Cardiovascular Research (2014) 102, 469–479 doi:10.1093/cvr/cvu052

Interferon regulatory factor 3 protects against adverse neo-intima formation Shu-Min Zhang 1,2†, Li-Hua Zhu 1,2†, Zuo-Zhi Li 3†, Pi-Xiao Wang 1,2, Hou-Zao Chen 3, Hong-Jing Guan 1,2, Ding-Sheng Jiang 1,2, Ke Chen 4, Xiao-Fei Zhang 4, Song Tian 1,2, Da Yang1,2, Xiao-Dong Zhang 3, and Hongliang Li 1,2* 1 Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Jiefang Road 238, Wuhan 430060, China; 2Cardiovascular Research Institute of Wuhan University, Wuhan, China; 3State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; and 4College of Life Sciences, Wuhan University, Wuhan, China

Received 21 October 2013; revised 19 February 2014; accepted 25 February 2014; online publish-ahead-of-print 4 March 2014 Time for primary review: 35 days

Aims

----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords

Neo-intima formation † Proliferation † IRF3 † PPARg

1. Introduction Neo-intima formation and inward remodelling are the most prominent pathological responses to mechanical injuries to arteries. Unfortunately, such injuries are inevitable during virtually all available surgical procedures, including angioplasty, stenting, atherectomy, and bypass surgery, that are used to treat patients with life-threatening coronary atherosclerosis. Thus, effective inhibition of neo-intima formation would be a promising strategy to sustain the long-term beneficial effects of these coronary interventions. Central to the pathophysiology of neo-intima formation is vascular smooth muscle cell (VSMC) proliferation.1 Upon arterial injury, endothelium is denuded, resulting in the loss of endothelium-derived growth-inhibiting factors, including NO and prostacyclin,2 and activation of platelets. Growth factors, such as platelet-derived growth factor (PDGF), are then released from activated platelets, leucocytes, and †

SMCs and stimulate VSMC to shift from a contractile phenotype to a proliferative phenotype.3,4 This adverse phenomenon is often accompanied by increased expression of proliferation markers and cyclins, such as proliferating cell nuclear antigen (PCNA) and cyclin D1, and decreased expression of proteins that are characteristic of differentiated SMCs, including smooth muscle actin (SMA), SM22a, and smoothelin.4 Alterations in these genes facilitate VSMC migration and proliferation, which culminate in excessive neo-intima formation and eventually arterial restenosis. Inflammatory cell infiltration and VSMC proliferation are critical, overlapping processes that are involved in intimal hyperplasia. In response to injury, several cell types, including VSMCs, secrete pro-inflammatory cytokines and chemokines, which lead to increased VSMC migration, proliferation, and subsequent intimal hyperplasia.5 Among the known cytokines, the interferon (IFN) family is a wellrecognized mediator of atherosclerosis and vascular remodeling.6

S.-M.Z., L.-H.Z., and Z.-Z.L. are co-first authors.

* Corresponding author. Tel: +86-27-88076990; fax: +86-27-88076990, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2014. For permissions please email: [email protected].

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Vascular smooth muscle cell (VSMC) proliferation is central to the pathophysiology of neo-intima formation. Interferon regulatory factor 3 (IRF3) inhibits the growth of cancer cells and fibroblasts. However, the role of IRF3 in vascular neointima formation is unknown. We evaluated the protective role of IRF3 against neo-intima formation in mice and the underlying mechanisms. ..................................................................................................................................................................................... Methods IRF3 expression was down-regulated in VSMCs after carotid wire injury in vivo, and in SMCs after platelet-derived growth factor (PDGF)-BB challenge in vitro. Global knockout of IRF3 (IRF3-KO) led to accelerated neo-intima formation and proand results liferation of VSMCs, whereas the opposite was seen in SMC-specific IRF3 transgenic mice. Mechanistically, we identified IRF3 as a novel regulator of peroxisome proliferator-activated receptor g (PPARg), a negative regulator of SMC proliferation after vascular injury. Binding of IRF3 to the AB domain of PPARg in the nucleus of SMCs facilitated PPARg transactivation, resulting in decreased proliferation cell nuclear antigen expression and suppressed proliferation. Overexpression of wild-type, but not truncated, IRF3 with a mutated IRF association domain (IAD) retained the ability to exert anti-proliferative effect. ..................................................................................................................................................................................... Conclusions IRF3 inhibits VSMC proliferation and neo-intima formation after vascular injury through PPARg activation.

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2. Methods 2.1 Animals The VSMC-specific IRF3 overexpressing transgenic mice (IRF3-TG) were generated by microinjection of the full-length homo-IRF3 cDNA under the transcriptional control of a constitutive SMC-specific promoter SM22a into fertilized embryos (C57BL/6 background). VSMC-specific PPARg overexpressing transgenic mice (PPARg-TG) were generated using a similar method (see Supplementary material online). All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 – 23, revised 1996) and were approved by the Animal Care and Use Committee of the Renmin Hospital of Wuhan University, China. For animal experiments, the adequacy of anaesthesia was confirmed by the absence of reflex response to foot squeeze.

