Atherosclerosis 234 (2014) 391e400

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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

Downregulation of the glucocorticoid-induced leucine zipper (GILZ) promotes vascular inflammation Rebecca T. Hahn a,1, Jessica Hoppstädter a,1, Kerstin Hirschfelder a, Nina Hachenthal a, Britta Diesel a, Sonja M. Kessler a, Hanno Huwer b, Alexandra K. Kiemer a, * a b

Department of Pharmacy, Pharmaceutical Biology, Saarland University, Saarbruecken, Germany Department of Cardiothoracic Surgery, Völklingen Heart Centre, Völklingen, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 August 2013 Received in revised form 28 February 2014 Accepted 23 March 2014 Available online 5 April 2014

Objective: Glucocorticoid-induced leucine zipper (GILZ) represents an anti-inflammatory mediator, whose downregulation has been described in various inflammatory processes. Aim of our study was to decipher the regulation of GILZ in vascular inflammation. Approach and results: Degenerated aortocoronary saphenous vein bypass grafts (n ¼ 15), which exhibited inflammatory cell activation as determined by enhanced monocyte chemoattractrant protein 1 (MCP-1, CCL2) and Toll-like receptor 2 (TLR2) expression, showed significantly diminished GILZ protein and mRNA levels compared to healthy veins (n ¼ 23). GILZ was also downregulated in human umbilical vein endothelial cells (HUVEC) and macrophages upon treatment with the inflammatory cytokine TNF-a in a tristetraprolin (ZFP36, TTP)- and p38 MAPK-dependent manner. To assess the functional implications of decreased GILZ expression, we determined NF-kB activation after GILZ knockdown by siRNA and found that NF-kB activity and inflammatory gene expression were significantly enhanced. Importantly, ZFP36 is induced in TNF-a-activated HUVEC as well as in degenerated vein bypasses. When atheroprotective laminar shear stress was employed, GILZ levels in HUVEC increased on mRNA and protein level. Laminar flow also counteracted TNF-a-induced ZFP36 expression and GILZ downregulation. MAP kinase phosphatase 1 (MKP-1, DUSP1), a negative regulator of ZFP36 expression, was distinctly upregulated under laminar shear stress conditions and downregulated in degenerated vein bypasses. Conclusion: Our data show a diminished expression of the anti-inflammatory mediator GILZ in the inflamed vasculature and indicate that GILZ downregulation requires the mRNA binding protein ZFP36. We suggest that reduced GILZ levels play a role in cardiovascular disease. Ó 2014 Published by Elsevier Ireland Ltd.

Keywords: Laminar shear stress mRNA stability Atherosclerosis Vein graft failure Toll-like receptor 2 (TLR2) Tristetraprolin (TTP, ZFP36) NF-kB

1. Introduction Atherosclerosis is a chronic inflammatory disease characterized by accumulation of inflammatory cells and lipids in the vascular wall of arteries. Vein graft remodeling is also characterized by inflammatory events [1,2]. The inflammatory activation of endothelial cells (EC) and macrophages (MF), triggered by different mediators such as tumor necrosis factor (TNF)-a, plays a central role within this process [3,4]. Generally, atherosclerotic plaques are localized in curvatures or bifurcations of vessels where static conditions as well as low and oscillatory shear stress occur [5]. These inflammatory conditions

* Corresponding author. Saarland University, Campus C2 2, P.O. Box 15 11 50, D-66041 Saarbruecken, Germany. Tel.: þ49 681 302 57301; fax: þ49 681 302 57302. E-mail address: [email protected] (A.K. Kiemer). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.atherosclerosis.2014.03.028 0021-9150/Ó 2014 Published by Elsevier Ireland Ltd.

promote the formation of atherosclerotic lesions by modification of gene and protein expression. In straight vessels the laminar blood flow is known as a main atheroprotective factor, which is important for the physiological function of the endothelium [5] via mechanisms called mechanotransduction [6]. Besides laminar shear stress as a physical inhibitor of vascular inflammation, other regulators antagonizing vascular inflammation are as yet poorly investigated. We hypothesized that glucocorticoid-induced leucine zipper (GILZ, synonymous TSC22D3) might represent a promising candidate. GILZ, an antiinflammatory protein inducible by glucocorticoids, was shown to be expressed in various cells of the immune system as well as in EC [7e9]. GILZ induction has been suggested to result in an inhibition of nuclear factor (NF)-kB and activator protein (AP)-1, thereby leading to diminished cytokine transcription [7,8]. We recently reported that the downregulation of GILZ upon TLR activation is critically involved in inflammatory macrophage (MF)

