Journal of Molecular and Cellular Cardiology 74 (2014) 284–294

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Original article

STAT4 deficiency protects against neointima formation following arterial injury in mice Lei Lv, Qiurong Meng, Meng Ye, Peng Wang ⁎, Guanhua Xue ⁎ Department of Vascular Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, China

a r t i c l e

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Article history: Received 3 March 2014 Received in revised form 4 June 2014 Accepted 6 June 2014 Available online 14 June 2014 Keywords: Apoptosis Migration Restenosis Signal transducer and activator of transcription 4 Vascular smooth muscle cell

a b s t r a c t Signal transducer and activator of transcription 4 (STAT4) has been associated with susceptibility to autoimmune diseases. Intriguingly, we previously reported that STAT4 might play a critical role in vascular smooth muscle cell (VSMC) proliferation. The present study therefore investigated the impact of STAT4 on VSMC migration, apoptosis and neointimal hyperplasia postinjury, as well as the underlying mechanisms. Guide-wire injury was associated with development of intimal neointima, STAT4 and phosphorylated STAT4 (p-STAT4) expressions were apparently up-regulated in the injured arteries. Neointima was greatly blocked in STAT4 knockout (KO) mice compared with wild type (WT) mice. A marked loss of inflammatory cells was identified in the vasculature postinjury in STAT4 KO mice. VSMC apoptosis was enhanced in the vasculature postinjury in STAT4 KO mice compared with WT mice. Cultured primary STAT4 KO VSMCs displayed reduced migration in comparison with WT controls. Mechanically, the deletion of STAT4 potently decreased the level of MCP-1, and its downstream targets MMP1 and MMP2. The effect of STAT4 on VSMC apoptosis was mainly mediated by the activation of the mitochondrial apoptotic pathway, as manifested by increased cytochrome c release and the activation of caspase-3. STAT4 therefore represents a promising molecular target to limit restenosis after artery intervention. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Angioplasty is widely used in clinical practice to treat various stenotic vascular disorders, but the postangioplasty reocclusion has been a big limit and the mechanism underlying the vascular remodeling remains poorly understood. Injury to the arterial wall triggers vascular smooth muscle cell (VSMC) proliferation, migration, and matrix secretion [1]. Defects in apoptotic pathways are also thought to occur in VSMCs during recurrent stenosis [2,3]. The resulting intimal hyperplasia is a common histological finding in restenosis after angioplasty [4]. Although the exact mechanism of these complicated events is not fully understood, the inhibition of VSMC proliferation and migration is considered a potential therapeutic interruption. We have previously reported that signal transducer and activator of transcription 4 (STAT4) gene knockdown overwhelmingly suppressed the proliferation of VSMCs. At the same time, VSMCs transduced by shSTAT4 lentivirus displayed more cells in G0/G1 phase and relatively low percentages of populations in S and G2/M-phases. An evident Abbreviations: DAPI, 4′-6-diamidino-2-phenylindole; DMEM, Dulbecco's modified eagle's medium; FBS, fetal bovine serum; H&E, hematoxylin-eosin; PBS, phosphate buffered saline; shRNA, short hairpin RNA; SMA, smooth muscle actin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VSMC, vascular smooth muscle cell. ⁎ Corresponding authors at: Department of Vascular Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 1630 Hao, Dongfang Road, Shanghai 200127, China. Tel.: +86 21 68383743; fax: +86 21 68383422. E-mail address: [email protected] (P. Wang).

http://dx.doi.org/10.1016/j.yjmcc.2014.06.001 0022-2828/© 2014 Elsevier Ltd. All rights reserved.

elevation in early apoptotic cells was noted following shSTAT4 lentivirus transfection [5]. However, an important gap exists regarding the fundamental molecular mechanisms mediated by STAT4 that is responsible for intimal hyperplasia. STAT4 is a member of the STAT family of molecules. These proteins are the molecular link from the cell surface cytokine receptors to the nucleus, where they serve as critical transcription factors. STAT4 expression is relatively restricted, with high expression in lymphoid and myeloid tissue, although more recent studies suggest that it might be expressed in other tissues as well. STAT4 is phosphorylated in response to IL-12 and IL-23 receptor activation, and is an important regulator of Th1 development [6,7]. STAT4 is also activated in response to type I interferons. So far, STAT4 has been associated mainly with autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus [8]. Guo et al. have concluded that STAT4 was a major regulator in proliferation of atherosclerosis-susceptible pigeon VSMCs [9]. In obese Zucker rats, lisofylline, an anti-inflammatory drug, reduced phosphorylated STAT4 (p-STAT4) activity in the vasculature and significantly attenuated neointimal responses to vascular injury in the carotid artery [10]. These observations highlight a critical role for STAT4 in peripheral arterial occlusive disease. Given that in the absence of STAT4, VSMC proliferation was inhibited, and VSMC apoptosis was prompted, we investigated whether STAT4 ablation modulated the migration of VSMCs as well, for the reason that VSMC migration also contributes to restenosis after angioplasty. Since it is unknown whether inhibited cell growth and promoted cell