2.2 Isolation and culture of VSMCs VSMCs were isolated from the thoracic aortas of 6- to 8-week-old mice by enzymatic dissociation. Briefly, the vessels were then incubated in 1 mL of DMEM containing 0.375 mg/mL of collagen Type II (C6885, 700.3 U/mg, Sigma) and

0.115 mg/mL of elastase Type I (E1250, 6 U/mg, Sigma) at 378C in a CO2 humidified chamber for 8 min. Then, the vessels were transferred to culture dishes containing 3 mL of DMEM/F12 with 0.2% BSA, the adventitia was gently removed, and the vessels were incised longitudinally. The vessels were minced with fine scissors, transferred to 2 mL of DMEM/F12 with 2% BSA, 1.36 mg/mL of collagen Type II and 0.092 mg/mL of elastase Type I, and incubated at 378C for 2–2.5 h. After the incubation, digested vessels were re-suspended in 3 mL of DMEM/F12, centrifuged at 200 g for 5 min, then re-suspended in 2.5 mL of DMEM with 20% FBS, and cultured in a six-well plate for 5 days. Rat aortic vascular smooth muscle cells (RAVSMCs) were isolated from the aortas of 200–250 g male Sprague– Dawley rats as described previously (see Supplementary material online).24

2.3 Statistical analysis Data are presented as the mean + standard deviation (SD). Comparisons between the two groups were performed using independent sample t-test. Differences in multiple groups were determined using analysis of a one-way analysis of variance (ANOVA) with least significant difference (LSD) or Tamhane’s T2 post hoc test. All in vivo and imaging studies were performed in a blinded manner. Values of P , 0.05 were considered significant.

3. Results 3.1 IRF3 is down-regulated in VSMCs upon vascular injury SMC proliferation is regarded as the principal mechanism underlying intimal thickening during atherosclerosis and vascular remodeling.25 The growth factor PDGF-BB is a critical regulator of neo-intima growth that enhances SMC proliferation and migration.26 Thus, to study whether IRF3 participates in VSMC proliferation, we first treated RAVSMCs and human aortic VSMCs (HAVSMCs) with PDGF-BB (20 ng/mL) and examined the temporal pattern of IRF3 expression using western blot analysis. We observed that the protein expression of IRF3 in both cultured cell lines was significantly induced at 6 h after PDGF-BB challenge, peaked at 24 h, and dramatically diminished at 48 h (Figure 1A and B), indicating that IRF3 may be involved in VSMC response to PDGF-BB. To gain further insight into the in vivo role of IRF3 in neo-intima formation, we utilized the left carotid artery (LCA) wire-injury model in C57/BL mice. The subsequent neo-intima formation was absent at the baseline and increased gradually from 7 to 28 days in wild-type (WT) mice (Figure 1C). This formation was further validated by the increased intimal area and intima/media ratio. VSMCs were the predominant cellular components in the neo-intimal area (Figure 1D). Interestingly, IRF3 expression was detected in and primarily confined to VSMCs that stained positive for the definitive SMC marker SMA either before or after the injury (Figure 1D). At 7–14 days, IRF3 expression gradually increased, but then returned to a level that was significantly below baseline at 28 days (Figure 1D). After wire injury, expressions of proliferation markers, namely PCNA and cyclin D1, were up-regulated, whereas expression of the differentiation marker SMA was reduced (Figure 1E). Finally, complementary results were obtained in western blotting experiments that analysed the protein levels of IRF3 and these marker proteins in the injured LCAs (Figure 1F). Taken together, these results validate the establishment of our vascular injury model and indicate a potential correlation between IRF3, VSMC proliferation, and neo-intima formation in mice.

3.2 IRF3 deficiency exacerbates neo-intima formation To directly study whether and how endogenous IRF3 impacts the extent of injury-induced intimal hyperplasia, we injured the LCAs of IRF3