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activation [9]. In stimulated MF, GILZ was actively downregulated via GILZ mRNA destabilization, a process, which required the mRNA binding protein tristetraprolin (TTP, ZFP36). These results implicate an inflammatory function of ZFP36 as does the finding that ZFP36 is expressed in EC and foam cells of atherosclerotic lesions [10]. Moreover, ZFP36 expression is inhibited downstream of the antiinflammatory dual-specificity protein phosphatase 1 (DUSP1, MKP1) [11], a potent inhibitor of p38 mitogen-activated protein kinase (MAPK) [12]. The role of DUSP1 in the pathophysiology of atherosclerotic plaques is controversial, as both anti-inflammatory [13,14] as well as pro-atherosclerotic actions have been suggested [15,16]. Our data suggest a profound decrease of GILZ expression in human EC and vein graft stenosis under inflammatory conditions. GILZ downregulation is mediated by ZFP36 upregulation and leads to an activation and translocation of NF-kB. These findings suggest that the disappearance of GILZ plays a key role in the development of vascular disease. 2. Materials and methods 2.1. Materials For Western blot analyses, anti-GILZ (sc-26518), anti-p65 (sc109), anti-p50 (sc-114), and anti-MKP-1 (sc-1199) antibodies were purchased from SantaCruz (Heidelberg, Germany), anti-ZFP36 (T5327) and anti-Tubulin (T9026) were obtained from Sigma (Taufkirchen, Germany), and anti-TLR2 (Cat # 3268-1) from Epitomics (Burlingame, USA). The IRdye-labeled secondary antibodies goat anti-mouse, goat anti-rabbit, and donkey anti-goat were from LI-COR Biosciences (Bad Homburg, Germany), and anti-GILZ antibody for IHC (FL-134) was obtained from SantaCruz (Heidelberg, Germany). RNAlater, Qiazol and the RNeasy mini kit were from Qiagen (Hilden, Germany). All primers, probes, and oligonucleotides were obtained from MWG (Ebersfeld, Germany). 5 HOT FIREPolÒ EvaGreenÒ qPCR Mix Plus was from Solis BioDyne (Tartu, Estonia). siGILZ, siZFP36 (siGENOME SMARTpool) and siControl were from Dharmacon (Nidderau, Germany). pcDNA3-p38a-dn and pcDNA3-p38b2-dn were a gift from Prof. Dr. Jian-Dong Li, University of Rochester Medical Center, USA. pGL4.32[luc2P/NF-kB-RE/Hygro] containing 5 repetitive elements of the NF-kB consensus sequence GGGAATTTCC was from Promega (Heidelberg, Germany). The plasmids pZeo-hTTP-sense (ZFP36-V) and pZeo-hTTPantisense (Co-V) were a kind gift from Hartmut Kleinert (Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany). All other materials were purchased from Sigma (Taufkirchen, Germany), Roth (Karlsruhe, Germany), MP Biomedicals (Heidelberg, Germany), and Merck (Darmstadt, Germany). 2.2. Vessel specimens Human healthy saphenous veins and degenerated aortocoronary saphenous vein bypass grafts were obtained from patients undergoing coronary bypass surgery and immediately transferred into RNA stabilization solution (RNAlater). All samples were obtained with the consent of patients, and permission was given by the local ethics committee (ref #102/09). Total RNA and proteins were isolated using Qiazol according to the manufacturer’s protocol. 2.3. Immunohistochemistry Paraffin-embedded sections of murine femoral arteries were stained for GILZ with the CSA II Kit (Dako, Hamburg, Germany) [17,18].

