DOI: 10.1111/exd.12562

Letter to the Editor

www.wileyonlinelibrary.com/journal/EXD

Electric current-induced lymphatic activation Kentaro Kajiya1, Yuko Matsumoto-Okazaki1, Mika Sawane1, Kaedeko Fukada1, Yuya Takasugi2, Tomonori Akai2, Naoki Saito1 and Yuichiro Mori1 1

Shiseido Research Center, Yokohama, Japan; 2Dai Nippon Printing, Tokyo, Japan Correspondence: Kentaro Kajiya, PhD, Shiseido Research Center, 2-2-1, Hayabuchi, Tsuzuki-ku, Yokohama 224-8558, Japan, Tel.: +81-45-590-6138, Fax: +81-45-590-6019, e-mail: [email protected] Abstract: The lymphatic system in skin plays important roles in drainage of wastes and in the afferent phase of immune response. We previously showed that activation of vascular endothelial growth factor receptor (VEGFR), specifically the VEGFC/VEGFR-3 pathway, attenuates oedema and inflammation by promoting lymphangiogenesis, suggesting a protective role of lymphatic vessels against skin inflammation. However, it remains unknown how physical stimuli promote lymphatic function. Here, we show that lymphatic endothelial cells (LECs) are activated by directcurrent (DC) electrical stimulation, which induced extension of actin filaments of LECs, increased calcium influx into LECs, and increased phosphorylation of p38 mitogen-activated protein kinase

(MAPK). An inhibitor of focal adhesion kinase, which plays a role in cellular adhesion and motility, diminished the DC-induced extension of F-actin and abrogated p38 phosphorylation. Timelapse imaging revealed that pulsed-DC stimulation promoted proliferation and migration of LECs. Overall, these results indicate that electro-stimulation activates lymphatic function by activating p38 MAPK.

Background

Fig. 1a. For time-lapse imaging of migrated cells, LECs were cultured on a fibronectin-coated ITO plate with a 660-lm gap in the middle, and pulsed DC was applied from side to side (Fig. S1). More methods are available in completed Materials and Methods of Data S1.

The lymphatic vascular system in skin, which is composed of a dense network of thin-walled capillaries, plays a major role in tissue fluid homoeostasis. Impairment of lymphatic function causes lymphoedema due to insufficient lymphatic transport and localized fluid retention and often leads to skin phenotypic changes, such as epidermal thickening, collagen and lipid deposition, progressive fibrosis and susceptibility to infections (1). Hereditary lymphoedema is associated with altered expression of many genes, including those encoding VEGFR-3, GATA2 and FOXC2 (2). We recently showed that overexpression of a lymphangiogenesis factor, angiopoietin-1, in epidermis attenuated oedema and inflammation by promoting lymphatic integrity, suggesting that normalization of lymphatic function protects against oedema formation (3,4). But, despite extensive studies of the action mechanisms of lymphangiogenic factors at the molecular level (2), little is known about how physical stimuli affect lymphatic function. It was recently reported that increase of interstitial fluid in mouse embryos resulted in VEGFR-3 phosphorylation and lymphangiogenesis (5). Moreover, low-frequency electric current stimulation has been used to treat lymphoedema patients (6), suggesting that physical stimuli could have an important influence on lymphatic function.

Questions addressed First, we investigated the response of lymphatic endothelial cells (LECs) to direct-current (DC) stimulation, using a specially designed electrode system. Second, we examined molecular changes associated with the electro-stimulation. Finally, migration and proliferation of LECs were investigated by prolonged pulsed-DC stimulation.

Experimental design Human dermal lymphatic endothelial cells (LECs) (7) were used for this study. For the application of DC, LECs were cultured on a fibronectin-coated indium tin oxide (ITO) electrode, and DC was introduced from a platinum electrode located above it as shown in

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Key words: calcium influx – direct current – lymphatic

Accepted for publication 5 October 2014

Results DC stimulation induced cytoskeleton extension of LECs To determine the functional effect of DC stimulation on LECs, we designed an electrical stimulation device in which LECs are seeded directly onto an ITO electrode. This allowed us to visualize the motility of LECs exposed to various DC voltages, using a microscope (Fig. 1a). Immunofluorescence analysis of phalloidin revealed that 2 V DC induced cytoskeleton extension as compared to control cells, whereas 1 V DC had no effect (Fig. 1b). LECs stimulated 2V DC show vertically long shape as compared to control cells (Insets in Fig. 1b). Next, to identify the downstream signals involved, we carried out Western blotting analyses. We found that DC stimulation voltage-dependently induced phosphorylation of ERK and p38. However, a 3 V DC stimulus resulted in loss of ERK and p38 protein and decreased cell viability (Fig. S2), indicating that this voltage is toxic to LECs (Fig. 1c). Calcium influx was observed in LECs stimulated with 2 and 2.5 V DC (Fig. 1d). Time-course examination showed that phosphorylation of p38 and ERK was detected after DC stimulation for 30 s; however, at 24 h, the effect was partially reduced (Fig. 1e).

