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The lignan (+)-episesamin interferes with TNF-a-induced activation of VSMC via diminished activation of NF-KB, ERK1/2 and AKT and decreased activity of gelatinases C. Freise1 and U. Querfeld2 1 Department of Pediatric Nephrology and Center for Cardiovascular Research, Charite - University Medicine, Campus Virchow Clinic, Berlin, Germany 2 Department of Pediatric Nephrology, Charite - University Medicine, Campus Virchow Clinic, Berlin, Germany

Received 4 August 2014, revision requested 3 September 2014, revision received 8 September 2014, accepted 24 September 2014 Correspondence: Dr. C. Freise, Ph.D., Center for Cardiovascular Research, Charite - University Medicine Berlin, Forschungshaus II, Hessische Str. 3-4, 10115 Berlin, Germany. E-mail: [email protected]

Abstract Aim: Activation of vascular smooth muscle cells (VSMC), a key event in the pathogenesis of atherosclerosis, is triggered by inflammatory stimuli such as tumour necrosis factor-alpha (TNF-a) causing a mitogenic VSMC response. The polyphenol (+)-episesamin (ES) was shown to counteract TNF-a-induced effects, for example in macrophages. Aiming for novel therapeutic options, we here investigated whether ES protects VSMC from TNF-a-induced growth and migration, which both contribute to the onset and progression of atherosclerosis. Methods: Human and murine VSMC were treated with combinations of ES and TNF-a. Expressions of mRNA were analyzed by RT-PCR. Enzymatic activities and proliferation were determined by specific substrate assays. Cell signalling was analyzed by Western blot and reporter gene assays. Migration was assessed by wound healing assays. Results: ES at 1–10 lM reduced basal and TNF-a-induced VSMC proliferation and migration due to impaired activation of extracellular signal-regulated kinases (ERK)1/2, Akt (protein kinase B), nuclear factorkappa B (NF-KB) and vascular cell adhesion molecule (VCAM)-1. This was accompanied by reduced expression and secretion of matrix metalloproteinases (MMP)-2/-9, which are known to promote VSMC migration. Specific inhibitors of Akt, NF-KB and MMP-2/-9 reduced TNF-a-induced VSMC proliferation, confirming ES-specific effects. Besides, ES reduced TNF-a- and H2O2-induced oxidative stress and in parallel induces antiinflammatory haem oxygenase (HO)-1 expression. Conclusion: ES interferes with inflammation-associated VSMC activation and subsequent decreased proliferation and migration due to anti-oxidative properties and impaired activation of NF-KB, known contributors to atherogenesis. These results suggest ES as a complemental treatment of VSMC specific vascular diseases such as atherosclerosis. Keywords atherosclerosis, (+)-episesamin, matrix metalloproteinases, NF-kappa B, TNF-a, vascular smooth muscle cells.

Atherosclerotic lesions are characterized by accumulation of cells and lipids within the arterial intima (Ross 1993). Chronic inflammation is an underlying process

of atherosclerosis including the release of growth factors and proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-a) by vascular smooth

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muscle cells (VSMC) and macrophages (Abedi & Zachary 1995, Clausell et al. 1995, Jovinge et al. 1997). A key event in the early stage of atherogenesis is the migration of VSMC from the arterial media to the intima. This requires remodelling of the surrounding extracellular matrix which occurs by proteolytic activity of matrix metalloproteinases (MMP)-2 and -9 (Newby & Zaltsman 2000). Expression of MMP-9 was found to be upregulated during atherosclerosis and MMP-2/-9 were shown to regulate VSMC proliferation/migration (Bendeck et al. 1994, Mason et al. 1999, Uzui et al. 2000, Cho & Reidy 2002, Kuzuya et al. 2003, Vigetti et al. 2006). While MMP-2 generally shows a constitutive expression, MMP-9 can be induced by TNF-a which includes activation of a functional NF-KB element (Sato et al. 1993), mitogen-activated protein kinases (MAPK) as well as of phosphoinositide-3-kinase (PI3K) and Akt signalling (Moon et al. 2004). Thus, VSMC activation by inflammatory signalling is a critical step in atherogenesis, and inhibition of VSMC activation appears an attractive target for prevention and/or pharmacological treatment of atherosclerosis. Another known important contributor to VSMC activation is oxidative stress which impacts the proliferation, migration or apoptosis of VSMC (Antoniades et al. 2009, Heistad et al. 2009). VSMC in atherosclerotic lesions exhibit high levels of reactive oxygen species (ROS) (Yokoyama et al. 2000, West et al. 2001) which promote acute inflammatory responses and subsequent vasculature dysfunction (Limon-Pacheco & Gonsebatt 2009, Sheehan et al. 2011). Thus, the suppression of oxidative stress and of arterial inflammation with subsequent block of VSMC proliferation and migration represents a rational approach in the treatment of atherosclerosis as also investigated by others (Kim et al. 2012). Several recent epidemiological studies have shown that greater dietary intake of polyphenols, especially from lignans, flavanols and hydroxybenzoic acids, is associated with a decreased risk of cardiovascular disease (CVD) (Covas et al. 2006, Tresserra-Rimbau et al. 2014). (+)-Episesamin (ES), an abundant lignan in sesame seed, has been identified as an active principle in an aqueous extract of Lindera obtusiloba (Japanese Spicebush) with a strong anti-inflammatory potential (Freise et al. 2010, Trowitzsch-Kienast et al. 2011). We have previously shown in vitro that ES decreases the activity of MMP-9 and the inflammatory response induced by TNF-a treatment in macrophages, preadipocytes and tumour cells (Freise et al. 2012b, 2013). However, the effects of ES on VSMC have not been studied previously. We hypothesized that the observed beneficial effects of lignans like ES could also provoke an inhibition of 2