2.4. Cell culture Preparation, cultivation, and characterization of HUVEC has been described previously [19,20]. HUVEC were used in passage three or four and grown in 6-well plates or on collagen (Roche, 1179179, 30 mg/ml in 0.2% acetic acid) coated glass slides in 4- well plates until confluence. Cultivation and preparation of primary human alveolar MFs have been described previously [9,21]. 2.5. Shear stress Confluent HUVEC grown on coated glass slides were exposed to laminar shear stress of 20 dyn/cm2 for 22 h in a parallel flow chamber modified after (Frangos et al., 1988) [22] and manufactured by upag AG (Vollersode, Germany). TNF-a (10 ng/ml) was added to the flow medium and flow was continued for another 2 h or 3.5 h. Flow was produced by a peristaltic pump (403U/VM purple/ white, Watson Marlow), and flow rates were adjusted to match a shear stress of 20 dyn/cm2. Silicon tubes were purchased from VWR (Darmstadt, Germany), and silicon mats for gasket construction were purchased from rfQ Medizintechnik (Tuttlingen, Germany). 2.6. Transfections HUVEC were grown until 80% confluency and transfected with siRNA (1 mM) or 20 mg/ml (luciferase vectors, p38dn) to 50 mg/ml (ZFP36 overexpression) plasmid DNA using AmaxaÒ NucleofectorÒ Technology according to the manufacturer’s instructions (Lonza, Basel, Switzerland). Experiments were performed 20 h or 24 h after transfection. 2.7. Luciferase reporter gene assay After transfection of HUVEC with pGL4.32[luc2P/NF-қB-RE/ Hygro] and siGILZ or siControl, luciferase activity was measured as described previously [9,19]. 2.8. Detection of mRNA Total RNA of cultivated cells was extracted with Qiazol or the RNEasy Mini kit. After residual DNA was removed (DNA-free kit, Applied Biosystems, Darmstadt, Germany), reverse transcription was carried out using the high capacity cDNA reverse transcription kit (Applied Biosystems, Darmstadt, Germany) and 0.25e1 mg total RNA. Real-time RT-PCR was performed in a Bio-Rad Cycler using gene-specific primers, dual-labeled probes (see online-only Data Supplement) or 5 HOT FIREPolÒ EvaGreenÒ qPCR Mix Plus [23]. cDNA cloned into pGEM-T Easy (Promega, Heidelberg, Germany) or a cDNA were used as a standard dilution series as described previously [21]. All samples and standards were analyzed in triplicate on each plate. 2.9. Western blot analysis Isolation of proteins of whole cell extracts was performed using Qiazol according to the manufacturer’s protocol or as reported previously [9,24]. Nuclear and cytosolic protein extracts were prepared as described [25]. Protein concentrations were determined by Bradford assay (Bio-Rad, Munich, Germany) or Pierce BCA Protein Assay Kit (Thermo Scientific, Bonn, Germany). Equal protein amounts were separated using 12% or 15% SDS/ PAGE gels. After electroblotting onto a PVDF membrane (Millipore GmbH, Schwalbach/Ts., Germany) GILZ, ZFP36, TLR2, DUSP1, p65,

