin gradient in a Boyden chamber system. Besides the inhibition of CASMC proliferation and the re-establishment of endothelial function, abrogating cellular migration is believed to be essential to prevent coronary restenosis. The precise mechanism remains elusive; however, it is known that IRF-1 can bind to the VCAM-1 promoter (37) and thus regulate integrin expression. Alternatively, CKI of the cip/kip family have been shown to interfere with smooth muscle cell migration (38). Therefore, induction of p21 may also be responsible for the inhibition of CASMC migration by IRF-1.

We were able to show that IRF-1 interferes with every single component of the triad of mechanisms that are necessary to prevent restenosis: inhibition of SMC proliferation, inhibition of SMC migration and potential improvement of endothelial function through NO induction. The IRF-1-mediated inhibition of mitogen-induced proliferation blocks the cell cycle in G1, a mechanism which is believed to have excellent therapeutic value because of its ultimate position in proliferative cellular signalling pathways (39). To verify the hypothesis that overexpression of IRF-1 in the vessel wall attenuates neointima formation, we applied the rat carotid injury model. We could demonstrate that overexpression of IRF-1 leads to significant reduction of neointimal hyperplasia. Therefore, we conclude that overexpression or induction of IRF-1, for example by interferon ß, which is also known to exhibit cell cycle inhibitory properties in vascular smooth muscle cells (40), may be an interesting strategy to prevent restenotic processes. Interestingly, it has been shown that recombinant overexpression of the interferon beta gene in a porcine PTCA model reduces neointima formation significantly (41).

To our knowledge, this is the first report which characterizes IRF-1 as a central inhibitor of restenosis. In conclusion, we demonstrate that: (i) IRF-1 is an important endogenous inhibitor of neointima formation in vivo; (ii) immunohistological assessment of human restenotic lesions suggests a role for IRF-1 in the limitation of neointimal hyperplasia in man; (iii) the anti-proliferative mechanisms initiated by IRF-1, which have been in part characterized previously in different cell systems, are evident in coronary artery smooth muscle cells. This includes cell cycle inhibition in G1 and induction of p21 and NO; (iv) further, IRF-1 inhibits mitogen-induced smooth muscle cell migration. Considering the low degree of neointima formation in wild-type animals and the significant increase of neointima formation in animals lacking the IRF-1 gene together with the consistently upregulated IRF-1 levels in human neointima, IRF-1 may be considered as a key endogenous inhibitor of neointimal hyperplasia. Eventually, interferon regulatory factors may be an interesting target for interventions to prevent neointimal hyperplasia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture
The cell lineage exclusively utilized were human coronary artery smooth muscle cells (CASMC), obtained from Clonetics/BioWhittaker, Walkersville, Maryland and used in passages lower than 10. Cells were maintained in SMBM® Medium (BioWhittaker) including 0.5 µg/ml hEGF, 5 mg/ml insulin, 1 µg/ml hFGF, 50 mg/ml Gentamicin and 10% FBS. For cell cycle synchronization, serum was withdrawn from culture medium for 72 h. Twenty-four hours prior to mitogen stimulation with 10% FCS and 10 ng/ml PDGF, cells were transduced by the appropriate adenovirus vector encoding for IRF-1 (Ad-IRF-1) or ß-galactosidase (Ad-lacZ) at a multiplicity of infection (MOI) of 100.

BrdU ELISA
Bromodeoxyuridine ELISA was performed using the Cell Proliferation ELISA BrdU kit (colorimetric) from Roche, Mannheim, Germany, according to the protocol of the manufacturer. The assay was performed 24 and 48 h post stimulation. Cells were exposed to a 10 µM BrdU pulse 12 and 1 h prior to analysis.

Flow cytometry
Cell cycle was monitored by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA) using propidium iodine (Cycle TestTM Plus DNA Reagent Kit, Becton Dickinson) according to the protocol of the manufacturer. Cells were harvested 24 and 48 h post-mitogen stimulation. Software based cell cycle analysis was performed by ModFit 2.0 software (Verity, Topsham, ME, USA).