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apoptosis indicated in vitro are relevant to neointimal formation in vivo, we also evaluated whether the neointimal hyperplasia was inhibited, the growth was abolished and the cell apoptosis was induced in the

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absence of STAT4 in a mouse model of vascular injury. Another goal of this study was to determine the mechanism by which STAT4 deficiency affected VSMC migration and apoptosis, as this remains unknown.

Fig. 1. STAT4 is activated in guide wire injured mouse femoral arteries. Expression of STAT4 or p-STAT4 in femoral arteries from sham and wire injured mice. Representative photographs of immunohistochemistry analysis are shown (magnification, ×400). (A) immunohistochemistry for α-SMA (b, e, h and k) and STAT4 (c and i) or p-STAT4 (f and l) was performed. Negativecontrol sections showed insignificant background staining (a, d, g and j). *, indicates the lumen. Representative blots of Western blotting analysis are shown (B). Tubulin served as an internal control. Bar graph shows relative densitometric values of Western blots (C and D). The enhanced STAT4 and p-STAT4 expression in intimal hyperplasia region was observed. Scale bar: 50 μm.

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Our study indicated for the first time that the deletion of STAT4 attenuated neointima formation through the inhibition of MCP-1 mediated pathway and the activation of mitochondrial apoptotic pathway. These findings support the idea that STAT4 is a key mediator of the VSMC migration and apoptosis, as well as help to identify STAT4 as a potentially novel drug target for vascular injury associated with atherosclerosis and restenosis.

mechanically dispersed by pipetting vigorously every 10 min for 40 to 60 min until N 90% of the cells were dispersed under the microscope. The cells were centrifuged at 1600 rpm for 5 min, then resuspended in 3 mL DMEM with 20% fetal calf serum (FCS), 2% penicillin–streptomycin, and cultured in plates or flasks. Cultured VSMCs showed 98% purity estimated by cell morphology and positive immunostaining with SM α-actin antibody.

2. Materials and methods

2.3. Adenovirus infection

2.1. Animals

STAT4 KO cells were grown in 6-well plates in DMEM containing 10% FBS. When the cells were nearly 50% confluent, the medium was changed to DMEM with 0.5% FBS. Adenoviruses containing the mouse MCP-1 gene (Ad-MCP-1), adenoviruses containing the mouse STAT4 gene (Ad-STAT4) and control adenoviruses (Ad-LacZ) were purchased from Genechem (Shanghai, China). The Ad-MCP-1, Ad-STAT4 or AdLacZ was added to the medium, respectively, at a multiplicity of infection (MOI) of 150. Ten hours later, the cells were washed three times with PBS, and the medium was changed to DMEM containing 10% FBS. Meanwhile, as a control (Con) in all experiments, an identical group of cells was left uninfected but was incubated for 10 h in serum-free DMEM.

All animal experiments were performed in accordance with and have been approved by the institutional ethical guidelines on animal care, Renji Hospital, Shanghai Jiaotong University, College of Medicine. STAT4 −/− mice on a BALB/c background (STAT4 KO) and wild-type BALB/c controls (WT) were purchased from Jackson Laboratories (Bar Harbor, Maine, USA). Mice were housed under specific pathogen-free conditions and maintained under standard conditions on a 12-h light, 12-h dark cycle with free access to water and food and used for experiments at 6–10 wk of age. 2.2. Isolation and culture of VSMCs