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Targeting the pathways related to the action of Type I and II IFNs might provide new therapies to reduce neo-intima formation. For instance, both IFN-b and IFN-g are negative regulators of VSMC proliferation in vitro.7,8 Furthermore, Hansson and Holm9 demonstrated that IFN-g inhibits the formation of arterial intimal lesion following balloon injury, and Stephan et al. 10 demonstrated that delivery of an adenoviral vector encoding IFN-b reduces VSMC proliferation and intima hyperplasia in vivo. The interferon regulatory factors (IRFs) were the first and are the best, characterized transcriptional regulators of IFN-induced signalling pathways.11 The mammalian IRF family consists of nine members, including IRF1–9. Compared with other IRFs, IRF3 is constitutively expressed in all cell types and is essential for the induction of Type I IFN genes, e.g. IFN-a and IFN-b; thus, it is involved in the host antiviral defence programme. Upon various stresses, IRF3 translocated into the nucleus, which leads to IFN gene transcription.12 Considering its well-understood role in IFN-b signalling, we anticipated that IRF3 may be involved in intima hyperplasia. However, antiinflammation strategies are difficult because systemic anti-inflammatory approaches have failed to prevent restenosis in several clinical trials. Interestingly, our recent studies have indicated that, even in the absence of IFNs, IRFs regulate a broad range of biological activities, including cardiovascular disease, stroke, and metabolic disorders.11,13 – 16 As transcription factors, IRFs can bind to interferon regulatory elements (IRF-Es) and regulate cell growth, differentiation, and apoptosis.17,18 Moreover, when binding to a specific DNA element, IRFs also bind to co-activators, such as HATs, and become modified;19,20 therefore, they can perform more critical and versatile functions. For example, Wesseley et al. showed that IRF1 is an endogenous inhibitor of neo-intimal growth by inducing p21, and that IRF1 regulates high glucose-induced proliferation of VSMCs.21,22 Notably, IRF3 inhibits the growth of cancer cells11 and fibroblasts,23 possibly through the induction of p53. Therefore, it is highly tempting to speculate that IRF3 has direct actions at the level of the vascular wall. We hypothesized that IRF3 is a direct inhibitor of VSMC growth and neo-intima formation. In the present study, we observed a significant reduction in IRF3 expression in a murine wire-injury model. As expected, VSMC-specific IRF3 transgenic mice exhibited mitigated intimal hyperplasia, whereas IRF3-deficient mice aggravated vascular injury. Mechanistically, we found that IRF3 protects against vascular remodelling through the activation of PPARg.

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Figure 1 Vascular injury reduces IRF3 expression. (A and B) Immunoblots showing changes in IRF3 expression at the indicated time points following PDGF-BB treatment (20 ng/mL) in (A) RAVSMCs or (B) HAVSMCs GAPDH served as the loading control. Right panels: quantification of normalized IRF3 levels (n ¼ 5). P-values were determined using the one-way ANOVA test. *P , 0.05 vs. PBS control; #P , 0.05 vs. cell culture after 6 h of PDGF-BB treatment; †P , 0.05 vs. cell culture after 24 h of PDGF-BB treatment. (C) Haematoxylin and eosin staining was performed on sections from WT mice at the indicated time points following injury. Arrows: internal elastic lamina. Scale bar: 50 mm. Bottom panel: quantification of intima area (left) and intima/media ratio at the indicated times (right) (n ¼ 9 – 11 at each time point). P-values were determined using the one-way ANOVA test. *P , 0.05 vs. Day 7; #P , 0.05 vs. Day 14. (D) Injury-induced changes in the expression of IRF3 (red), and SMA (green) were assessed by immunostaining in the left carotid arteries of WT mice. Scale bar: 50 mm. Bottom panel: quantification of IRF3 immunostaining at the indicated time points (n ¼ 4). (E) Co-staining of PCNA (upper), cyclin D1 (middle), and SMA (bottom) with nucleus (DAPI, blue). Scale bar: 50 mm. Bottom panel: quantification of PCNApositive cells (left), and expression of cyclin D1 (middle) and SMA (right) (n ¼ 5 – 9). P-values were determined using the one-way ANOVA test. *P , 0.05 vs. sham; #P , 0.05 vs. Day 14. (F) Immunoblots of the indicated proteins after wire injury. GAPDH served as the loading control. Bottom panel: quantification of the indicated proteins normalized to GAPDH. Blots are representative of three independent experiments. P-values were determined using the one-way ANOVA test. *P , 0.05 vs. sham; #P , 0.05 vs. Day 7; †P , 0.05 vs. Day 14 in (D) and (F ). Data represent the mean + SD.

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3.3 VSMC-specific IRF3 transgenic mice exhibit reduced neo-intima formation Given the robust expression of IRF3 in VSMCs, we hypothesized that IRF3 primarily targets SMCs to exert its function in regulating smooth muscle proliferation. To this end, we generated several lines of SMC-specific IRF3 overexpressing TG under the control of the SM22a promoter (Figure 3A). TG4 transgenic line (hereafter referred to as IRF3-TG mice), in which VSMCs stably express the highest IRF3