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VSMC activation. Therefore, we here investigated in vitro whether ES protects human and murine VSMC from TNF-a-induced mitogenic effects.

Materials and methods All chemicals and toxins were obtained from SigmaAldrich (Munich, Germany), if not stated otherwise, and of the highest purity available.

Cell culture Human aortic VSMC (Life technologies, Karlsruhe, Germany) were cultured in phenol red containing Medium 231 supplemented with the smooth muscle growth supplement (Life technologies). The murine VSMC cell line (MOVAS-1) was purchased from ATCC (ATCCâ CRL-2797TM) and cells were cultured in a humidified atmosphere at 37°C and 5% CO2. The standard culture medium consisted of phenol red free DMEM (Gibcoâ, Life technologies) with 862 mg l1 L-alanyl-L-glutamine, 1.0 g l1 glucose, 50 lg ml1 streptomycin, 50 units ml1 penicillin, supplemented with 10% heatinactivated foetal bovine serum (FBS; Biochrom, Berlin, Germany).

Isolation of (+)-episesamin from Lindera obtusiloba ES was isolated as described (Trowitzsch-Kienast et al. 2011) and was stored in aliquots of a stock solution of ES in 100% ethanol with a concentration of 7.14 mM.

Analysis of VSMC proliferation Cell cycle-synchronized human and murine VSMC were treated as indicated with or without TNF-a and rising concentrations of ES for 24 h in 96-well plates. During the last 4 h of incubation, 10% (v/v) AlamarBlue (Biozol, Eching, Germany) was added, and the absorbance at 570 nm/600 nm was measured using an ELISA reader (Bio-Rad, Munich, Germany).

Analysis of VSMC apoptosis Apoptosis in murine and human VSMC was quantified from caspase-3/7 activity using the SensoLyteTM Homogenous AFC Caspase-3/7 Assay Kit (AnaSpec, San Jose, CA, USA) as described (Freise et al. 2010) (see Data S1).

Analysis of VSMC migration Migration of human and murine VSMC was assessed by a wound healing assay as described (Freise et al.

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2012a). Briefly, artificial wounds were created in confluent cell layers using a sterile 1000-ll pipette tip. After washing with PBS, the cells were treated as indicated with TNF-a alone and together with rising concentrations of ES for 22 h. The wound diameters were photographed at the beginning and at the end of the experiment using a KEYENCE digital microscope. Diameters of three randomly chosen fields were measured for each well using the Image J software (version 1.41; National Institutes of Health, Bethesda, MA, USA).

Western blot measurements Vascular smooth muscle cells were treated as indicated for 10 min with TNF-a, ES and TNF-a+ES, washed with PBS and lysed in buffer containing 50 mM TrisHCl pH 7.4, 2.25 M urea, 1.4% sodium dodecyl sulphate, 100 mM dithiothreitol and per 10 ml buffer one tablet of Complete Mini Protease Inhibitor cocktail (Roche, Penzberg, Germany). Equal protein amounts were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) using a tank blot apparatus (Hoefer, Holliston, MA, USA). Membranes blocked for 1 h with 5% skim milk powder in a buffer containing 10 mM Tris, 154 mM NaCl, 0.1% Tween 20 were incubated over night at 4°C with the following specific primary antibodies against Akt, p-Akt, Erk1/2, pErk1/2 (all from Cell Signaling, Beverly, MA, USA), (a)

(b)

(c)

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· Episesamin blocks activation of VSMC

HO-1, VCAM-1 (Santa Cruz, Santa Cruz, CA, USA) and b-Actin (Sigma-Aldrich). After washing, membranes were incubated 1 h with horseradish peroxidase-labelled secondary antibodies (Dako, Hamburg, Germany). Bands were detected by enhanced chemiluminescence (GE Healthcare, Munich, Germany) using the Luminescent Image Analyser LAS-4000 (Fujifilm, D€ usseldorf, Germany) and were quantified using Image J.

Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) Cell synchronized VSMC at approx. 90% confluency were treated 24 h as indicated with TNF-a, the Akt-inhibitor Ly294002 and ES alone or ES in combination with the other stimulators. Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and transcribed into cDNA with the High Capacity RNA to DNA kit (Applied Biosystems, Foster City, CA, USA). The cDNA concentrations were determined by a NanoDrop ND-1000 device (NanoDrop Technologies, Wilmington, NC, USA). Quantitative PCR using SYBR Green Master Mix (Applied Biosystems) and subsequent melting curve analysis was performed using the Mx3000p system (Stratagene/Agilent Technologies, Waldbronn, Germany). Relative RNA amounts were calculated using the 2DDCt method and normalized to mRNA expressions of the

Figure 1 ES decreases basal and TNF-a induced VSMC proliferation. Effects of ES or TNF-a alone and of combinations thereof on the proliferation of cell cycle-synchronized murine (a, c) and human (b, d) VSMC after 24 h treatment as indicated. Shown are means  SD of four independent measurements. *P < 0.05, **P < 0.01, *** P < 0.001. © 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12400

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housekeeping genes YWAHZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) or HPRT (hypoxanthine-phophoribosyl transferase). The primer sequences used are available on demand.

Measurement of reactive oxygen species Intracellular oxidative stress was determined as previously described [25]. Briefly, VSMC in black 96 wells (Greiner Bio-One GmbH, Frickenhausen, Germany) were incubated 24 h with ES, TNF-a, H2O2 and/or N-acetyl cysteine (NAC), a known reactive oxygen species (ROS) inhibitor, which all were added 60 min before addition of 5-(6-)-chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate acetylester (CM-H2DCFDA, 20 lmol L1; Life technologies, Karlsruhe, Germany).

(a)

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DCF is a widely used probe for detecting intracellular oxidative stress requiring a catalyst to be oxidized by hydrogen peroxide or superoxide (Vrbacky et al. 2007, Wrona et al. 2008), thus H2DCF oxidation functions as an indication of general oxidative stress. After replacement of the supernatants by fresh basal medium, fluorescence (kex: 485 nm, kem: 525 nm) was measured after 45 min at 37°C.

Determination of MMP-activity in VSMC supernatants Activities of MMP-2 and MMP-9 were measured by cleavage of 0.01 mg ml1 dye-quenched DQ-gelatin (Molecular Probes, Life Technologies GmbH, Darmstadt, Germany) as described (Freise et al. 2012a). Cells were treated for 24 h as indicated with TNF-a, the Aktinhibitor Ly294002 and ES alone or ES in combination

(b)

(c)

Figure 2 ES decreases basal and TNF-a induced VSMC migration and induces VCAM-1 expression. Wounded confluent layers of murine (a) and human (b) VSMC were treated as indicated. After 22 h, wound diameters were determined as described in the materials and methods section. Shown are means  SD of three individual experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (compared to treatment with TNF-a). (c) Murine VSMC were treated 24 h as indicated. VCAM-1 protein expression was determined by Western blot and band intensities were normalized to b-actin expression. Shown are bands of one representative experiment and means  SD of three individual experiments. *P ≤ 0.05 (compared to treatment with TNF-a).

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with the other stimulators. Afterwards, the medium was replaced by serum-free growth medium for 20 h. Supernatants (50 ll) were transferred to black 96-well plates (Greiner Bio-One) and mixed with 150 ll DQgelatin with or without addition of the MMP activator

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4-aminophenyl mercuric acetate (APMA; 67 lM) which allows the measurement of total gelatinolytic activity including proMMPs. Fluorescence signals were monitored for 120 min at 30°C using a Victor3 microplate reader (Perkin Elmer, Rodgau, Germany).

(a)

(b)

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Figure 3 Effects of ES are mediated by inhibition of ERK1/2 and Akt activation. (a) Phosphorylation of ERK1/2 and Akt was determined in murine VSMC treated as indicated for 10 min by specific Western blots. Shown are representative blots from one out three independent measurements and the respective means  SD from quantification of band intensities (ratio pERK1/2 to ERK1/2). (b) Effects of the specific ERK1/2 and Akt inhibitors, UO126 and Ly294002, respectively, on murine VSMC cell growth (n = 4). (c) Effects of UO126 and Ly294002 alone and in combination with ES on murine VSMC migration. Shown are means  SD of three independent measurements. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (compared to treatment with TNF-a). © 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12400

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Transient transfection of VSMC and assessment of NF-KB activation

ES blocks basal and TNF-a-induced activation of ERK1/ 2 and Akt

Using the FuGENEâ HD Transfection Reagent (Promega, Mannheim, Germany), VSMC were transiently transfected in white 96-well plates (NUNC, Roskilde, Denmark) with reporter plasmids (Promega) for NF-KB (pGL4.32[luc2P/NF-KB-RE/Hygro]) and a renilla vector (pGL4.74 [hRluc/TK]). After 24 h, the cells were treated as indicated with TNF-a, ES or TNF-a+ES using different ES concentrations. One treatment group also received 15 nM of the HSP90-inhibitor 17-Dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG, InvivoGen, San Diego, CA, USA) as a positive control for inhibition of NF-KB activity (Hertlein et al. 2010). After additional 24 h, using the Dual-GloâLuciferase Assay System (Promega) and a Victor3 microplate reader (Perkin Elmer), NF-KB activities were estimated as relative luminescence units (RLU) after normalization to the transfection of the renilla control reporter plasmid.