2.4. Scratch-induced wound healing assay and transwell migration assay To isolate primary cells, the mice were given an intraperitoneal (i.p.) injection of sodium pentobarbital (150 mg/kg) before death, which was confirmed by the absence of a heartbeat. Cultured WT and STAT4 KO VSMCs were isolated from the mouse thoracic aortas. The thoracic aortas were excised, washed in phosphate-buffered saline, and incubated in DMEM containing 1 mg/mL of Collagenase type II (Worthington Biochemical Corp) for 10 to 15 min. Then, under microscopic guidance, the adventitia was removed with fine forceps, the vessels were incised longitudinally, and the endothelial cells were gently scraped off. The vessels were then transferred to culture dishes containing DMEM with 1 mg/mL of Collagenase type I (Worthington Biochemical Corp) and 0.125 mg/mL of Elastase type III (Sigma), minced with scissors, incubated in 37 °C, 5% CO2 humidified incubator, and the cells were

VSMCs were plated at an initial density of 1 × 105 cells/mL to form a monolayer. Then, cells were wounded by scraping with a pipette tip to make a gap in the cell monolayer. The images of cell migration were observed at post scratching hour 0 (immediately after scratching) and 24, and photographed at 5 marked locations on each dish using a phasecontrast microscope (×100). The number of migrated cells was counted and averaged. The analysis of VSMC invasion involved the use of transwell plates that contained polycarbonate 8-μm pore membrane filters. VSMCs that were harvested using 0.25% trypsin were seeded in the upper wells (1 × 105 cells in 200 μL of serum-free DMEM containing 1% FBS), whereas the lower wells were filled with 600 μL DMEM containing 10% FBS. After 12 h of incubation, all nonmigrant cells

Fig. 2. Ablation of STAT4 suppresses VSMC migration. The confluent VSMC monolayers were wounded by scraping. Cell migration to the wound surface was monitored from 0 to 24 h (A). The number of migrated cells was counted and averaged (B). Migration assay was also carried out with transwell culture chambers (C). Five different areas of migrated cells were counted for each data point (D). Values expressed as mean ± SEM from six independent experiments; *P b 0.05 compared to WT.

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were removed from the upper face of the transwell membrane with a cotton swab and migrant cells were fixed and stained with 0.1% hexamethylpararosaniline chloride in 4% paraformaldehyde. Migration was quantified by counting the number of stained cells from 5 randomly chosen fields under a phase-contrast microscope and photographed (×100). All experiments were carried out in triplicate and repeated at least six times. 2.5. Preparation of cytosolic extract The cytochrome c apoptosis assay kit (Biovision, CA, USA) was used for cytosolic extract in this experiment. VSMCs were homogenized with the cytosol extraction buffer provided in the kit and then centrifuged at 1400 g for 10 min at 4 °C to remove debris. The supernatant was then centrifuged at 10,000 g for 30 min at 4 °C, and stored at − 80 °C in preparation for Western blot. 2.6. Western blot analysis Protein was extracted from the femoral arteries (after careful removal of adventitial tissue) of each group (five mice were used in each group) or cultured VSMCs. The supernatant was used for Western

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blot analysis as previously described [11]. The expression of α-SMA, STAT4, p-STAT4, MCP-1, MMP1, MMP2, cytochrome c, caspase-3, Fas, tumor necrosis factor receptor 1 (TNF-R1) and Fas-associated death domain (FADD) was analyzed by Western blotting. The antibodies against α-SMA, STAT4, p-STAT4, MMP1, cytochrome c, caspase-3, Fas and FADD were purchased from abcam (abcam, Cambridge, UK). The antibodies against MCP-1, MMP2 and TNF-R1 were purchased from Cell Signaling Technology, Inc. (Danvers, MA). The antibody against Tubulin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).] The expression level of Tubulin served as an internal control for protein loading. 2.7. Arterial injury models Each mouse was anesthetized by intraperitoneal injection of 50 mg/kg of pentobarbital diluted in 0.9% sodium chloride solution. Guide wire injury of the femoral artery was performed by three passages of a 0.014-inch guide wire (Radius X-TRa Support PTCA GUIDEWIRE; Radius Medical Technologies, Maynard, MA, USA) as described previously [12]. Briefly, the exposed muscular branch artery was dilated by topical application of one drop of 1% lidocaine hydrochloride. Transverse arteriotomy was performed in the muscular

Fig. 3. Effect of STAT4 on related proteins. Protein extracted from cultured VSMCs was resolved by SDS-PAGE, transferred to a PVDF membrane and blotted with anti-MCP-1, anti-MMP1, anti-MMP2, anti-cytochrome c, anti-caspase-3 or anti-tubulin antibodies to analyze protein expression. Representative blots are shown (A, C, E). Bands were quantified by densitometric analysis, and the results were shown as relative density compared with control (B, D, F). Values expressed as mean ± SEM from three independent experiments; *P b 0.05 compared to WT.