level [5.0-fold compared with non-transgenic (NTG) mice] was used for further experiments (Figure 3B; see Supplementary material online, Figure S1). Intimal hyperplasia was not observed in sham arteries in IRF3-TG and control mice (NTG, data not shown). The neo-intimal area in IRF3-TG mice was 44.8 and 63.1% lower than that in NTG mice at 14 and 28 days, respectively (P , 0.05; Figure 3C). However, the medial area of wire-injured LCAs did not differ between groups. Thus, the intima/media ratio was markedly lower in IRF3-TG mice compared with NTG mice at both time points (Figure 3C). After wire injury, IRF3-TG mice had lower expression levels of PCNA, cyclin D1 than NTG mice (Figure 3D and E). The decreased proliferation in IRF3-TG mice was accompanied with an increase in the expression of differentiation markers, including SMA and SM22a, when compared with NTG controls, as determined by immunostaining (Figure 3F) and western blotting (Figure 3G). Similarly, IRF3 overexpression (4.6-fold) led to decreased SMC proliferation induced by PDGF-BB (20 ng/mL) (Figure 3H), reduced expression levels of PCNA and cyclin D1, and elevated expression levels of SMA, SM22a, and smoothelin (Figure 3I). Taken together, these data support the hypothesis that VSMC-derived IRF3 is crucial for vascular maintenance and the prevention of excessive intimal growth.

3.4 IRF3 transactivates PPARg to reduce SMC proliferation Although we verified the anti-proliferative role of IRF3 in VSMCs, the underlying mechanism remains to be elucidated. Increasing evidence indicates that peroxisome proliferator-activated receptors (PPARs), in particular PPARg, inhibit VSMC proliferation and neo-intima formation at the vascular level.27,28 Recently, we identified IRFs as potential co-activators of the PPAR family in the peripheral tissues.29 Thus, we next investigated whether IRF3 regulates PPARg in SMCs. PPARg initiates transcription of target genes by binding to a consensus DNA sequence AGGTCA, termed the PPAR response element (PPRE). To do so, we examined PPARg transactivation using a dual luciferase reporter gene assay driven by PPREs in primary RAVSMCs. Intriguingly, although overexpression of IRF3 (AdIRF3) alone only marginally increased PPRE luciferase activity, co-infection with adenovirus encoding PPARg significantly augmented PPRE activity (Figure 4A). Conversely, co-infection of AdIRF3 and PPARg robustly diminished PCNA transcription, as demonstrated by the insufficient induction of PCNA promoter activity in RAVSMCs following PDGF-BB (20 ng/mL) challenge (Figure 4B). Taken together, these results indicate that IRF3 may repress PCNA-mediated SMC proliferation by facilitating activation of

Figure 2 Neo-intima formation is accelerated in IRF3-KO mice. Carotid arteries were harvested from WT and IRF3-KO mice 14 and 28 days after injury. (A) Haematoxylin and eosin staining was performed on sections from WT and IRF3-KO mice at the indicated time points before and after injury. Arrows: internal elastic lamina. Scale bar: 50 mm. Right panels: quantification of intima area (upper) and intima/media ratio at the indicated times (lower) (n ¼ 6 –15 at each time point). (B) Co-staining of PCNA (upper) and cyclin D1 (lower) with nucleus (DAPI, blue). Scale bar: 50 mm. Right panels: quantification of PCNA-positive cells (upper) and expression of cyclin D1 (lower) (n ¼ 5 – 6). (C) Immunoblotting of PCNA and cyclin D1 at Day 28 after injury. GAPDH served as the loading control. Right panel: quantification of normalized protein levels. Blots are representative of three independent experiments. (D) Co-staining of SMA (upper), smoothelin (middle), and OPN (lower) with nucleus (DAPI, blue). Scale bar: 50 mm. Right panels: quantification of the indicated proteins (n ¼ 4 – 8 at each time points). (E) Immunoblotting of SMA, SM22a, and smoothelin at Day 28 after injury. GAPDH served as the loading control. Right panel: quantification of the normalized protein levels. Blots are representative of three independent experiments. (F ) VSMCs were harvested from WT and IRF3-KO mice, and stimulated with PDGF-BB (20 ng/mL) for 48 h. WT and IRF3-KO VSMC proliferation was assessed by BrdU incorporation and is shown as values of absorbance at 370 nm (n ¼ 6). P-values were determined using independent sample t-test. *P , 0.05 vs. PDGF-BB treated WT mice. (G) Western analysis (left) and quantification (right) of levels of the indicated proteins in WT and IRF3-KO VSMCs stimulated with PDGF-BB (20 ng/ml) for 24 or 48 h (n ¼ 5). P-values were determined using the independent sample t-test. *P , 0.05 vs. WT mice in (A – G). Data represent the mean + SD.