The ES mediated inhibition of cell growth and migration of VSMC can, at least in part, be referred to a diminished basal and TNF-a-induced activation of ERK1/2 and Akt in the presence of ES (Fig. 3a). This assumption was confirmed by applying the specific inhibitors of ERK1/2 and Akt, U0126 and Ly294002 respectively, in proliferation and migration experiments. When applied alone, both inhibitors only slightly reduced cell growth (Fig. 3b) and migration (Fig. 3c) of VSMC. In combinations with TNF-a, the TNF-a-induced proliferation was attenuated by both inhibitors (Fig. 3b), whereas only Ly294002 significantly reduced TNF-a-induced migration (Fig. 3c).

Statistical analyses Data sets were analysed by one-way ANOVA followed by the Tukey post hoc test using GraphPad PRISM, version 5.01 (GraphPad Software, La Jolla, CA, USA), and differences with P values ≤0.05/≤0.01/≤0.001 (*/**/***) were considered to be statistically significant.

Results ES attenuates basal and TNF-a-induced cell growth and migration of VSMC Murine (Fig. 1a) and human (Fig. 1c) VSMC showed a concentration-dependent reduced basal growth due to the presence of 10 lM ES by 465% and 35  5% respectively. In addition, enhanced VSMC growth induced by TNF-a was reduced by ES to the level of the control in murine (Fig. 1b) and human (Fig. 1d) cells. ES also reduced the TNF-a-induced migration of murine (Fig. 2a) and human (Fig. 2b) cells to the level of control treated VSMC. Regarding a possible mode of action of ES, in the migration experiments a 1.9  0.4-fold induction of protein expression of the adhesion molecule VCAM-1 by TNF-a in the murine VSMC was observed, which was reduced to the expression level of the control in the presence of 10 lM ES (Fig. 2c). Applying the same experimental conditions as in the proliferation and migration experiments, no elevated activities of apoptosis specific caspases-3/-7 in the VSMC were observed (see Fig. S1).

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ES blocks basal and TNF-a-induced MMP-2/-9 mRNA expression and secretion RT-PCR analysis showed that treatment with TNF-a induced a 3.11  0.18-fold upregulation of MMP-9 and a somewhat lower 1.61  0.11-fold upregulation of MMP-2 mRNA expression, while ES at 10 lM repressed the mRNA expression of MMP-9 to an only 1.33  0.09-fold upregulation and of MMP-2 to an only 1.08  0.06-fold upregulation (Fig. 4a). As expected from Fig. 3b and literature data, treatment with the Akt-inhibitor Ly294002 provoked a similarly reduced MMP-expression (Fig. 4a, b). In line with the mRNA expression pattern, supernatants of TNF-a treated cells exhibited elevated gelatinolytic activities compared to control, indicating enhanced secretion of MMPs. These were almost reduced to the basal level when increased concentrations of ES were applied (Fig. 4b). The same was observed after activation of all secreted (pro)MMPs in the supernatants with APMA, also indicating that the total amount of secreted MMPs was decreased by ES. In line with the preceding experiments, Akt inhibition by Ly294002 also inhibited the release of MMPs into supernatants of VSMC (Fig. 4b).

ES blocks TNF-a-induced activation of NF-KB Besides Akt, activation of the transcription factor NFKB is known to regulate MMP-expression in VSMC. Figure 5a shows that ES at 10 lM reduced the TNF-ainduced 2.35  0.12-fold enhanced activation of NF-KB to an only 1.27  0.12-fold enhancement, suggesting that this inhibitory effect contributes to the reduced release of MMP-2/-9 by murine VSMC. To confirm possible effects of the ES mediated inhibition of NF-KB and MMP-activity on VSMC growth, we again performed proliferation experiments. As shown

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(a)

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Figure 4 Effects of ES on basal and TNF-a-induced gene expression and secretion of MMP-2 and MMP-9 by VSMC. (a) Cell cycle-synchronized murine VSMC were treated as indicated for 24 h. Expressions of MMP-2 and MMP-9 mRNA were calculated in relation to controls. Shown are means  SD of four independent measurements. (b) Enzymatic activities of MMP-2 and MMP-9 in supernatants of VSMC which were treated 24 h as indicated with or without addition of APMA. Shown are means  SD from four independent experiments with three parallel measurements. ***P < 0.001 (compared to treatment with TNF-a). aP < 0.001, cP < 0.05, nsnot significant (compared to control).

in Fig. 5b, both inhibition of NF-KB by 17-DMAG (17D) and pharmacological inhibition of MMP-2/-9 by the specific inhibitor Ro28-2653 (Ro28) diminished the TNF-a-induced proliferation of murine VSMC.