Role of focal adhesion kinase (FAK) in cytoskeleton extension To investigate the mechanism of DC-induced cytoskeleton extension, phalloidin staining was performed after DC stimulation in the presence or absence of a FAK inhibitor, PF573228. Cytoskeleton extension was inhibited by PF573228 (Fig. 2a). LECs after the stimulation of 2V DC show vertically long shape as compared to cells in the presence of PF573228 (Insets in Fig. 2a). Western blot analyses confirmed that the DC-dependent phosphorylation of p38 was, in

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 922–941

Letter to the Editor

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Figure 1. DC stimulation induces cytoskeleton extension, Ca2+ increase and phosphorylation of p38 MAPK. (a) Schematic illustration of application of electrical stimulation. Human lymphatic endothelial cells (LECs) were cultured on a fibronectin-coated ITO (indium tin oxide) plate. A platinum electrode was placed above. Note the 1-mm gap between the electrodes. (b) Direct current (DC) induced cytoskeleton extension. Insets show representative cells. Scale bar: 100 lm. (c) Stimulation with 2 V DC induced phosphorylation of ERK and p38 on LEC, whereas 1 V DC had no effect. 3 V DC resulted in loss of ERK and p38 protein. Quantitative analyses confirmed 2 V DC phosphorylates p38 and ERK. (d) 2 and 2.5 V DC increased the calcium concentration. DRatio shows significant calcium increase after 2 and 2.5 V stimulation. ***P < 0.001. (e) 2 V DC induced phosphorylation of ERK and p38 from 15 min to 4 h after application, but at 24 h, the effect was reduced. Quantification evaluation confirmed time-dependent change of p38 and ERK phosphorylation.

part, inhibited in the presence of PF573228. Moreover, neither ERK inhibitor PD98059 nor PI3 kinase inhibitor LY294002 had any effect on the DC-induced phosphorylation of p38 (Fig. 2b). In the absence of calcium in the medium, the DC-induced calcium increase was diminished (Fig. 2c). Tetrodotoxin (1 lM), a voltage-dependent Na+ channel inhibitor, diminished both p38 phosphorylation and cytoskeleton extension (Fig. 2d,e), while nifedipine, a voltagedependent calcium channel blocker, had no effect on the DC-induced calcium increase (data not shown).

Pulsed DC induced proliferation and migration of LECs To investigate the activation of LECs by prolonged exposure to DC, pulsed DC was applied to LECs, and proliferation assay was performed. The results indicated that pulsed-DC stimulation for 5 or 10 min increased the proliferation of LECs (Fig. 2f). Moreover, pulsed DC-induced proliferation was blocked in the presence of PF573228 (Fig. 2g). Finally, time-lapse imaging of LECs under

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 922–941

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Figure 2. DC stimulation induces cytoskeleton extension via the FAK pathway and migration and proliferation of LECs. (a) A FAK inhibitor, PF573228, had no effect on cytoskeleton organization in the absence of electrical stimulation, whereas it decreased cytoskeleton extension induced by 2 V DC stimulation. Insets show representative cells. (b) Phosphorylation of p38 MAPK was inhibited in the presence of the FAK inhibitor, whereas ERK inhibitor PD573228 and P13K inhibitor LY294002 had no effect. Quantification evaluation confirmed partial decrease of p38 phosphorylation. (c) In the absence of calcium or in the presence of tetrodotoxin, the cytoskeleton extension induced by DC was inhibited. (d, e) DCinduced calcium influx, (d) and DC-induced phosphorylation of p38 (e) were diminished in the absence of calcium or in the presence of tetrodotoxin. (f) PulsedDC promoted proliferation of LECs. (g) Pulsed DC-induced proliferation was inhibited in the presence of PF573228. (h, i) The analyses of migratory trajectory of control and pulsed-DC stimulated LECs (h) revealed the significant directional migration of LECs towards +X (X-axis) which is the cathode (i). (j) Shear stress induced cytoskeleton extension of LECs. The arrow shows the direction of laminar flow. *P < 0.05. **P < 0.01 and ***P < 0.001, scale bar: 100 lm.

pulsed-DC stimulation revealed that LECs migrated towards the cathode, as compared to control cells (Fig. S1, Movies S1 and S2). Computer-assisted migratory trajectory analyses demonstrated that pulsed-DC stimulation resulted in the significant directional migration of LECs towards the cathode (h, i). Interestingly, these data are consistent with a previous report that microvascular endothelial cells migrated towards the cathode (8). Finally, to determine a physiological role of lymphatic endothelial activation including cytoskeleton extension by DC, laminar shear stress was introduced to LECs, revealing the prominent cytoskeleton extension, suggesting one of possible role of DC in lymphatic activation. One could speculate that cell membrane depolarization of LECs induced by electric stimuli could trigger calcium influx, leading to lymphatic activation like proliferation and migration, although more study is needed for deciphering the mechanism.