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knockout (IRF3-KO) mice and their WT littermates. Various parameters were assessed at 14 and 28 days post-injury. Haemodynamic parameters, including heart rate and blood pressure, were comparable between groups before and after wire injury (data not shown). No evidence of intimal hyperplasia was observed in sham-operated arteries of both genotypes (Figure 2A). Quantitative morphometry of the injured vessels revealed an accelerated increase in the neo-intima size in IRF3-KO mice compared with WT mice, which was apparent at 14 days and progressed by 28 days {[intima area, 14 days (20.86 + 3.4) × 103 vs. (13.27 + 2.08) × 103, respectively, P , 0.05; 28 days (30.33 + 5.99) × 103 vs. (21.35 + 2.33) × 103, respectively, P , 0.05; intima/media ratio, 14 days 0.46 + 0.04 vs. 0.33 + 0.07, respectively, P , 0.05; 28 days 0.86 + 0.19 vs. 0.57 + 0.10, respectively, P , 0.05; Figure 2A]}. We next determined whether this advanced neo-intima formation was attributable to enhanced VSMC proliferation. Quantification of immunostaining and western blot analysis demonstrated that IRF3-KO mice had significantly increased expression levels of PCNA and cyclin D1 at both time points after wire injury (Figure 2B and C ). Concomitantly, IRF3 deficiency resulted in a lower expression of differentiation markers (SMA, SM22a, and smoothelin) and increased expression of SMC proliferation-associated osteopontin (OPN) compared with that observed in the WT mice (Figure 2D and E). Thus, we reasoned that IRF3 might directly regulate SMC proliferation. To address this hypothesis, we measured DNA synthesis in these cells using BrdU incorporation. Murine aortic SMCs were isolated from IRF3-KO and littermate control mice. Stimulation with PDGF-BB (20 ng/mL) for 48 h increased WT-derived SMC proliferation by 3.59-fold relative to controls (Figure 2F). We observed that PDGF-BB-induced proliferation was 51.0% higher in IRF3-KO-derived SMCs than in SMCs from their WT littermates (Figure 2F). Accordingly, the expression levels of PCNA and cyclin D1 were significantly higher, whereas the expression levels of SMA, SM22a, and smoothelin were lower in IRF3-KO-derived SMCs than in WT SMCs (Figure 2G). Therefore, the loss of IRF3 may instigate injury-induced VSMC proliferation, thus enhancing intimal hyperplasia.

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the PPARg signalling pathway. Notably, IRF3 overexpression does not alter PPARg promoter activity (Figure 4C). To further validate whether IRF3-mediated anti-proliferative effect is PPARg-dependent, conditional PPARg deletion in smooth muscle cells (hereafter referred to as PPARg-KO mice) was established as described previously.30 Primary SMCs derived from PPARg-KO and WT control mice were infected with either AdIRF3 or AdGFP controls prior to PDGF-BB challenge. Following PDGF-BB treatment, IRF3 overexpression largely restored otherwise decreased PPRE luciferase activity in the presence (WT controls), but not in the absence, of WT PPARg (SMC-PPARg-KO) (Figure 4D). Similarly, AdIRF3 failed to reduce PDGF-BB-induced PCNA transcription (Figure 4E) and SMC proliferation (Figure 4F) upon PPARg ablation. Therefore, these data indicate that PPARg is necessary for IRF3-mediated anti-proliferative effect. Next, we knocked down the expression of IRF3 using RNA interference (AdshIRF3). PPARg overexpression (SMC-PPARg-TG) sufficiently compensated the adverse effects of IRF3 interference (AdshIRF3) on PPRE luciferase activity (see Supplementary material online, Figure S2A), PCNA transcription activity (see Supplementary material online, Figure S2B), and SMC proliferation (see Supplementary material online, Figure S2C). Taken together, these results showed that PPARg is both required and sufficient for IRF3-mediated attenuation of SMC proliferation.

Since IRF3 does not impact on PPARg transcription (Figure 4C), we next determined whether IRF3 functions as a co-activator of PPARg. Co-immunoprecipitation (Co-IP) assays showed that IRF3 readily interacted with PPARg, and vice versa (Figure 5A), which was further confirmed by a GST pull-down assay (Figure 5B). Accordingly, immunofluorescence microscopy demonstrated that IRF3 and PPARg were predominantly co-localized in the nucleus of primary RAVSMCs in the presence of a PDGF-BB (20 ng/mL) challenge (Figure 5C). It should be noted that the existence of IRF3 was primarily confined to cytoplasm under normal condition (Figure 5C). Next, we performed Co-IP of IRF3 and PPARg to study whether PDGF-BB is required for this interaction. Although IRF3 binding to PPARg was relatively weak when treated with PBS control, this interaction was largely enhanced in the presence of PDGF-BB challenge (see Supplementary material online, Figure S3A and B). These results indicate that IRF3 transactivates PPARg after injury. Next, we examined which protein domains are critical for the physical association between IRF3 and PPARg by generating a series of Myc-tagged truncated PPARg mutants (Figure 5D). Various