ES attenuates TNF-a and H2O2 induced oxidative stress Reactive oxygen species (ROS) are known contributors to VSMC activation including activation of NF-KB.

Figure 6a shows a 1.98  0.07-fold increase of ROS compared to control due to treatment with TNF-a in VSMC which was reduced by 10 lM ES to an only 1.24  0.12-fold and, as a positive control, likewise reduced to an only 1.02  0.03-fold increased ROS measurement in the presence of the known anti-oxidant substance N-acetylcysteine (NAC). This anti-oxidative activity of ES was similarly observed after induction of ROS with H2O2, which was almost neutralized in the

(a)

Figure 5 Effects of ES on TNF-ainduced activation of NF-KB and growth of VSMC. (a) Activation of the transcription factor NF-KB was measured in VSMC which were treated as indicated. Shown are means  SD of four independent measurements. (b) Effects of activation and inhibition of NF-KB and of MMP-2/-9 enzymatic activities on VSMC cell growth were determined by proliferation measurements. Shown are means  SD of four independent measurements. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (compared to treatment with TNF-a). aP < 0.001, bP < 0.01, c P < 0.05 (compared to control).

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(c) Figure 6 Effects of ES on intracellular oxidative stress in VSMC induced by TNF-a and H2O2. (a+b) Anti-oxidative effects of ES were determined after 2 h treatment as indicated in cell cycle-synchronized VSMC. Shown are means  SD of four independent experiments with three parallel measurements. ***P ≤ 0.001 (compared to treatment with TNF-a or H2O2). aP < 0.001, b P < 0.01, cP < 0.05 (compared to control). (c) Effects of ES on the protein expression of anti-oxidative HO-1 in VSMC after 24 h treatment. Band intensities were normalized to b-actin expression. Shown are representative blots and means  SD out of three individual experiments. ***P ≤ 0.001 (compared to treatment with TNF-a).

presence of ES and NAC (Fig. 6b). These data are in accordance with Western blot experiments showing that treatment of VSMC with 10 lM ES induced a strong 2.57  0.45-fold upregulation of the expression of potent anti-oxidant haem oxygenase (HO)-1 (Fig. 6c), which is believed to protect against atherosclerotic disease (Stocker & Perrella 2006), while TNFa-treated cells shown no changed HO-1 expression compared to control. 8

Discussion We here report that the lignan (+)-episesamin (ES) prevents vascular smooth muscle cells (VSMC) from TNF-a-induced activation of ERK1/2, Akt and NFjB signalling and exerts anti-oxidative effects in VSMC. Mitogenic and/or pro-inflammatory cytokines such as TNF-a can induce VSMC activation characterized

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by enhanced proliferation and migration. VSMC migration and subsequent accumulation of VSMC within the intimal layer cause intimal hyperplasia and progressive stenosis of the vessel (Ward et al. 2000, Dzau et al. 2002, Lacolley et al. 2012) rendering the inhibition of VSMC activation as an therapeutic approach for therapeutic intervention in atherosclerosis (Jung et al. 2000, Tulis 2006). ES interfered with TNF-a-induced activation of MAPK and PI3K/Akt pathways, which both are known to impact atherogenesis (Goetze et al. 1999, Lee et al. 2009). Further, the TNF-a-induced expression of the adhesion molecule VCAM-1, known be enhanced in atherosclerotic lesions and to impact vascular inflammation (Davies et al. 1993, Cybulsky et al. 2001), was reduced by ES. Besides affecting migration of VSMC, a TNF-ainduced upregulation of VCAM-1 expression was also shown to increase the binding of T lymphocytes to VSMC, thus, acting as a co-stimulator of immune cells (Gamble et al. 1995). Therefore, the antimitogenic effects of ES on TNF-a-induced VSMC growth and migration and the reduction of VCAM-1 expression suggest an inhibitory potential of ES to block VSMC activation and the subsequent progression of vascular lesions. In the early stage of atherogenesis, the remodelling of the extracellular matrix by matrix metalloproteinases (MMP) such as MMP-9 is a prerequisite for the migration of VSMC from the arterial media to the intima (Newby & Zaltsman 2000). Abnormal matrix degradation due to dysregulated MMP-9 expression can promote the progression of atherosclerosis (Guo et al. 2014) and both gelatinases MMP-2 and -9 promote VSMC proliferation (Uzui et al. 2000, Cho & Reidy 2002) and migration (Mason et al. 1999, Vigetti et al. 2006). A known regulator of MMP-9 expression is the PI3K/Akt-pathway (Moon et al. 2003, Lee et al. 2009) which amongst others is activated by pro-inflammatory TNF-a. The suppressive effects of ES on TNF-a-induced activation of Akt along with the subsequent reduced mRNA expression and secretion of MMP-2 and MMP-9 in VSMC due to ES treatment seem to be an important mode of action of ES as also suggested for other polyphenolic compounds (Zhai et al. 2014). Besides, also activation of the transcription factor NF-KB in response to TNF-a and oxidative stress promotes proliferation, migration and the upregulation of MMPs in VSMC (Ward et al. 2000, Bond et al. 2001, Zhang et al. 2013). Conversely, the induction of antiinflammatory and/or anti-oxidative proteins such as haem-oxygenase (HO)-1 attenuates VSMC activation. Therefore, in line with other natural drugs such as quercetin, ochnaflavone or genistein (Moon et al. 2003, Suh et al. 2006, Wang et al. 2008), the ES-