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Letter to the Editor

Further research is needed to determine the functional role of lymphatic vascular electrotaxis in vivo and to investigate possible applications, including stem cell maintenance (9).

Conclusions Direct-current electrical stimulation promoted both cytoskeleton extension of LECs via the FAK pathway and calcium influx, leading to induction of proliferation and migration of LECs to the cathode in vitro. Electric stimuli could be used to control lymphatic function and its related diseases.

Acknowledgements We would like to thank Suin Kyo for her technical assistance. K.K and Y.M designed, performed the research study and analysed the data. M.S, K.F, N.S and Y.M performed the research and analysed the data. K.K wrote the paper, Y.T and T.A contributed the development of DC application instrument for the study.

Conflict of interests The authors have declared no conflicting interests.

References 1 Rockson S G. J Am Coll Cardiol 2008: 52: 799– 806. 2 Tammela T, Alitalo K. Cell 2010: 140: 460–476. 3 Kajiya K, Kidoya H, Sawane M et al. Am J Pathol 2012: 180: 1273–1282. 4 Sawane M, Kajiya K. Exp Dermatol 2012: 21(Suppl 1): 22–25. 5 Planas-Paz L, Strilic B, Goedecke A et al. EMBO J 2012: 31: 788–804. 6 Jahr S, Schoppe B, Reisshauer A. J Rehabil Med 2008: 40: 645–650.

7 Kajiya K, Hirakawa S, Ma B et al. EMBO J 2005: 24: 2885–2895. 8 Bai H, McCaig C D, Forrester J V et al. Arterioscler Thromb Vasc Biol 2004: 24: 1234–1239. 9 Zhao Z, Qin L, Reid B et al. Stem Cell Res 2012: 8: 38–48.

Supporting Information

Figure S1. Schematic illustration of DC application for migration assay of LECs. Figure S2. 3 V of DC application resulted in the decrease of cell viability. Movie S1. Control cells without pulsed DC. Movie S2. Exposure to pulsed DC induced migration of LECs towards the cathode.

Additional supporting data may be found in the supplementary information of this article. Data S1. Complete materials and methods.

DOI: 10.1111/exd.12563 www.wileyonlinelibrary.com/journal/EXD

Letter to the Editor

DAMP molecules S100A9 and S100A8 activated by IL-17A and house-dust mites are increased in atopic dermatitis Shan Jin1,2,3, Chang Ook Park1, Jung U Shin1, Ji Yeon Noh1, Yun Sun Lee1, Na Ra Lee1,2, Hye Ran Kim1, Seongmin Noh1, Young Lee4, Jeung‑Hoon Lee4 and Kwang Hoon Lee1,2 1

Department of Dermatology & Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Korea; 2Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea; 3Department of Dermatology, Yanbian University Hospital, Yanji, Jilin, China; 4Department of Dermatology and Research Institute for Medical Sciences, Chungnam National University, Daejeon, Korea Correspondence: Kwang Hoon Lee, MD, PhD, Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea, Tel.: 82-2-2228-2080, Fax: 82-2-393-9157, e-mail: [email protected] Shan Jin and Chang Ook Park contributed equally to this work. Abstract: S100A9 and S100A8 are called damage-associated molecular pattern (DAMP) molecules because of their proinflammatory properties. Few studies have evaluated S100A9 and S100A8 function as DAMP molecules in atopic dermatitis (AD). We investigated how house-dust mites affect S100A9 and S100A8 expression in Th2 cytokine- and Th17 cytokine-treated keratinocytes, and how secretion of these molecules affects keratinocyte-derived cytokines. Finally, we evaluated expression of these DAMP molecules in AD patients. S100A9 expression and S100A8 expression were strongly induced in IL-17A- and Dermatophagoides (D.) farinae-treated keratinocytes, respectively. Furthermore, co-treatment with D. farinae and IL-17A strongly increased expression of S100A9 and S100A8 compared with

D. farinae-Th2 cytokine co-treatment. The IL-33 mRNA level increased in a dose-dependent manner in S100A9-treated keratinocytes, but TSLP expression did not change. S100A8/A9 levels were also higher in the lesional skin and serum of AD patients, and correlated with disease severity. Taken together, S100A9 and S100A8 may be involved in inducing DAMPmediated inflammation in AD triggered by IL-17A and house-dust mites.

Background

(PAMP) molecules and endogenous damage-associated molecular pattern (DAMP) molecules. DAMP molecules are intracellular and function primarily in maintaining cell homeostasis, but they may

It has become increasingly evident that innate inflammatory response is triggered by pathogen-associated molecular pattern

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Key words: atopic dermatitis – Dermatophagoides farinae – IL-17A – IL33 – S100A8 – S100A9

Accepted for publication 5 October 2014

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 922–941

Electric current-induced lymphatic activation.

The lymphatic system in skin plays important roles in drainage of wastes and in the afferent phase of immune response. We previously showed that activ...
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