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Fig. 4. The protein levels in both WT and STAT4 KO mice under different conditions. Protein extracted from the femoral arteries was resolved by SDS-PAGE, transferred to a PVDF membrane and blotted with anti-MCP-1, anti-MMP1, anti-MMP2, anti-cytochrome c, anti-caspase-3 or anti-tubulin antibodies to analyze protein expression. Representative blots of mouse femoral arteries without endothelial damage (A, C, E) or with endothelial damage (B, D, F) are shown. The histogram (right panel) represents quantification of immunoblots. Values expressed as mean ± SEM from three independent experiments; *P b 0.05 compared to WT-mice.

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branch, microsurgery forceps were used to extend the arteriotomy through which the wire was carefully inserted into the femoral artery for more than 5 mm toward the iliac artery. Control sham-operated arteries underwent dissection, temporary clamping without passage of the wire. Surgery was carried out using a dissecting microscope. After removal of the wire, the arteriotomy site was ligated. None of the mice exhibited signs of ischemia to their hind limbs and all mice had full use of the limb immediately post-surgery. Following euthanasia using an overdose of sodium pentobarbital, the femoral artery was harvested at day 14, since STAT4 and p-STAT4 expression was induced and peaked in femoral artery at day 14 post injury. 2.8. Morphometric analysis Femoral arteries harvested at 14 days after wire injury were examined histologically for evidence of neointimal hyperplasia using routine hematoxylin eosin (HE) and Elastica van Gieson staining (EVG). Digital

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images were collected with light microscopy using an Olympus BHT microscope (Melville, NY) with 400× objective. Six evenly-spaced sections through each femoral artery were morphometrically analyzed. Intimal area (I) and medial area (M) were measured (arbitrary units) using ImageJ software (National Institutes of Health, Bethesda, MD). 2.9. Immunohistochemical staining A universal immunoenzyme polymer method was used for immunostaining. Sections were cut from formalin-fixed, paraffinembedded tissue blocks, mounted on polylysine-coated slides, dewaxed in xylene, and rehydrated through a graded ethanol series. After deparaffinization, antigen retrieval treatment was performed at 121 °C for 15 min in 10 mM sodium citrate buffer (pH 6.0), and was then treated with 3% hydrogen peroxide in methanol solution for 20 min to quench endogenous peroxidase activity. To block intrinsic avidin–biotin binding, the tissue slides were treated with avidin–biotin blocking kit reagents (Vectastain

Fig. 5. Effect of overexpression of MCP-1 on the migration of STAT4 KO VSMCs and related proteins. The confluent VSMC monolayers were wounded by scraping. Cell migration to the wound surface was monitored from 0 to 24 h (A). The number of migrated cells was counted and averaged (B). Migration assay was also carried out with transwell culture chambers (C). Five different areas of migrated cells were counted for each data point (D). Values expressed as mean ± SEM from six independent experiments. Protein extracted from VSMCs infected with Ad-LacZ or Ad-MCP-1 was resolved by SDS-PAGE, transferred to a PVDF membrane and blotted with anti-MCP-1, anti-MMP1, anti-MMP2 or anti-tubulin antibodies to analyze protein expression. Representative blots are shown (E). Bands were quantified by densitometric analysis, and the results were shown as relative density compared with control (F). Values expressed as mean ± SEM from three independent experiments.*P b 0.05 compared to Con/Ad-LacZ.

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Elite ABC kit, Vector Laboratories, Burlingame, CA) for 15 min. AntiSTAT4, anti-p-STAT4, anti-PCNA (Santa Cruz Biotechnology, Santa Cruz, Calif), anti-CD68, anti-CD45 (Dako, Carpinteria, Calif) and anti-CD4 (BD Pharmingen, San Diego, USA) antibodies were used as the primary antibodies. The final products were visualized using the 3-3′diaminobenzidine tetrahydrochloride (DAB) detection system (Dako Cytomation, Glostrup, Denmark). Percent positivity was calculated as number of positive cells/number of total nuclei. All experiments were performed in triplicate.

the manufacturer's protocol. Nucleus was counterstained with DAPI. Confocal microscopy was performed with the Confocal Laser Scanning Microscope Systems (Leica). The percentage of apoptosis was calculated by counting 6 times the number of apoptotic cells in all cells of each section.