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induced protein expression of HO-1 with subsequent anti-oxidative activity contributes to the ES-mediated inhibition of growth and MMP-expression/secretion in VSMC. Regarding a potential (complemental) ES rich diet, ES together with its epimer sesamin are contained in commercially available sesamin supplements at a approx. 1 : 1 ratio and also in refined sesame oil. Compared to sesamin, ES seems to be the more potent drug which might be due to a significantly different metabolism (Yasuda et al. 2012). Our study suggests beneficial effects of ES in atherosclerosis and implies a new possibility to prevent inflammation-triggered activation of VSMC during the onset and progression of vascular diseases such as atherosclerosis. Further studies, for example in animal models of atherosclerosis, should be performed to investigate the therapeutic potential of ES.

Conflict of interest The authors declare that they have no competing interests. The authors thank Kerstin Sommer for excellent technical assistance and the Berliner Sparkassenstiftung Medizin for financial support of this study.

References Abedi, H. & Zachary, I. 1995. Signalling mechanisms in the regulation of vascular cell migration. Cardiovasc Res 30, 544–556. Antoniades, C., Antonopoulos, A.S., Bendall, J.K. & Channon, K.M. 2009. Targeting redox signaling in the vascular wall: from basic science to clinical practice. Curr Pharm Des 15, 329–342. Bendeck, M.P., Zempo, N., Clowes, A.W., Galardy, R.E. & Reidy, M.A. 1994. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res 75, 539–545. Bond, M., Chase, A.J., Baker, A.H. & Newby, A.C. 2001. Inhibition of transcription factor NF-kappaB reduces matrix metalloproteinase-1, -3 and -9 production by vascular smooth muscle cells. Cardiovasc Res 50, 556– 565. Cho, A. & Reidy, M.A. 2002. Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury. Circ Res 91, 845–851. Clausell, N., de Lima, V.C., Molossi, S., Liu, P., Turley, E., Gotlieb, A.I., Adelman, A.G. & Rabinovitch, M. 1995. Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy. Br Heart J 73, 534–539. Covas, M.I., Nyyssonen, K., Poulsen, H.E., Kaikkonen, J., Zunft, H.J., Kiesewetter, H., Gaddi, A., de la Torre, R., Mursu, J., Baumler, H., Nascetti, S., Salonen, J.T., Fito, M., Virtanen, J. & Marrugat, J. 2006. The effect of

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Episesamin blocks activation of VSMC

· C Freise and U Querfeld

polyphenols in olive oil on heart disease risk factors: a randomized trial. Ann Intern Med 145, 333–341. Cybulsky, M.I., Iiyama, K., Li, H., Zhu, S., Chen, M., Iiyama, M., Davis, V., Gutierrez-Ramos, J.C., Connelly, P.W. & Milstone, D.S. 2001. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest 107, 1255–1262. Davies, M.J., Gordon, J.L., Gearing, A.J., Pigott, R., Woolf, N., Katz, D. & Kyriakopoulos, A. 1993. The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and Eselectin in human atherosclerosis. J Pathol 171, 223–229. Dzau, V.J., Braun-Dullaeus, R.C. & Sedding, D.G. 2002. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med 8, 1249–1256. Freise, C., Erben, U., Neuman, U., Kim, K., Zeitz, M., Somasundaram, R. & Ruehl, M. 2010. An active extract of Lindera obtusiloba inhibits adipogenesis via sustained Wnt signaling and exerts anti-inflammatory effects in the 3T3-L1 preadipocytes. J Nutr Biochem 21, 1170–1177. Freise, C., Ruehl, M., Erben, U., Farndale, R., Somasundaram, R. & Heimesaat, M. 2012a. The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 promotes enzymatic activity of matrixmetalloproteinase-2 in vitro. Eur J Microbiol Immunol 2, 186–191. Freise, C., Trowitzsch-Kienast, W., Ruehl, M., Erben, U., Seehofer, D., Kim, K., Zeitz, M. & Somasundaram, R. 2012b. (+)-Episesamin exerts anti-neoplastic effects in human hepatocellular carcinoma cell lines via suppression of nuclear factor-kappa B and inhibition of MMP-9. Invest New Drugs 30, 2087–2095. Freise, C., Trowitzsch-Kienast, W., Erben, U., Seehofer, D., Kim, K.Y., Zeitz, M., Ruehl, M. & Somasundaram, R. 2013. (+)-Episesamin inhibits adipogenesis and exerts antiinflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling, down-regulation of PPARgamma and induction of iNOS. J Nutr Biochem 24, 550–555. Gamble, J.R., Bradley, S., Noack, L. & Vadas, M.A. 1995. TGF-beta and endothelial cells inhibit VCAM-1 expression on human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 15, 949–955. Goetze, S., Xi, X.P., Kawano, Y., Kawano, H., Fleck, E., Hsueh, W.A. & Law, R.E. 1999. TNF-alpha-induced migration of vascular smooth muscle cells is MAPK dependent. Hypertension 33, 183–189. Guo, L., Ning, W., Tan, Z., Gong, Z. & Li, X. 2014. Mechanism of matrix metalloproteinase axis-induced neointimal growth. J Mol Cell Cardiol 66, 116–125. Heistad, D.D., Wakisaka, Y., Miller, J., Chu, Y. & PenaSilva, R. 2009. Novel aspects of oxidative stress in cardiovascular diseases. Circ J 73, 201–207. Hertlein, E., Wagner, A.J., Jones, J., Lin, T.S., Maddocks, K.J., Towns, W.H. 3rd, Goettl, V.M., Zhang, X., Jarjoura, D., Raymond, C.A., West, D.A., Croce, C.M., Byrd, J.C. & Johnson, A.J. 2010. 17-DMAG targets the nuclear factor-kappaB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition. Blood 116, 45–53 Epub 2010 Mar 29. Jovinge, S., Hultgardh-Nilsson, A., Regnstrom, J. & Nilsson, J. 1997. Tumor necrosis factor-alpha activates smooth