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and p50 were detected using specific antibodies and the ODYSSEYÒ Infrared Imaging System (LI-CORÒ, LI-COR Biosciences, Bad Homburg, Germany) as described previously [26]. Relative protein amounts were determined using either Odyssey or ImageJ software. 2.10. Statistical analysis All experiments were performed with HUVEC or MF from at least two different donors and cell preparations. All other experiments were repeated at least three times. Data are expressed as mean þ SEM and statistical significance was determined by Student’s t-test. 3. Results 3.1. GILZ expression in degenerated vein bypasses Degenerated vein bypasses were identified as inflamed tissue due to significantly increased mRNA expression of the inflammatory markers CCL2 (MCP1) [18,19] and TLR2 [20] compared to healthy veins (Fig. 1A). Importantly, inflamed veins showed a significantly reduced GILZ mRNA expression (Fig. 1A). Similar results were observed when analyzing GILZ and TLR2 on protein level (Fig. 1B). In order to identify GILZ expressing cells, we stained murine histological samples and detected a distinct localization of GILZ protein in the endothelial layer of vessels (Fig. 1 in online-only Data Supplement). 3.2. Inflammatory response in EC and MF Both immunohistochemistry as well as data previously presented by ourselves and others suggested a distinct expression of GILZ in EC and MFs [8,9]. As shown in Fig. 1C and D, GILZ mRNA as well as protein was downregulated under inflammatory conditions, i.e. after TNF-a treatment in both cell types. Inflammatory cell activation was confirmed by an induction of TLR2 and CCL2 (MCP1) mRNA in HUVEC (Fig. 1E). Interestingly, the mRNA binding protein ZFP36, known to destabilize GILZ mRNA in MFs [9], was strongly induced by TNF-a (Fig. 2A). Early after TNF-a treatment, ZFP36 was present in its lowphosphorylated, low molecular weight form, which is known to be the active, but instable variant. At later time points, the phosphorylated high-molecular weight isoform of ZFP36, which is inactive but stable [27], predominated (Fig. 2A). In order to examine the functional links between GILZ and ZFP36 expression in HUVEC, an siRNA-mediated knockdown of ZFP36 was performed. In HUVEC transfected with control siRNA (siCo), ZFP36 was upregulated upon TNF-a treatment, which was paralleled by reduced GILZ expression. In contrast, GILZ downregulation was abrogated in siZFP36transfected cells (Fig. 2BeD). To support these findings, we transiently overexpressed ZFP36 in HUVEC (Fig. 2E), which indeed resulted in reduced GILZ levels. Taken together, these results suggest a key role for ZFP36 in the regulation of GILZ expression. 3.3. Regulation of GILZ and ZFP36 by anti-inflammatory laminar shear stress While inflammatory conditions downregulated GILZ, laminar shear stress as an anti-inflammatory and anti-atherosclerotic stimulus elevated GILZ mRNA levels in HUVEC (Fig. 3A). The same finding was observed on protein level (Fig. 3B). The antiinflammatory activation state was confirmed by elevated HO1 (heme oxygenase 1) mRNA expression (Fig. 3C).

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While ZFP36 was induced by TNF-a on the transcriptional level, its expression tended to decrease during laminar flow (Fig. 3D). A combination of laminar flow and TNF-a completely abrogated GILZ downregulation, which we typically observed upon TNF-a treatment in statically cultured HUVEC (Fig. 3E). Concordantly, ZFP36 induction by TNF-a was abrogated in cells exposed to laminar flow (Fig. 3F), suggesting that the lack of ZFP36 induction contributes to the elevated GILZ expression in TNF-a-treated shear stressed cells. Interestingly, ZFP36 levels were elevated in degenerated, inflamed venous bypasses (Fig. 3G).

3.4. Mechanisms of GILZ downregulation in inflammation Our data suggest an inverse regulation of ZFP36 and GILZ in inflammation and under anti-inflammatory conditions in HUVEC. In fact, ZFP36 has been reported by ourselves to be a destabilizer of GILZ mRNA in macrophages [9] and has been shown to be regulated by DUSP1 [11], which inhibits mitogeneactivated protein kinases, most importantly p38 MAPK [12]. To assess the influence of p38 MAPK activation on ZFP36 and GILZ expression, p38 phosphorylation in HUVEC was inhibited by pretreatment with the p38 MAPK inhibitor SB203580 prior to TNFa challenge. p38 inhibition antagonized TNF-a-mediated ZFP38 induction both on mRNA and protein level (Fig. 4A, B and E). Similar data were obtained after transfection of HUVEC with a dominant negative mutant of p38 (Fig. 2 in online-only Data Supplement). Reduced ZFP36 expression was accompanied by an abrogation of GILZ downregulation (Fig. 4C, D and F), indicating that p38 inhibition enhances GILZ expression by reducing ZFP36 levels. Interestingly, a significant downregulation of DUSP1 protein expression in degenerated vein bypasses could be detected (Fig. 4G). In cultivated HUVEC, an upregulation of DUSP1 mRNA levels by laminar shear stress and downregulation by TNF-a was observed (Fig. 4H).