Kinase assay
Cyclin A was immunoprecipitated using the polyclonal antibody T310 as described previously (42). Immunoprecipitations and western blotting techniques have been described elsewhere (42). Kinase assays were performed at 30°C for 30 min in kinase assay buffer (50 mM Tris–HCl at pH 7.2, 10 mM MgCl2) containing histone H1, ATP (200 µM), {gamma}[-32P]ATP, as described earlier (42).

p21 cotransfection and western blotting
Coronary artery smooth muscle cells were transfected with a reporter plasmid vector containing luciferase under the control of the p21 promoter using Fugene® transfection reagent (Roche, Mannheim, Germany). Lucerifase expression assay and western blotting were carried out as previously described (43). The following antibodies were used: IRF-1 catalog no. sc-497 (rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, Ca, USA), p21 catalog no. 610233 (mouse monoclonal, BD Biosciences, San Diego, Ca, USA).

Migration assay
The Chemicon (Temecula, CA, USA) QCMTM-FN Assay was utilized to measure cell migration towards an immobilized extracellular matrix protein gradient towards fibronectin (haptotaxis) in a Boyden chamber system. The kit was applied according the protocol of the manufacturer using 2.5x104 cells per well in the assay. Twenty-four hours after incubation cells on the bottom side of the chamber were fixed and stained with 1% crystal violet solution. Cells were counted on an inverted microscope within ten randomly assigned 100x power fields per chamber.

NO assay
A colorimetric Nitric Oxide Synthase Assay Kit (Calbiochem, San Diego, CA, USA) was used according to the protocol of the manufacturer.

Mouse carotid flow cessation model
To induce neointimal hyperplasia in IRF-1 knockout (22) and wild-type mice of the same genetic background (C57BL/6J), the carotid cessation model was used as described (12). For histomorphometry, mice were killed 28 days after ligation of the common carotid artery which was further embedded in paraffin. Morphometry was carried out 600 µm proximal of the ligation suture in the common carotid artery.

Rat carotid injury model
To determine the effect of IRF-1 overexpression on the prevention of neointimal hyperplasia, the rat carotid injury model was used as previously described (44). Recombinant adenovirus was directly injected into the temporarily ligated common carotid artery at a volume of 0.2 ml and a concentration of 1010 PFU virus. After 20 min incubation time, the ligatures were released and the external carotid artery was ligated. Fourteen days later, the common carotid artery was perfusion fixed with 10% formalin at physiological pressure and excised. For morphometry, Elastica-van-Giesson stained histology slides were scanned by a Zeiss Axiovert 100 system. Morphometric analysis was performed by SigmaScan 5.0 software (SPSS Inc., Chicago, IL, USA). All animal experiments were performed after approval according to section 15, paragraph 1, ‘Deutsches Tierschutzgesetz’ (German animal protection law).

Immunohistochemistry
Immunohistochemistry was carried out as previously described (12). Briefly, formalin-fixed, paraffin-embedded sections were deparaffinized and stained with polyclonal rabbit anti-IRF1 (1:1000, catalog no. sc-497, Santa Cruz Biotechnology) and mouse anti-p21 (1:80, catalog no. M7202, DAKO, Hamburg, Germany) antibodies, using an avidin-biotin-peroxidase/alkaline phosphatase complex technique (12). Human tissue was stained with the IRF1-polyclonal rabbit anti-IRF1 antibody (1:80), mouse anti-p21 (1:80; DAKO catalog no. M7202) and anti-Ki67 antibodies (1:250; DAKO catalog no. M7240). Specific binding of the antibodies was detected by Fast Red or DAB (DAKO). Negative controls were carried out by omission of the primary and secondary antibody as well as of a nonspecific antibody with identical immunoglobulin-subtype.

Statistical analysis
Unless mentioned otherwise, results are expressed as mean plus/minus standard error. The significance of variability amongst the means of the experimental groups was determined by one or two way analysis of variance (ANOVA), using SPSS for Windows V10.0 software. Differences among experimental groups were considered to be statistically significant when P<0.05.