2.10. Immunofluorescent staining and confocal microscopy Paraffin-embedded sections were incubated with anti-α-actin (abcam, Cambridge, UK) and further stained with appropriate TRITCconjugated secondary antibodies (Santa Cruz, CA). Apoptotic vascular cells were identified by TUNEL assay (Chemicon, USA) according to

Fig. 6. Lesion formation in WT and STAT4 KO mice after injury of the femoral artery. Wiremediated vascular injury was produced in WT and STAT4 KO mice. The femoral arteries were excised at 14 days after injury. Sample sections were stained with H&E (magnification, ×400) or Elastica-van-Gieson (magnification, ×400), and neointimal formation was evaluated. Scale bar: 50 μm. Representative photographs are shown (A and B). *, indicates the lumen. Bar graphs show the I/M ratio (C) quantified by Image-Pro Plus 6.0 software. Values expressed as mean ± SEM of 10–12 independent experiments. *P b 0.05 versus sham.

Fig. 7. Reduced inflammation in injured arteries of STAT4 deficient mice. Staining for inflammatory cell markers CD68 (macrophages) and CD45 (leukocytes) of wire-injured arteries from STAT4 KO mice (n = 8) or WT mice (n = 8) was performed. Macrophages/ leukocytes as a percentage of the total number of cells (nuclei) within the injured arteries were shown (A, B). Very few CD68-positive or CD45-positive cells were observed in the STAT4 deficient injured arteries. Staining for CD4+ cells of wire-injured arteries from STAT4 KO mice (n = 8) or WT mice (n = 8) was also performed. CD4+ cells as a percentage of the total number of cells (nuclei) within the injured arteries were shown (C). Very few CD4-positive cells were observed in the STAT4 deficient injured arteries. Data are expressed as mean ± SEM. *P b 0.05 versus WT.

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2.11. Data analysis Data are presented as the mean + SEM. The normal distribution was tested using the D'Agostino and Pearson omnibus normality test. If values were normally distributed, differences between two groups were tested by Student's t-test for unpaired means, differences between more than two groups use ANOVA followed by Bonferroni's multiple comparison test. If results did not follow a normal distribution, differences were compared using the Mann–Whitney or Kruskall–Wallis test followed by Dunn's post hoc analysis, respectively. Statistical significance was assumed when P reached a value b 0.05. 3. Results 3.1. Expression of STAT4 and p-STAT4 in neointima of injured femoral arteries To evaluate whether STAT4 could play roles in restenosis, we examined whether guide wire injured femoral arteries of mice exhibited an increased expression of STAT4 and activated STAT4 (P-STAT4). Mild to moderate intimal hyperplasia developed in guide wire injured mouse femoral arteries postinjury. The augmentation of STAT4 expression was observed in injured compared to sham-operated arteries 14 days after injury. Importantly, both immunohistochemical and Western blotting analysis demonstrated a clear increase of p-STAT4 expression in wire injured arteries compared with sham, thus implying that STAT4 might be involved in the development of neointimal lesions after wire injury in mice (Fig. 1). 3.2. STAT4 depletion in VSMCs retards migratory capacity To characterize the effect of STAT4 on VSMC migration, we used primary VSMCs isolated from WT and STAT4 KO mice. Western blot analysis verified that STAT4 and p-STAT4 were expressed in WT cells but not in STAT4 KO cells (Supplemental Fig. 1). We also evaluated the expression of STAT1/p-STAT1 and STAT3/p-STAT3, and no major differences were observed in WT cells and STAT4 KO cells (data not shown). Wound healing assay was employed to determine the effect of STAT4 deletion on the migrative capability of VSMCs. As shown in Figs. 2A and B, STAT4 deletion led to a marked decrease in migratory capacity. With transwell migration assay, a similar migration status was observed (Figs. 2C and D). These results indicated that STAT4 ablation could decrease the VSMC migratory capability. 3.3. STAT4 deficiency abolishes VSMC migration through regulation of MCP-1, MMP1 and MMP2 MCP-1 belongs to a C–C chemokine superfamily of small proteins that are important in recruiting and activating leukocytes during inflammation. In vitro studies have demonstrated that numerous types of cells including VSMCs are capable of expressing MCP-1 in the presence of serum or specific stimuli [13]. The migration of VSMCs is also mediated by chemokines [14]. Therefore, we tested whether MCP-1 participated in the regulation of STAT4 ablation. The protein expression of MCP-1 was investigated by Western blot in STAT4 KO cells. As shown in Figs. 3A and B, the protein level of MCP-1 was lower in STAT4 KO cells than that of the WT cells. Additionally, since MMP1 and MMP2 are induced by MCP-1 as evidence by a previous