3.5. Functional implications of GILZ downregulation Finally, we aimed to determine whether GILZ downregulation has functional implications in inflammatory activation of HUVEC. GILZ knockdown was shown to increase MF activation, as assessed by TNF-a induction and NF-kB activation [9], whereas GILZ overexpression inhibited NF-kB activation in EC [8]. We therefore knocked down GILZ in HUVEC by siRNA resulting in reduced GILZ mRNA and protein levels (Fig. 5A, B). GILZ knockdown induced the nuclear translocation of the NF-kB subunits p65 and p50 (Fig. 5C). Using a luciferase reporter gene under an NF-kB promoter, we showed that GILZ knockdown significantly increased NF-kB activity compared to control transfected cells (Fig. 5D). The absence of GILZ after double-transfection with GILZ siRNA and the luciferase plasmid was confirmed using Western blot analysis (data not shown), whereas functionality of the luciferase assay was verified measuring TNF-a-induced NF-kB activity (Fig. 3 in onlineonly Data Supplement). These findings showed that disappearance of GILZ can liberate NF-kB and induce its activation and translocation. We then assessed whether downregulation of GILZ had any influence on TLR2 [20] and adhesion molecule expression [28]. We found indeed that the TNF-a-induced expression of TLR2, ICAM1 and SELE (E-selectin) was enhanced in siGILZ-transfected HUVEC compared to equally treated siCo-transfected controls (Fig. 5E), suggesting that the absence of GILZ drives a proinflammatory response.

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Fig. 1. GILZ mRNA and protein expression in clinical samples, macrophages, and EC. Panel A: GILZ, TLR2, and CCL2 mRNA expression in human veins. mRNA expression in saphenous veins (n ¼ 23) and degenerated aortocoronary saphenous vein bypass grafts (n ¼ 15) was measured by real-time RT-PCR using ACTB for normalization. Data are presented as individual values (black squares) as well as 25th and 75th percentiles as boxes within geometric medians (line), arithmetic medians (square), 10th and 90th percentiles as whiskers, and ends of values (cross). Panel B: GILZ and TLR2 protein expression in human veins. Equal protein amounts were assessed by Western blot analysis using tubulin as loading control. One representative blot out of 4 independent experiments with 11 healthy and 12 degenerated samples is shown. Signal intensities were measured relative to tubulin values, and values for healthy controls were set as one. Panel C-E: TNF-a response. Primary human MF (C) or HUVEC (D, E) were treated with 10 ng/ml TNF-a for 2 h or for the indicated time points. Protein levels were measured by Western blot analysis using tubulin as loading control. mRNA levels were determined by real-time RT-PCR using ACTB for normalization. For HUVEC, results of three independent experiments are shown (duplicates). For MF, data represent means of two independent experiments performed in triplicate.

4. Discussion The anti-inflammatory mediator GILZ is inducible by glucocorticoids and acts as an inhibitor of NF-kB and AP-1 by direct binding and inhibition of their nuclear translocation [7]. Expression of GILZ

has been described for many human tissues and cell types [7e9]. These include EC and MF, suggesting a potential role for GILZ in the pathogenesis of atherosclerosis. We previously reported that TLR activation leads to GILZ downregulation in primary human MF and lung tissue of LPS-

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Fig. 2. ZFP36-dependent GILZ downregulation in EC. Panel A: Induction of ZFP36 by TNF-a. HUVEC were treated with 10 ng/ml TNF-a for the times indicated and protein levels of ZFP36 were assessed by Western blot analysis using tubulin as loading control (n ¼ 3, duplicates). Values for untreated cells were set as one hundred percent or one, *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated cells. Panel B-D: Influence of ZFP36 knockdown on TNF-a-induced GILZ downregulation. HUVEC were transfected with ZFP36 siRNA (siZFP36) or control siRNA (siCo). 20 h after transfection, cells were treated with TNF-a (10 ng/ml). ZFP36 and GILZ protein expression were analyzed by Western blot using tubulin as loading control. One representative blot out of 3 independent experiments (A) and data of three independent experiments (duplicates) are shown (BeD). Panel E: Influence of ZFP36 overexpression on GILZ expression. HUVEC were transfected with either control (Co-V) or ZFP36 expression (ZFP36-V) vector and harvested after 24 h. ZFP36 and GILZ expression were assessed by western blot analysis. Tubulin served as a loading control. Values indicate relative signal intensities of 3 independent experiments performed in duplicate after normalization to tubulin values. Values for Co-V transfected cells were set as one. *p < 0.05, **p < 0.01, compared to Co-V transfected cells.

treated mice. [9] Here, we present evidence that diminished GILZ expression is a deleterious response in degenerated vein bypasses. A GILZ downregulation or even absence in inflammatory diseases, such as chronic rhinosinusitis, Crohn’s disease, or tuberculosis has been reported in the literature [9,29,30], indicating that the absence of GILZ is a general phenomenon in inflammation.