4. Discussion Our group and others have provided evidence of the emerging role that IRFs play in peripheral tissues and organs other than the immune system. The biological function of IRF3 in cardiovascular diseases, however, remains largely unexplored. The present study is the first direct demonstration that IRF3 is a critical endogenous regulator of VSMC proliferation, and that IRF3 is a novel co-regulator of PPARg, at least in VSMCs. The major findings of this study can be summarized as follows. First, VSMC-derived IRF3 constitutes the majority of IRF3 expression in the vascular wall, and this expression is reduced following wire injury. Secondly, IRF3 deficiency leads to enhanced VSMC proliferation and aggravated neo-intima formation, whereas IRF3 overexpression exerts a protective effect in the vasculature. Finally, we identified IRF3 as a novel activator of PPARg and determined that PPARg is indispensable for the anti-proliferative function of IRF3. Cell proliferation in response to injury is a principal feature in a broad range of pathophysiological conditions, including oncogenesis and proliferative vascular diseases. IRF3 was initially identified as a transcriptional regulator of Type I IFNs; recently, IRF3 was characterized as a regulator of cell proliferation and apoptosis.11 Kim et al. 31 demonstrated that ectopic IRF3 expression inhibits cell growth in several normal cell lines, such as REF52 and NIH3T3. Additionally, IRF3 transcriptionally activates TRAIL, which is involved in virus-induced apoptosis.32

Figure 3 VSMC-specific IRF3 overexpression alleviates neo-intima formation. (A) Schematic representation of the transgene construct. (B) Western blot (left) and densitometric (right) analysis of IRF3 expression in common carotid artery (CCA) lysates from the founder TG and NTG mice lines (n ¼ 5). (C) Haematoxylin and eosin staining was performed on sections from NTG and IRF3-TG mice at the indicated time points following injury. Arrows: internal elastic lamina. Scale bar: 50 mm. Right panels: quantification of intima area (upper) and intima/media ratio at the indicated times (lower) (n ¼ 6 – 14 at each time point). (D) Co-staining of PCNA (upper) and cyclin D1 (lower) with nucleus (DAPI, blue). Scale bar: 50 mm. Right panels: quantification of PCNA-positive cells (upper) and expression of cyclin D1 (lower) (n ¼ 4 – 6). (E) Immunoblot analysis of PCNA, cyclin D1 at Day 28 after injury. GAPDH served as the loading control. Right panel: quantification of normalized protein levels. Blots are representative of three independent experiments. (F) Co-staining of SMA (upper) and SM22a (lower) with nucleus (DAPI, blue). Scale bar: 50 mm. Right panel: quantification of the indicated proteins (n ¼ 4 – 5). (G) Immunostaining for SMA, SM22a, and smoothelin at Day 28 after injury. GAPDH served as the loading control. Right panel: quantification of the normalized protein levels. Blots are representative of three independent experiments. (H ) VSMCs were harvested from NTG and IRF3-TG mice, and stimulated with PDGF-BB (20 ng/mL) for 48 h. NTG and IRF3-TG VSMC proliferation was assessed by BrdU incorporation and is shown as values of absorbance at 370 nm (n ¼ 6). (I ) Western blot analysis (left) and quantification (right) of the levels of the indicated proteins in NTG and IRF3-TG VSMCs stimulated with PDGF-BB (20 ng/mL) for 24 or 48 h (n ¼ 5 – 6). P-values were determined using the independent sample t-test. *P , 0.05 vs. NTG mice in (C and I ). Data represent the mean + SD.

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3.5 The anti-proliferative effect of IRF3 is dependent on IRF3 –PPARg interaction

PPARg deletion mutants were then co-transfected with Flag-tagged IRF3, followed by co-immunoprecipitation. The results repeatedly revealed that the N-terminal A/B domain of PPARg but not the DNAbinding domain (DBD, C domain), the hinge region (D domain), or the ligand-binding domain (LBD, E/F domain) retained the ability to interact with IRF3 (Figure 5D). We next generated a series of IRF3 truncation mutants and found that neither the N-terminal DBD nor the less conservative intermediate region associated with PPARg could interact with PPARg; only the C-terminal IRF association domain (IAD) was capable of this interaction (Figure 5E). More importantly, overexpression of truncated IRF3 with a mutated IAD domain (Admutant) failed to enhance PPRE activity (see Supplementary material online, Figure S3C) or suppress PCNA transcription (see Supplementary material online, Figure S3D). Indeed, AdIRF3 but not Admutant sufficiently abrogated PDGF-BB-induced VSMC proliferation, as shown by the BrdU incorporation assay (see Supplementary material online, Figure S3E). We therefore concluded that, at least in vitro, the interaction with PPARg is essential for the IRF3-mediated suppression of PCNA expression and SMC proliferation.