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report [15], we also examined the expression of MMP1 and MMP2 in STAT4 KO cells. As expected, the expression of both MMP1 and MMP2 significantly decreased in STAT4 KO cells when compared with WT cells. Furthermore, in mouse femoral arteries with or without endothelial damage, the expression of MCP-1, MMP1 and MMP2 was substantially decreased in femoral arteries from STAT4 KO mice as compared with WT mice (Figs. 4A and B). Interestingly, impaired VSMC migration in STAT4 KO was rescued by the overexpression of MCP-1 (Figs. 5A–D). We also found that the overexpression of MCP-1 indeed caused enhanced expression of both MMP1 and MMP2 (Figs. 5E and F), suggesting that MCP-1 may require both MMP1 and MMP2 to promote migration in VSMCs. To explore further whether the MCP-1/MMP signaling may play a role in VSMC migration mediated by STAT4, we infected VSMCs with Ad-STAT4 and noticed that the expression of MCP-1, MMP1 and MMP2 was increased significantly in VSMCs (Supplemental Figs. 2A and B). Concurrently, the migration of VSMCs infected with Ad-STAT4 was stimulated (Supplemental Figs. 2E and F). 3.4. Deletion of STAT4 induces VSMC apoptosis in association with cytochrome c release and caspase activation Previously, we demonstrated that the deletion of STAT4 induced cell cycle arrest and apoptosis in vitro [5]. Here, to determine the molecular basis for VSMC apoptosis affected by STAT4 ablation, we measured the changes in cytochrome c and caspase-3. Interestingly, an increase in the amount of cytochrome c in the cytosolic fraction was seen in STAT4 KO cells when compared with WT cells (Figs. 3C and D). Furthermore, we assessed whether leakage of cytosol cytochrome c could lead to the activation of caspase-3 in STAT4 KO cells. The cleaved fragment of activated caspase-3 was found to be more pronounced and the level of procaspase-3 expression was decreased in comparison with those in WT cells, indicating the activation of caspase-3 (Figs. 3E and F). Furthermore, similar results for the release of cytochrome c and caspase-3 activation could also be observed in mouse femoral arteries with or without endothelial damage (Figs. 4C–F). The other apoptosis pathway, the death receptor pathway, which is also termed the extrinsic apoptosis pathway, is mediated by death receptors of TNF receptor superfamily. In this study, the expression of Fas, TNF-R1 and FADD was unchanged in STAT4 KO cells (Supplemental Fig. 3), thus eliminating the involvement of the death receptor pathway. 3.5. STAT4 deficiency attenuates postinjury neointima formation in the vasculature A guidewire was used to injure the femoral artery of age- and gender-matched WT or STAT4 KO mice. Both groups were from the same colony. Histological examination revealed that the neointima was markedly decreased in STAT4 KO mice compared with WT mice (Figs. 6A and B). The ratio of I/M is N2-fold greater in the WT mice than in STAT4 KO mice (Fig. 6C). 3.6. Deletion of STAT4 causes reduced accumulation of inflammatory cells within injured arteries Given the effects of STAT4 and MCP-1 on leukocytes, we examined whether STAT4 deficiency affected key inflammatory events associated with vascular injury. The recruitment of both macrophages