GILZ was recently shown to exert anti-inflammatory actions in different animal models of inflammatory disease [31e33], although the observed effects were primarily attributed to interference with leukocyte functions. In addition to leukocytes, EC play a key role in vascular inflammation [8]. Investigations on the role of GILZ in EC were mostly carried out after GILZ overexpression. In this context,

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Fig. 3. Inflammatory responses in HUVEC and aortocoronary saphenous vein bypass grafts. Panel A, B, C, D, E, F: GILZ, HO1, and ZFP36 expression under inflammatory and antiinflammatory conditions. HUVEC were treated with 10 ng/ml TNF-a under static conditions or exposed to 24 h laminar flow (20 dyn/cm2) as indicated. GILZ (A), HO1 (C), and ZFP36 (D) mRNA levels were determined by real-time RT-PCR using ACTB for normalization. Values for untreated cells were set as one, **p < 0.01, ***p < 0.001 compared to untreated cells under static conditions. Data were obtained from 4 independent experiments performed in duplicate. GILZ (B, E) and ZFP36 (F) protein levels were measured by Western blot analysis using tubulin as loading control ((B, F) n ¼ 6; (E) n ¼ 8 derived from 4 independent experiments). Values for untreated cells were set as one as indicated, **p < 0.01, ***p < 0.001, þp ¼ 0.051 compared to untreated cells. Data represent means of 4 independent experiments performed in duplicate. Panel G: ZFP36 expression in human veins. Equal protein amounts were assessed by Western blot analysis using tubulin as loading control. One representative blot out of 3 independent experiments with 8 healthy and 10 degenerated samples is shown. Signal intensities were measured relative to tubulin values, and values for healthy samples were set as one.

GILZ was shown to reduce the capacity of EC to support leukocyte interactions, i.e. rolling, adhesion, and transmigration [8]. Although a growing body of evidence has accumulated regarding the importance of GILZ in EC, little is known about the role of endogenous GILZ in EC and the mechanisms regulating its expression. Herein, we confirm a substantial expression of GILZ in EC [8,34,35] and demonstrate that GILZ expression is considerably decreased in TNF-a-challenged HUVEC and MF. In contrast, atheroprotective laminar shear stress induced GILZ mRNA and protein in EC when compared to static culture conditions, as previously suggested by a microarray approach [35].

Blood flow influences atherosclerosis by exerting shear stress on vascular endothelium, which differs in magnitude and direction depending on the vascular anatomy and blood pressure. Shear stress alters the phenotype of EC, which respond to it via mechanoreceptors that translate mechanical distortions (see Data Supplement, Fig. 4) into various molecular signals [36]. Correspondingly, EC were shown to respond to laminar shear stress by glucocorticoid receptor translocation [37]. As multiple glucocorticoid responsive elements are present in the GILZ promoter [7], GILZ induction by laminar shear stress might be a result of glucocorticoid receptor activation.

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Fig. 4. Mechanisms of ZFP36 regulation. Panels A-F: ZFP36 and GILZ expression after inhibition of p38 MAPK activity. HUVEC were pretreated with solvent control DMSO or SB203580 (10 mM), followed by treatment with 10 ng/ml TNF-a for 2 h (AeB, EeF) or 4 h (CeD). Protein levels were measured by Western blot analysis using tubulin as loading control (AeD). mRNA levels were determined by real-time RT-PCR using ACTB for normalization (EeF). Values for cells pretreated with the solvent control DMSO, either in the presence (B, E) or absence (D, F) of TNF-a were set as one hundred percent. Data show means of three (AeD) or two (EeF) independent experiments performed in triplicates, *p < 0.05, **p < 0.01, ***p < 0.001, n.s: not significant. Panel G: DUSP1 protein expression in human veins. Equal protein amounts were assessed by Western blot analysis using tubulin as loading control. One representative blot out of 4 independent experiments with 11 healthy and 12 degenerated samples is shown. Signal intensities were measured relative to tubulin values, and values for samples from healthy tissues were set as one. Panel H: DUSP1 expression under pro- and anti-inflammatory conditions. HUVEC were treated with 10 ng/ml TNF-a under static conditions or exposed to 24 h laminar flow (20 dyn/cm2) as indicated. mRNA levels were determined by real-time RT-PCR using ACTB for normalization. Values for untreated cells under static conditions were set as one, **p < 0.01, ***p < 0.001 compared to untreated cells. Data represent means of 4 independent experiments performed in duplicate.