   ACKNOWLEDGEMENTS
 
This study was supported by a grant awarded to R.W. (We 1811/3-1 and 3-2) from the Deutsche Forschungsgemeinschaft (DFG). R.B. was also supported by a DFG grant (Br 1583/1-2). We are indebted to Angelika Dötterl, Renate Hegenloh and Erika Schneider for expert technical assistance. We gratefully acknowledge the expertise of Michael Weber in genotyping IRF-1-deficient mice. We thank J.L. Stein and P. Vaughan for IRF-1 cDNA, T. Mak for IRF-1 knockout mice and B. Vogelstein for the p21 promoter construct and p53 expression plasmids.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Deutsches Herzzentrum, Lazarettstrasse 36, 80636 München, Germany. Tel: +49 8912181514; Fax: +49 8912184013; Email: rwessely{at}dhm.mhn.de Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Tunstall-Pedoe, H., Kuulasmaa, K., Mahonen, M., Tolonen, H., Ruokokoski, E. and Amouyel, P. (1999) Contribution of trends in survival and coronary-event rates to changes in coronary heart disease mortality: 10-year results from 37 WHO MONICA project populations. Monitoring trends and determinants in cardiovascular disease. Lancet, 353, 1547–1557.[CrossRef][ISI][Medline]

  2. Topol, E.J. and Serruys, P.W. (1998) Frontiers in interventional cardiology. Circulation, 98, 1802–1820.[Free Full Text]

  3. Fishman, D.L., Leon, M.B. and Baim, D.S. (1994) A randomized comparison of coronary stent placement and ballon angioplasty in the treatment of coronary artery disease. New Engl. J. Med., 17, 758–769.

  4. Schwartz, S.M., deBlois, D. and O'Brien, E.R.M. (1995) The intima—soil for artherosclerosis and restenosis. Circul. Res., 77, 445–465.[Free Full Text]

  5. Komatsu, R., Ueda, M., Naruko, T., Kojima, A. and Becker, A.E. (1998) Neointimal tissue response at sites of coronary stenting in humans. Macroscopic, histological and immunohistochemical analysis. Circulation, 98, 224–233.[Abstract/Free Full Text]

  6. Hamon, M., Bauters, C., McFadden, E.P., Wernert, N., Lablanche, J.M., Dupuis, B. and Bertrand, M.E. (1995) Restenosis after coronary angioplasty. Eur. Heart J., 16, 33–48.

  7. Kastrati, A., Schomig, A., Elezi, S., Schuhlen, H., Dirschinger, J., Hadamitzky, M., Wehinger, A., Hausleiter, J., Walter, H. and Neumann, F.J. (1997) Predictive factors of restenosis after coronary stent placement. J. Am. Coll. Cardiol., 30, 1428–1436.[Abstract]

  8. Miyamoto, M., Fujita, T., Kimura, Y., Harada, H., Sudo, Y., Miyata, T. and Taniguchi, T. (1988) Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-beta gene regulatory elements. Cell, 54, 903–913.[CrossRef][ISI][Medline]

  9. Vaughan, P.S., van Wijnen, A.J., Stein, J.L. and Stein, G.S. (1997) Interferon regulatory factors: growth control and histone gene regulation—it's not just interferon anymore. J. Mol. Med., 75, 348–359.[CrossRef][ISI][Medline]

  10. Horiuchi, M., Yamada, H., Akishita, M., Ito, M., Tamura, K. and Dzau, V. (1999) Interferon regulatory factors regulate interleukin-1beta-converting enzyme expression and apoptosis in vascular smooth muscle cells. Hypertension, 33, 162–166.[Abstract/Free Full Text]

  11. Spink, J. and Evans, T. (1997) Binding of the transcription factor interferon regulatory factor-1 to the inducible nitric-oxide synthase promoter. J. Biol. Chem., 272, 24417–24425.[Abstract/Free Full Text]

  12. Zohlnhofer, D., Richter, T., Neumann, F., Nuhrenberg, T., Wessely, R., Brandl, R., Murr, A., Klein, C.A. and Baeuerle, P.A. (2001) Transcriptome analysis reveals a role of interferon-gamma in human neointima formation. Mol. Cell, 7, 1059–1069.[CrossRef][ISI][Medline]

  13. McGill, C.J. and Brooks, G. (1995) Cell cycle control mechanisms and their role in cardiac growth. Cardiovasc. Res., 30, 557–569.[CrossRef][ISI][Medline]

  14. Hengst, L. and Reed, S.I. (1998) Inhibitors of the Cip/Kip family. Curr. Top. Microbiol. Immunol., 227, 25–41.[ISI][Medline]

  15. Lallemand, C., Bayat-Sarmadi, M., Blanchard, B. and Tovey, M.G. (1997) Identification of a novel transcriptional regulatory element common to the p53 and interferon regulatory factor 1 genes. J. Biol. Chem., 272, 29801–29809.[Abstract/Free Full Text]