Fig. 8. STAT4 deficiency suppresses cell proliferation and promotes cell apoptosis in vivo.PCNA staining of wire-injured arteries from STAT4 KO mice (n = 10) or WT mice (n = 10) is shown, magnification, ×400 (A). PCNA-positive cells appear brown–black, and the number of PCNA-positive cells in the arteries from STAT4 KO mice was lower than that from WT mice. Quantification of PCNA-positive cells of femoral arteries from either the STAT4 KO mice or WT mice is shown (B). Immunohistochemical analysis of arterial sections for apoptosis by TUNEL assay is shown, magnification, ×400 (C). The TUNEL positive cells were stained green, smooth muscle α-actin was stained red and nuclei were stained blue by DAPI. Quantitation of the number of apoptotic VSMCs in the medial or intima layer of femoral artery is shown (D). DAPI-stained nuclei that were also positive for TUNEL staining were counted from randomly selected images and expressed as percentage of TUNEL-positive cells as compared to total cells. Significantly more apoptotic VSMCs were found in the STAT4 KO mice (n = 10) compared to WT mice (n = 10). Scale bar: 50 μm. Data are expressed as mean ± SEM. *P b 0.05 versus WT. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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(CD68-positive cells) and leukocytes (CD45-positive cells) was significantly reduced in STAT4 KO mice compared to WT controls (Figs. 7A and B) at 14 days of injury. We expanded the inflammatory cell analysis

by immunostaining with cell-specific markers. STAT4 deficiency also resulted in a dramatic reduction in the accumulation of CD4+ cells (Fig. 7C).

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3.7. Lack of STAT4 suppresses cell proliferation and promotes cell apoptosis in the vasculature postinjury Both VSMC proliferation and apoptosis contribute to the development of neointimal formation [16]. Previously, we confirmed that STAT4 affected VSMC proliferation and apoptosis in vitro [5], subsequently, here we performed in vivo experiments to determine whether STAT4 mediated cell proliferation and apoptosis. To assess VSMC growth, arterial sections were stained with anti-PCNA antibody and analyzed. STAT4 deficiency significantly suppressed cell proliferation, as indicated by a reduced number of PCNA-positive cells 14 days after femoral artery injury (Figs. 8A and B). TUNEL assay was also performed to determine the level of cellular apoptosis in neointima or media. Arterial walls in the WT mice after femoral artery injury contained few nucleus-stained cells. More apoptotic cells appeared in arterial walls of the STAT4 KO mice after femoral artery injury (Fig. 8C). As shown in Fig. 8D, the apoptosis rates in both intima and media in STAT4 KO mice were significantly increased when compared to WT mice (P b 0.05). 4. Discussion The major finding in this report is the remarkable effect that STAT4 has in the development of neointimal hyperplasia following arterial injury. Four key and novel observations are presented. First, the genetic ablation of STAT4 in mice results in almost complete inhibition of restenosis following endothelial denudation. Second, STAT4 exerts important effects on the migration of VSMCs via the inhibition of MCP-1 and matrix metallopeptidases (MMPs). Third, STAT4 deficiency is associated with reduced infiltration of inflammatory cells into the damaged vessel within 14 days after injury. Fourth, STAT4 deficiency inhibited cell growth as well as enhanced cell apoptosis in the vasculature postinjury in vivo, and its role of amplifying apoptosis may connect mitochondria to caspase cascades. The mechanism of restenosis can be described as the complex involvement of several growth factors and cytokines that induce the proliferation and migration of vascular smooth muscle cells (VSMCs) and the degradation of extracellular matrix (ECM) by MMPs [17]. In response to vascular injury, VSMC migration is in part, critical for neointima formation. Since we have previously addressed the important role of STAT4 in VSMC proliferation [5], it is reasonable to test whether STAT4 also affects VSMC migration. Intriguingly, our data showed for the first time that STAT4 knockdown abolished VSMC migration. There is evidence implicating that chemokines are involved in vascular diseases [18]. MCP-1, a CC chemokine, historically known to play an important role in leukocyte trafficking and activation, was among the first chemokines studied. Extensive research demonstrated its effect on VSMC migration [19,20]. In our study, concomitant with the changes that STAT4 ablation inhibited VSMC migration, the key proinflammatory cytokine MCP-1 level was decreased. Moreover, impaired VSMC migration in STAT4 KO was rescued by the overexpression of MCP-1. Thus, we proposed that MCP-1 might be associated with the depletion of STAT4 expression and play a primary role in reduced migration of VSMCs. Evidence suggests the relationship between MCP-1 and MMPs. It is reported that recombinant human MCP-1 induced MMP1 and MMP2 in primary human skin fibroblasts and a stable fibroblast cell line [15]. We have shown that both MMP1 and MMP2 levels significantly suppressed in parallel with the down-regulation of MCP-1 in STAT4 KO VSMCs. MMPs are believed to play a crucial role in connective tissue remodeling in a variety of physiological processes such as angiogenesis and wound healing [21]. The migration of VSMCs may require the degradation or remodeling of ECM surrounding the cells [22]. For ECM degradation or remodeling, a number of in vivo and in vitro studies have been established to relate MMPs [23]. Both MMP1 and MMP2 were thought essential for facilitating cell migration [24,25]. These observations and our current data once again supported the contribution