Most interestingly, a TNF-a challenge failed to diminish GILZ levels in HUVEC exposed to laminar shear stress. We previously reported a correlation between GILZ downregulation and upregulation of the mRNA-binding protein ZFP36 in MF [9]. In line with these findings, GILZ downregulation was paralleled by a rapid and extensive induction of ZFP36 in TNF-a-treated HUVEC, suggesting a possible role of ZFP36 in GILZ repression. Both ZFP36 overexpression and knockdown experiments confirmed this

assumption. Accordingly, ZFP36 induction was also seen in atherosclerotic vessels by others and ourselves [10], but not under anti-inflammatory conditions, i.e. laminar shear stress. ZFP36 upregulation is usually considered to be an anti-inflammatory feedback loop, since inflammatory cytokines, such as TNF-a, represent the most extensively studied ZFP36 targets [38,39]. Therefore, ZFP36 upregulation has been suggested to be an atheroprotective process [10]. It has to be noted, however, that ZFP36-

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Fig. 5. GILZ downregulation drives pro-inflammatory responses in EC. Panel A, B: GILZ knockdown. HUVEC were transfected with GILZ siRNA (siGILZ) or control siRNA (siCo). Cells were harvested after 20 h (A, B) or 24 h (A). GILZ protein expression was analyzed by Western blot using tubulin as loading control (A) and mRNA expression was quantified by realtime RT-PCR using ACTB for normalization (B). One representative blot out of 4 independent experiments (A) and data of three independent experiments (duplicates) are shown. (B) Panel C: NF-kB translocation after GILZ knockdown. HUVEC were nucleofected with GILZ siGILZ or siCo and harvested after 20 h. Equal protein amounts were assessed by Western blot analysis using tubulin as loading control. Results of 4 independent experiments are shown. *p < 0.05, #p ¼ 0.065 compared to siCo transfected cells. Panel D: NF-kB activity after GILZ knockdown. HUVEC were transfected with either siCO or siGILZ and an NF-kB driven luciferase reporter construct. Cells were harvested 20 h post transfection. NF-kB activity was determined by measuring luciferase activity. Data represent means of 4 independent experiments performed in quinticate. Values for siCo were set as one, ***p < 0.001 compared to siCo transfected cells. Panel E: Impact of GILZ knockdown in HUVEC on TNF-a-induced expression of TLR2 and adhesion molecules. HUVEC were transfected with siGILZ or siCo. 20 h after transfection, TNF-a was added for an additional 4 h mRNA expression was quantified by real-time RT-PCR using ACTB for normalization. Values obtained for siCo transfected cells were set as one hundred percent. *p < 0.05, ***p < 0.001 compared to siCo transfected cells.

associated mRNAs encode a broad spectrum of proteins engaged in various cellular processes, including the anti-inflammatory mediator IL-10 [40]. Therefore, additional factors might be needed to orchestrate ZFP36 actions. These trigger factors may include other mRNA-binding proteins, whose binding might be further modulated by miRNAs [41]. Up to date, however, the identity of other mRNA-binding proteins and/or miRNAs interacting with GILZ mRNA remains elusive. In addition, we can not rule out the possibility that GILZ expression might also be affected by other mechanisms, such as proteasomal degradation [42]. The p38 MAPK pathway has been shown to promote ZFP36 upregulation in human and murine MFs by increasing ZFP36 expression [11,43,44]. In line with these reports, we found that p38 inhibition also markedly reduced ZFP36 levels in HUVEC, whereas GILZ downregulation upon TNF-a-treatment was abrogated, indicating that p38 regulates GILZ expression via a mechanism involving ZFP36. DUSP1, which can be induced in EC by laminar shear stress, has been suggested to protect arteries from inflammation, mainly by