  16. Coccia, E.M., Del Russo, N., Stellacci, E., Orsatti, R., Benedetti, E., Marziali, G., Hiscott, J. and Battistini, A. (1999) Activation and repression of the 2-5A synthetase and p21 gene promoters by IRF-1 and IRF-2. Oncogene, 18, 2129–2137.[CrossRef][ISI][Medline]

  17. Chang, M.W., Barr, E., Lu, M.M., Barton, K. and Leiden, J.M. (1995) Adenovirus-mediated overexpression of the cyclin/cyclin-dependent kinase inhibitor p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J. Clin. Invest., 96, 2260–2268.[ISI][Medline]

  18. Tanaka, N., Ishihara, M., Lamphier, M.S., Nozawa, H., Matsuyama, T., Mak, T.W., Aizawa, S., Tokino, T., Oren, M. and Taniguchi, T. (1996) Cooperation of the tumor suppressors IRF-1 and p53 in response to DNA damage. Nature, 382, 816–818.[CrossRef][Medline]

  19. Bassenge, E. and Busse, R. (1988) Endothelial modulation of coronary tone. Prog. Cardiovasc. Dis., 30, 349–380.[CrossRef][ISI][Medline]

  20. Tanner, F.C., Meier, P., Greutert, H., Champion, C., Nabel, E.G. and Luscher, T.F. (2000) Nitric oxide modulates expression of cell cycle regulatory proteins: a cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation. Circulation, 101, 1982–1989.[Abstract/Free Full Text]

  21. von der Leyen, H., Gibbons, G.H., Morishita, R. and Dzau, V.J. (1994) In vivo gene transfer of nitric oxide synthase inhibits neointima formation in injured rat carotid arteries. (Abtract.) Eur. Heart J., 15, 426.

  22. Tanaka, N., Ishihara, M., Kitagawa, M., Harada, H., Kimura, T., Matsuyama, T., Lamphier, M.S., Aizawa, S., Mak, T.W. and Taniguchi, T. (1994) Cellular commitment to oncogene-induced transformation of apoptosis is dependent on the transcription factor IRF-1. Cell, 77, 829–839.[CrossRef][ISI][Medline]

  23. Prost, S., Bellamy, C.O., Cunningham, D.S. and Harrison, D.J. (1998) Altered DNA repair and dysregulation of p53 in IRF-1 null hepatocytes. FASEB J., 12, 181–188.[Abstract/Free Full Text]

  24. Willman, C.L., Sever, C.E., Pallavicini, M.G., Harada, H., Tanaka, N., Slovak, M.L., Yamamoto, H., Harada, K., Meeker, T.C., List, A.F. et al. (1993) Deletion of IRF-1, mapping to chromosome 5q31.1, in human leukemia and preleukemic myelodysplasia. Science, 259, 968–971.[Abstract]

  25. Doherty, G.M., Boucher, L., Sorenson, K. and Lowney, J. (2001) Interferon regulatory factor expression in human breast cancer. Ann. Surg., 233, 623–629.[CrossRef][ISI][Medline]

  26. Nozawa, H., Oda, E., Nakao, K., Ishihara, M., Ueda, S., Yokochi, T., Ogasawara, K., Nakatsuru, Y., Shimizu, S., Ohira, Y. et al. (1999) Loss of transcription factor IRF-1 affects tumor susceptibility in mice carrying the Ha-ras transgene or nullizygosity for p53. Genes Devl., 13, 1240–1245.[Abstract/Free Full Text]

  27. Kroger, A., Ortmann, D., Krohne, T.U., Mohr, L., Blum, H.E., Hauser, H. and Geissler, M. (2001) Growth suppression of the hepatocellular carcinoma cell line Hepa1-6 by an activatable interferon regulatory factor-1 in mice. Cancer Res., 61, 2609–2617.[Abstract/Free Full Text]

  28. Yonemitsu, Y., Kaneda, Y., Tanaka, S., Nakashima, Y., Komori, K., Sugimachi, K. and Sueishi, K. (1998) Transfer of wild-type p53 gene effectively inhibits vascular smooth muscle cell proliferation in vitro and in vivo. Circ. Res., 82, 147–156.[Abstract/Free Full Text]

  29. Kumar, A. and Lindner, V. (1997) Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow. Arterioscler. Thromb. Vasc. Biol., 17, 2238–2244.[Abstract/Free Full Text]