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Intriguingly, IRF3-mediated control of cell growth and apoptosis may not be attributed to IFN-b signalling alone. For example, IFN-b is not activated by IRF3 in U87MG and U-2OS cancer cells.33 Weaver et al. 34 suggested that IRF3 enhances apoptosis in several cell lines and primary cells independently of IFN. Notably, IRF3 overexpression decreases cell growth and increases cell senescence in human fibroblast cells via p53 activation.23 Furthermore, IRF3 may play a role as a tumour suppressor because IRF3 can restrain the growth of several cancer cell lines in vivo and in vitro in a p53-dependent manner.31,35 Some data have suggested crosstalk between pathways involved in cancer and proliferative vascular diseases. Induction of the tumour-suppressor gene p16 by PPARa,36 for example, may exert beneficial effects on proliferative vascular diseases. However, the precise role of IRF3 in these diseases remains unknown. IRF3 induction by viral infection in immune cells is the best characterized pathway,11 but it can be activated or deregulated in response to various stimulations, such as Chlamydia pneumoniae, endoplasmic reticulum stress,37 oncogenesis, and lymph node hyperplasia.38 Although IRF3 promotes C. pneumoniae-induced foam cell formation39 and regulates endothelial cell proliferation,40 the biological function of IRF3 in vascular disease remains poorly understood. To evaluate the role that IRF3 plays

in mechanical injury-induced neo-intima formation, we examined the expression pattern and distribution of IRF3 in murine carotid arteries after wire injury. We observed that the early induction of IRF3 levels at 7 and 14 days after wire injury does not parallel with an increased level of proliferation markers. It is possible that this increase in IRF3 levels may be a compensatory response towards such an acute stress. As shown in Figure 1D, IRF3 expression increased to a maximum of two-fold of its baseline level at 14 days. This mild increase in IRF3 level may be insufficient to counteract the pro-proliferative effect of wire injury. Indeed, a 4.6-fold of IRF3 overexpression led to lower PCNA and cyclin D1 expressions compared with NTG-derived SMCs as early as 24 h following PDGF-BB treatment (Figure 3I). Moreover, although the underlying mechanisms is unclear, we found that the expression of vascular IRF3 diminishes at a later phase of arterial wound repair. IRF3 could be down-regulated or degraded by several factors, such as RBCK1,41 Npro,42 and Ro52.43 Interestingly, RBCK1, an E3 ubiquitin ligase that catalyses the ubiquitination and degradation of IRF3, also drives cell cycle progression and cancer cell proliferation. Whether RNCK1-mediated IRF3 degradation was involved in VSMC proliferation is unknown. Thus, the mechanism of sequential IRF3

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Figure 4 IRF3 transactivates PPARg to reduce SMC proliferation. (A and C) Adenoviruses encoding PPRE luciferase (A), PCNA luciferase (B), and PPARg luciferase (C) together with adenoviruses harbouring PPARg and IRF3 were used to infect primary RAVSMCs jointly or separately as indicated. Cultured cells were subjected to control or PDGF-BB treatment before luciferase activity was measured (n ¼ 5). P-values were determined using the one-way ANOVA test (A and B) and independent sample t-test (C). *P , 0.05 vs. AdGFP; #P , 0.05 vs. AdIRF3. (D and E) Adenoviruses encoding PPRE luciferase (D) and PCNA luciferase (E) together with adenoviruses harbouring GFP or IRF3 were used to infect primary VSMCs derived from PPARg-KO or PPARgfloxed control mice as indicated. Cultured cells were subjected to control or PDGF-BB treatment before luciferase activity was measured (n ¼ 5). P-values were determined using the one-way ANOVA test. (F ) VSMC proliferation was assessed by BrdU incorporation in VSMCs under the same conditions described in (D and E), n ¼ 5. P-values were determined using the one-way ANOVA test. *P , 0.05 vs. AdGFP/PPARg-floxed. Data represent the mean + SD.

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Figure 5 The anti-proliferative effect of IRF3 is dependent on IRF3 – PPARg interaction. (A) Co-immunoprecipitation of IRF3 and PPARg. HEK293T cells were transfected with Myc-tagged PPARg and Flag-tagged IRF3, or Flag -tagged PPARg and Myc-tagged IRF3. Lysates were immunoprecipitated with anti-Myc and analysed by immunoblotting using anti-Flag antibodies. (B) Immunoblot analysis of GST pull-down of Flag-tagged PPARg with GST-IRF3 or GST. (C) Co-localization of IRF3 (red) and PPARg (green) in the nucleus of RAVSMCs treated with PBS control or PDGF-BB (20 ng/mL); nuclei were stained with DAPI. (D and E) Schematic representation of PPARg deletion mutants and IRF3 deletion mutants (upper panels). (D) HEK293T cells were transfected with Flag-tagged IRF3, and Myc-tagged PPARg deletion mutants were immunoprecipitated from lysates with anti-Myc and immunoblotted with anti-Flag. Mapping of the IRF3-binding region of PPARg is shown (lower panel). (E) Alternatively, HEK293T cells were transfected with Flag-tagged PPARg, and Myc-tagged IRF3 deletion mutants, and lysates were immunoprecipitated with anti-Myc and immunoblotted with anti-Flag. Mapping of the PPARg-binding region of IRF3 is shown (lower panel). Data represent the mean + SD.

up-regulation and down-regulation following vascular injury should be investigated in future studies. Intriguingly, double immunostaining of IRF3 and SMA revealed that, instead of inflammatory cells, VSMCs appear to be the main source of IRF3 in the vasculature. Although this

expression may partly be because of the predominance of SMCs in neo-intima, it is highly tempting to speculate that IRF3 plays a critical role in the major pathological processes of SMCs during vascular wound repair. Indeed, upon IRF3-KO, we observed enhanced

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Supplementary material Supplementary material is available at Cardiovascular Research online.