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Acta Physiol 2014 muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol 17, 490–497. Jung, F., Haendeler, J., Goebel, C., Zeiher, A.M. & Dimmeler, S. 2000. Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death. Cardiovasc Res 48, 148–157. Kim, J.-Y., Park, H.-J., Um, S.H., Sohn, E.-H., Kim, B.-O., Moon, E.-Y., Rhee, D.-K. & Pyo, S. 2012. Sulforaphane suppresses vascular adhesion molecule-1 expression in TNF-a-stimulated mouse vascular smooth muscle cells: involvement of the MAPK, NF-jB and AP-1 signaling pathways. Vascul Pharmacol 56, 131–141. Kuzuya, M., Kanda, S., Sasaki, T., Tamaya-Mori, N., Cheng, X.W., Itoh, T., Itohara, S. & Iguchi, A. 2003. Deficiency of gelatinase a suppresses smooth muscle cell invasion and development of experimental intimal hyperplasia. Circulation 108, 1375–1381. Lacolley, P., Regnault, V., Nicoletti, A., Li, Z. & Michel, J.B. 2012. The vascular smooth muscle cell in arterial pathology: a cell that can take on multiple roles. Cardiovasc Res 95, 194–204. Lee, E.J., Kim, D.I., Kim, W.J. & Moon, S.K. 2009. Naringin inhibits matrix metalloproteinase-9 expression and AKT phosphorylation in tumor necrosis factor-alphainduced vascular smooth muscle cells. Mol Nutr Food Res 53, 1582–1591. Limon-Pacheco, J. & Gonsebatt, M.E. 2009. The role of antioxidants and antioxidant-related enzymes in protective responses to environmentally induced oxidative stress. Mutat Res 674, 137–147. Mason, D.P., Kenagy, R.D., Hasenstab, D., Bowen-Pope, D.F., Seifert, R.A., Coats, S., Hawkins, S.M. & Clowes, A.W. 1999. Matrix metalloproteinase-9 overexpression enhances vascular smooth muscle cell migration and alters remodeling in the injured rat carotid artery. Circ Res 85, 1179–1185. Moon, S.K., Cho, G.O., Jung, S.Y., Gal, S.W., Kwon, T.K., Lee, Y.C., Madamanchi, N.R. & Kim, C.H. 2003. Quercetin exerts multiple inhibitory effects on vascular smooth muscle cells: role of ERK1/2, cell-cycle regulation, and matrix metalloproteinase-9. Biochem Biophys Res Commun 301, 1069–1078. Moon, S.K., Cha, B.Y. & Kim, C.H. 2004. ERK1/2 mediates TNF-alpha-induced matrix metalloproteinase-9 expression in human vascular smooth muscle cells via the regulation of NF-kappaB and AP-1: involvement of the ras dependent pathway. J Cell Physiol 198, 417–427. Newby, A.C. & Zaltsman, A.B. 2000. Molecular mechanisms in intimal hyperplasia. J Pathol 190, 300–309. Ross, R. 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362, 801–809. Sato, H., Kita, M. & Seiki, M. 1993. v-Src activates the expression of 92-kDa type IV collagenase gene through the AP-1 site and the GT box homologous to retinoblastoma control elements. A mechanism regulating gene expression independent of that by inflammatory cytokines. J Biol Chem 268, 23460–23468.