suppressing the activities of p38 and JNK MAP kinases in the vascular endothelium [14]. Investigations on LPS-induced ZFP36 expression in MF obtained from DUSP1 knockout mice or in epithelial and macrophage-like cell lines after siRNA-mediated DUSP1 silencing recently revealed a reverse correlation between DUSP1 and ZFP36 expression: lack of DUSP1 was shown to result in enhanced ZFP36 expression, whereas p38 inhibition had opposing effects. This led to the conclusion that DUSP1 suppressed ZFP36 expression by abrogating p38 activity [11]. Our data showing DUSP1 induction in HUVEC exposed to laminar shear stress, paralleled by diminished ZFP36 levels and elevated GILZ expression, suggest a similar mechanism. In line with this finding, we found that DUSP1 was expressed in healthy, but not in atherosclerotic vessels. To assess functional implications of GILZ downregulation under inflammatory conditions, we used siRNA to knockdown GILZ in HUVEC and subsequently measured NF-kB activity. NF-kB is a major pro-inflammatory transcription factor, whose translocation results in the expression of various cytokines, growth factors, and adhesion

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molecules [28,45]. Endothelial cell-specific NFekB inhibition has been reported to protect mice from atherosclerosis and vascular remodeling [46,47], emphasizing the critical role of NF-kB in the pathogenesis of atherosclerosis. We found that the absence of GILZ enhanced NF-kB activity, i.e. nuclear translocation of p65 and p50 and NF-kB-dependent transcription. Correspondingly, NF-kB activation has been reported to be diminished after GILZ overexpression in several cell types [30,48e50]. Moreover, GILZ knockdown has been shown to activate airway epithelial cells by increased cytokine expression [50] and to amplify NF-kB activation in LPS-treated MFs [9]. A recent report by Cheng et al. [8] contrasts our findings by showing that GILZ siRNA did not modify TNF-a-induced endothelial cell responses, i.e. leukocyte rolling, transmigration, and IL-6 release. However, these results can hardly be compared to our studies, since both treatment schemes and the readout parameters differ. The fact that TNF-a treatment alone results in diminished GILZ expression might complicate matters further, as it might be hard to distinguish between the effects of the naturally occurring GILZ downregulation and the siRNA-mediated GILZ knockdown. Our data on GILZ siRNA-transfected HUVEC support a functional importance of enhanced NF-kB signaling. We found indeed that several pro-inflammatory mediators, including TLR2, SELE and ICAM1 [20,28], were upregulated in TNF-a-treated siGILZtransfected HUVEC compared to equally treated siCo-transfected controls. These findings indicate that GILZ downregulation might be a critical step in atherogenesis. 5. Conclusion Taken together, our data show that the expression of GILZ in human EC prevents vascular inflammation by suppressing NF-kB activation. This assumption is supported by the decrease of GILZ levels found in atherosclerotic vessels, suggesting upregulation of GILZ as a potential target for the treatment of the inflamed endothelium. Acknowledgments We would like to thank Theo Ranssweiler for excellent technical assistance and Nicolas Frank as well as Susanne Schütz for help in the preparation of histological samples. We also thank PD Dr. Dieter Mink from the Klinikum Saarbrücken for supply of umbilical cords and Dr. Matthias Engel for providing the NF-kB plasmid. Finally, we thank Christian and Alexander Hahn for support in development and construction of parallel flow chambers. The work was supported, in part, by the Deutsche Forschungsgemeinschaft (KI 702). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.03.028. References [1] Karper JC, de Vries MR, van den Brand BT, Hoefer IE, Fischer JW, Jukema JW, et al. Toll-like receptor 4 is involved in human and mouse vein graft remodeling, and local gene silencing reduces vein graft disease in hypercholesterolemic APOE*3Leiden mice. Arterioscler Thromb Vasc Biol 2011;31:1033e40. [2] McPhee JT, Nguyen LL, Ho KJ, Ozaki CK, Conte MS, Belkin M. Risk prediction of 30-day readmission after infrainguinal bypass for critical limb ischemia. J Vasc Surg 2013;57:1481e8. [3] McKellar GE, McCarey DW, Sattar N, McInnes IB. Role for TNF in atherosclerosis? Lessons from autoimmune disease. Nat Rev Cardiol 2009;6:410e7.

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