Acknowledgements We greatly thank Dr Tadatsugu Taniguchi for generously providing IRF3 knockout mice. We also appreciate RIKEN BRC for shipping IRF3 knockout mice through the National BioResource Project of the MEXT, Japan, and Xiang Jie-Kong for performing carotid arterial injuries. Conflict of interest: none declared.

Funding This work was supported by the National Natural Science Foundation of China (nos 81170086, 81330005, 81370365, and 81370209), the National Science and Technology Support Project (nos 2011BAI15B02, 2012BA I39B05, 2013YQ030923-05, and 2014BAI02B01), the National Basic Research Program of China (no. 2011CB503902), the Ministry of Education New Century Outstanding Talents Support Program (no. NCET-10-0641), and the Independent Scientific Research Project of Wuhan University (no. 2012302020215).

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aggravation in neo-intima formation at a later phase post-injury than that observed at an earlier phase. Because the inflammatory response is generally considered an initiative event, the anti-proliferative function of IRF3 in the vascular wall may be largely attributable to SMCs. Indeed, VSMC-specific overexpression of IRF3 was sufficient to counteract injury-induced intimal growth. The present study elucidated a previously unknown intracellular signalling pathway induced by IRF3. In addition to the intrinsic transactivation potential of IRFs, these proteins may acquire specific functions by directly binding to another IRF member or other transcriptional factors, including PU.1, E47, and Stat, with their IAD.44 In the present study, we identified PPARg as a novel IRF3-interacting protein. PPARg is a member of the nuclear hormone receptor family best known for its systemic effects on atherogenic dyslipidemia.45 Moreover, PPARg transactivation inhibits VSMC proliferation at the vascular wall level,27 although its molecular mechanism is not fully understood. Here, we added that the binding of IRF3 to the AB domain of PPARg is necessary for the activation of PPRE luciferase activity and inhibition of SMC proliferation. In agreement with our study, several other co-activators, namely SCAN domain protein 1 (SDP1), CBP, tip60, and p300, also interact with and regulate the function of the AB domain of PPARg.46 – 48 We recently showed that IRF9 activates PPARa target genes by interacting with the C, D, and EF domains but not the AB domain.29 Thus, IRFs likely tightly regulate the activation of individual PPARs by recognizing specific domains. Interestingly, the application of PPARg agonists or PPARg gene transfer causes decreased PCNA expression in tumour cells49 and VSMCs,27 as was observed upon IRF3 overexpression in our study. PCNA is well known as a cell cycle marker and a requisite for chromosomal DNA replication, where it acts as a DNA-sliding clamp. Once recruited by IRF3, PPARg binds to the PPRE region of the PCNA gene, thereby negatively regulating its transcription. This speculation was confirmed in a rescue experiment in which overexpression of WT IRF3 instead of truncated IRF3 with a mutant IAD recapitulated the anti-proliferative function of IRF3. Manipulation of IRF3 had no effect on the expression and promoter activity of PCNA and SMC proliferation in sham-operated carotid arteries and control primary SMC cultures. Indeed, despite the persistent existence of PPARg in the nucleus of SMCs, as shown in Figure 5C and reported by Law et al.,50 we showed that IRF3 translocates into the nucleus upon PDGF-BB stimulation (Figure 5C). These observations further indicate that PPARg is a bona fide target of IRF3. Furthermore, PPARg has been implicated in SMC proliferation, migration, and apoptosis in response to mechanical vascular injury. We also observed that IRF3 positively regulates differentiation markers in our model, although whether this regulation is a direct effect of the IRF3–PPARg axis will be explored in future studies. Although we cannot rule out the possible involvement of all these events in the protective function of the IRF3– PPARg axis, the present study demonstrates that the anti-proliferative effect at least partially contributes to IRF3–PPARg complex and strengthens the hypothesis that PPARg acts directly at the vascular wall level. In summary, our data provide a new insight into the role of IRF3 in vascular wound repair as an inhibitor of VSMC proliferation after mechanical injury. We also identified a novel signalling pathway in which IRF3 directly binds to and serves as a previously undescribed co-activator of PPARg. Therefore, IRF3 activators may be of therapeutic value for treating restenosis after stent implantation, angioplasty, or bypass surgery.

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IRF3 inhibits neo-intima formation

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