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Acta Physiol 2014 Sheehan, A.L., Carrell, S., Johnson, B., Stanic, B., Banfi, B. & Miller, F.J. Jr 2011. Role for Nox1 NADPH oxidase in atherosclerosis. Atherosclerosis 216, 321–326. Stocker, R. & Perrella, M.A. 2006. Heme oxygenase-1: a novel drug target for atherosclerotic diseases? Circulation 114, 2178–2189. Suh, S.J., Jin, U.H., Kim, S.H., Chang, H.W., Son, J.K., Lee, S.H., Son, K.H. & Kim, C.H. 2006. Ochnaflavone inhibits TNF-alpha-induced human VSMC proliferation via regulation of cell cycle, ERK1/2, and MMP-9. J Cell Biochem 99, 1298–1307. Tresserra-Rimbau, A., Rimm, E.B., Medina-Remon, A., Martinez-Gonzalez, M.A., de la Torre, R., Corella, D., SalasSalvado, J., Gomez-Gracia, E., Lapetra, J., Aros, F. et al. 2014. Inverse association between habitual polyphenol intake and incidence of cardiovascular events in the PREDIMED study. Nutr Metab Cardiovasc Dis 24, 639–647. Trowitzsch-Kienast, W., Ruehl, M., Kim, K.Y., Emmerling, F., Erben, U., Somasundaram, R. & Freise, C. 2011. Absolute configuration of antifibrotic (+)-episesamin isolated from lindera obtusiloba BLUME. Z Naturforsch C, 66c, 460–464. Tulis, D.A. 2006. Methods for identifying cardiovascular agents: a review. Recent Pat Cardiovasc Drug Discov 1, 47–56. Uzui, H., Lee, J.D., Shimizu, H., Tsutani, H. & Ueda, T. 2000. The role of protein-tyrosine phosphorylation and gelatinase production in the migration and proliferation of smooth muscle cells. Atherosclerosis 149, 51–59. Vigetti, D., Moretto, P., Viola, M., Genasetti, A., Rizzi, M., Karousou, E., Pallotti, F., De Luca, G. & Passi, A. 2006. Matrix metalloproteinase 2 and tissue inhibitors of metalloproteinases regulate human aortic smooth muscle cell migration during in vitro aging. FASEB J 20, 1118– 1130. Vrbacky, M., Drahota, Z., Mracek, T., Vojtiskova, A., Jesina, P., Stopka, P. & Houstek, J. 2007. Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria. Biochim Biophys Acta 1767, 989–997. Wang, J., Zhang, R., Xu, Y., Zhou, H., Wang, B. & Li, S. 2008. Genistein inhibits the development of atherosclerosis

C Freise and U Querfeld

· Episesamin blocks activation of VSMC

via inhibiting NF-kappaB and VCAM-1 expression in LDLR knockout mice. Can J Physiol Pharmacol 86, 777–784. Ward, M.R., Pasterkamp, G., Yeung, A.C. & Borst, C. 2000. Arterial remodeling. Mechanisms and clinical implications. Circulation 102, 1186–1191. West, N., Guzik, T., Black, E. & Channon, K. 2001. Enhanced superoxide production in experimental venous bypass graft intimal hyperplasia: role of NAD(P)H oxidase. Arterioscler Thromb Vasc Biol 21, 189–194. Wrona, M., Patel, K.B. & Wardman, P. 2008. The roles of thiol-derived radicals in the use of 20 ,70 -dichlorodihydrofluorescein as a probe for oxidative stress. Free Radic Biol Med 44, 56–62. Yasuda, K., Ikushiro, S., Wakayama, S., Itoh, T., Yamamoto, K., Kamakura, M., Munetsuna, E., Ohta, M. & Sakaki, T. 2012. Comparison of metabolism of sesamin and episesamin by drug-metabolizing enzymes in human liver. Drug Metab Dispos 40, 1917–1926. Yokoyama, M., Inoue, N. & Kawashima, S. 2000. Role of the vascular NADH/NADPH oxidase system in atherosclerosis. Ann N Y Acad Sci, 902, 241–247; discussion 247–8. Zhai, X., Chi, J., Tang, W., Ji, Z., Zhao, F., Jiang, C., Lv, H. & Guo, H. 2014. Yellow wine polyphenolic compounds inhibit matrix metalloproteinase-2, -9 expression and improve atherosclerotic plaque in LDL-receptorknockout mice. J Pharmacol Sci 125, 132–141. Zhang, H., Wang, Z.W., Wu, H.B., Li, Z., Li, L.C., Hu, X.P., Ren, Z.L., Li, B.J. & Hu, Z.P. 2013. Transforming growth factor-beta1 induces matrix metalloproteinase-9 expression in rat vascular smooth muscle cells via ROSdependent ERK-NF-kappaB pathways. Mol Cell Biochem 375, 11–21.

Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Analysis of VSMC apoptosis Figure S1. Effects of TNF-a and ES on apoptosis in VSMC.

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12400

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2 and AKT and decreased activity of gelatinases.

Activation of vascular smooth muscle cells (VSMC), a key event in the pathogenesis of atherosclerosis, is triggered by inflammatory stimuli such as tu...
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