  30. Horiba, M., Kadomatsu, K., Nakamura, E., Muramatsu, H., Ikematsu, S., Sakuma, S., Hayashi, K., Yuzawa, Y., Matsuo, S., Kuzuya, M. et al. (2000) Neointima formation in a restenosis model is suppressed in midkine-deficient mice. J. Clin. Invest., 105, 489–495.[ISI][Medline]

  31. Harmon, K.J., Couper, L.L. and Lindner, V. (2000) Strain-dependent vascular remodeling phenotypes in inbred mice. Am. J. Pathol., 156, 1741–1748.[Abstract/Free Full Text]

  32. Sata, M. and Walsh, K. (2000) Fas ligand-deficient mice display enhanced leukocyte infiltration and intima hyperplasia in flow-restricted vessels. J. Mol. Cell. Cardiol., 32, 1395–1400.[CrossRef][ISI][Medline]

  33. Kroef, M.J., Willemze, R. and Landegent, J.E. (1993) Dinucleotide repeat polymorphism in the interferon regulating factor 1 (IRF1) gene. Hum. Mol. Genet., 2, 1748.[Free Full Text]

  34. Harada, H., Kitagawa, N., Tanaka, N., Yamamoto, H., Harada, K., Ishihara, M. and Taniguchi, T. (1993) Anti-oncogenic and oncogenic potentials of interferon regulatory factor -1 and -2. Science, 259, 971–974.[Abstract]

  35. Bachmaier, K., Neu, N., Pummerer, C., Duncan, G.S., Mak, T.W., Matsuyama, T. and Penninger, J.M. (1997) iNOS expression and nitrotyrosine formation in the myocardium in response to inflammation is controlled by the interferon regulatory transcription factor 1. Circulation, 96, 585–591.[ISI][Medline]

  36. Quyyumi, A.A., Dakak, N., Andrews, N.P., Gilligan, D.M., Panza, J.A. and Cannon, R.O., III (1995) Contribution of nitric oxide to metabolic coronary vasodilation in the human heart. Circulation, 92, 320–326.[Abstract/Free Full Text]

  37. Neish, A., Read, M., Thanos, D., Pine, R., Maniatis, T. and Collins, T. (1995) Endothelial interferon regulatory factor 1 cooperates with NF-kappa B as a transcriptional activator of vascular cell adhesion molecule 1. Mol. Cell. Biol., 15, 2558–2569.[Abstract]

  38. Poon, M., Marx, S.O., Gallo, R., Badimon, J.J., Taubman, M.B. and Marks, A.R. (1996) Rapamycin inhibits vascular smooth muscle cell migration. J. Clin. Invest., 98, 2277–2283.[ISI][Medline]

  39. Sriram, V. and Patterson, C. (2001) Cell cycle in vasculoproliferative diseases: potential interventions and routes of delivery. Circulation, 103, 2414–2419.[Abstract/Free Full Text]

  40. Mintzer, R.J., Croze, E., Rubanyi, G.M. and Johns, A. (1998) Differential effects of IFN-beta1b on the proliferation of human vascular smooth muscle and endothelial cells. J. Interferon Cytokine Res., 18, 939–945.[ISI][Medline]

  41. Stephan, D., San, H., Yang, Z.Y., Gordon, D., Goelz, S., Nabel, G.J. and Nabel, E.G. (1997) Inhibition of vascular smooth muscle cell proliferation and intimal hyperplasia by gene transfer of beta-interferon. Mol. Med., 3, 593–599.[ISI][Medline]

  42. Hengst, L., Dulic, V., Slingerland, J., Lees, E. and Reed, S. (1994) A cell cycle-regulated inhibitor of cyclin-dependent kinases. Proc. Natl. Acad. Sci. USA, 91, 5291–5295.[Abstract/Free Full Text]

  43. Wessely, R., Henke, A., Zell, R., Kandolf, R. and Knowlton, K.U. (1998) Low-level expression of a mutant coxsackieviral cDNA induces a myocytopathic effect in culture: an approach to the study of enteroviral persistence in cardiac myocytes. Circulation, 98, 450–457.[Abstract/Free Full Text]

  44. Clowes, A.W., Reidy, M.A. and Clowes, M.M. (1983) Mechanisms of stenosis after arterial injury. Lab. Invest., 49, 208–215.[ISI][Medline]


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