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of MMPs to aggressive VSMC migration. The precise mechanism underlying inhibited migration of VSMCs in the absence of STAT4 is not clear, but an indirect effect via MCP-1/MMP signaling is conceivable. Restenosis is associated with high resistance to apoptosis in the intima, reflecting the failure of the apoptotic pathway in the cells [26,27]. We have reported that STAT4 silencing induced the apoptosis of VSMCs in vitro in our earlier preliminary study. Here we demonstrated that STAT4 ablation could trigger VSMC apoptosis in the vasculature postinjury, which echoed its apoptosis stimulating effect in vivo. It is well known that apoptosis is executed by caspases, which are activated by two mechanisms, the Fas/TNF-R1 death receptor pathway and the mitochondria pathway [28,29]. We observed that in VSMCs, the depletion of STAT4 expression was associated with increased release of cytochrome c from mitochondria and caspase-3 activation, indicating that the ablation of STAT4 resulted in the release of cytochrome c from the mitochondria into the cytosol, activated caspase-3, and eventually caused apoptosis. It is not known whether other signaling pathways take part in induced apoptosis in the absence of STAT4. Therefore this aspect will be the subject of future in-depth studies. As we know that neointimal hyperplasia is the imbalance between cell proliferation and apoptosis, most of the studies indicate that the apoptosis of VSMCs is involved in atherosclerotic plaque vulnerability. It is suggested that VSMC apoptosis is sufficient to accelerate atherosclerosis, promote plaque calcification and medial degeneration, prevent expansive remodeling, and promote stenosis in atherosclerosis [30,31]. However, we demonstrated that increased apoptosis of VSMCs was protective in neointimal hyperplasia after vascular injury. Some recent studies support our findings. For example, Liu et al. demonstrated that n-butylidenephthalide inhibited neointimal hyperplasia in balloon injured rat carotid artery due to its dual effects of proliferative inhibition and apoptotic induction on VSMCs [32]. Kamenz et al. suggested that there was an insufficient increase in VSMC apoptosis concomitant with intimal hyperplasia [33]. This contradiction may be explained by two facts. First, although the pathogenesis of intimal thickening is similar to that of atherosclerosis, the difference of pathomechanism between them may exist. Second, there are pathological discrepancies between early and advanced stages of atherosclerosis or intimal hyperplasia. The adhesion and recruitment of inflammatory cells to the site of vascular injury are essential for the initiation and progression of neointimal formation [34,35]. STAT4 is a critical regulator of inflammation and T helper type I (Th1) lineage development. STAT4 KO mice lack many IL-12-stimulated responses, including the induction of IFN-γ secretion and the differentiation of Th1 cells. The phenotype of STAT4 KO mice is very similar to that of IL-12 KO mice, resulting in the failure to produce Th1 CD4+ T cells [36,37]. In our study, inflammation, determined as the percentage of CD68 positive macrophages and CD45 positive leukocytes, was decreased in injured arteries from STAT4 KO mice compared with arteries from WT mice. Importantly, STAT4 deficiency was also associated with a significant reduction in the recruitment of CD4+ T cells. Therefore, our results help to explain the mechanism by which STAT4 contributes to vascular inflammatory response and neointimal progression after vascular injury. Furthermore, we applied a femoral artery injury model that resembled human restenosis after angioplasty to STAT4 KO mice and showed that the STAT4 deficiency significantly prevented restenosis. Our demonstration that STAT4 depletion may prevent neointima formation highlights the potential of targeting STAT4 as an applicable therapeutic tool to ameliorate intimal hyperplasia. Considering that STAT4 has significant effects on biological remodeling in response to vascular injury, including VSMC proliferation, migration and apoptosis, our studies add new knowledge in relation to molecular mechanisms of vascular remodeling in hypertension, atherosclerosis and restenosis. These studies may have clinical relevance to prevention or treatment of vascular damage associated with neointima formation by regulating STAT4 gene.

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STAT4 deficiency protects against neointima formation following arterial injury in mice.

Signal transducer and activator of transcription 4 (STAT4) has been associated with susceptibility to autoimmune diseases. Intriguingly, we previously...
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