International Journal of Molecular Sciences

Article Visfatin Promotes through the Activation of ERK1/2 and JNK1/2 Pathway

Byung-Cheol Lee 1 , Jisun Song 1, Arim Lee 1, Daeho Cho 2 and Tae Sung Kim 1,* 1 Department of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea; [email protected] (B.-C.L.); [email protected] (J.S.); [email protected] (A.L.) 2 Institute of Convergence Science, Korea University, Seoul 136-701, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2-3290-3416; Fax: +82-2-3290-3921

 Received: 24 October 2018; Accepted: 15 November 2018; Published: 19 November 2018 

Abstract: Visfatin, a member of the adipokine family, plays an important role in many metabolic and stress responses. The mechanisms underlying the direct therapeutic effects of visfatin on wound healing have not been reported yet. In this study, we examined the effects of visfatin on wound healing in vitro and in vivo. Visfatin enhanced the proliferation and migration of human dermal fibroblasts (HDFs) and keratinocytes the expression of wound healing-related vascular endothelial growth factor (VEGF) in vitro and in vivo. Treatment of HDFs with visfatin induced activation of both extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases 1 and 2 (JNK1/2) in a time-dependent manner. Inhibition of ERK1/2 and JNK1/2 led to a significant decrease in visfatin-induced proliferation and migration of HDFs. Importantly, blocking VEGF with its neutralizing antibodies suppressed the visfatin-induced proliferation and migration of HDFs and human keratinocytes, indicating that visfatin induces the proliferation and migration of HDFs and human keratinocytes via increased VEGF expression. Moreover, visfatin effectively improved wound repair in vivo, which was comparable to the wound healing activity of epidermal growth factor (EGF). Taken together, we demonstrate that visfatin promotes the proliferation and migration of HDFs and human keratinocytes by inducing VEGF expression and can be used as a potential novel therapeutic agent for wound healing.

Keywords: visfatin; wound healing; VEGF; MAPK

1. Introduction The three layers of skin contain the human dermal fibroblasts (HDFs), epidermal keratinocytes, immune cells, nerves, and intradermal adipocytes [1–3]. The constant communication between the different cellular constituents of various compartments of the skin and its (ECM) plays a critical role in maintaining physiological homeostasis of the body [4–6]. The most essential resident cell of the dermis is HDF which is involved in wound healing through the production of ECM [4,5]. HDFs communicate with each other and with other cell types, including keratinocytes, immune cells, and adipocytes, thus playing a crucial role in regulating skin physiology and tissue repair [2,7]. Tissue repair immediately starts after the skin gets damaged. Wound repair is a tightly regulated complex process, which includes hemostasis, inflammation, proliferation, and re-epithelialization [8,9]. During wound healing, proliferation and migration of resident HDFs into the damaged area results in the recovery of skin wounds [10,11]. Visfatin, also known as pre-B cell colony-enhancing factor (PBEF) or nicotinamide phosphoribosyl- (NAMPT), was originally cloned from activated peripheral blood lymphocytes in 1994, as a highly conserved intracellular and extracellular 52 kDa adipokine [12–16]. Visfatin is released from various cell types and tissues of higher organisms, including peripheral-blood

Int. J. Mol. Sci. 2018, 19, 3642; doi:10.3390/ijms19113642 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 3642 2 of 15 monocytes, lymphocytes, dendritic cells, macrophages, adipose tissues, in particular visceral adipose tissue and is expressed in various malignant tumors, including colon, stomach, pancreas, liver, prostate, and breast cancers [13,15,17–24]. To date, two forms of visfatin have been identified: intracellular and extracellular. Intracellular visfatin plays a key role in maintaining the activity of nicotinamide adenine dinucleotide (NAD)-dependent and is implicated in the regulation of cellular metabolism. A recent study has reported that both intracellular and extracellular visfatin have been shown to be involved in tumor development [25]. Additionally, many studies have demonstrated that visfatin promotes angiogenesis by enhancing the proliferation, migration, formation of functional neovessels, and invasion of vascular endothelial cells in vitro and in vivo [26–29]. Further, visfatin has been shown to exhibit anti-apoptotic and proinflammatory properties [30]. Previous studies have reported the potential effects of visfatin on the proliferation and collagen synthesis in rat cardiac tissue and on induction of vascular smooth muscle cells [31]. Vascular endothelial cell growth factor (VEGF) is an angiogenic factor and the most important regulator of cell proliferation, migration, and permeabilization [32–35]. Many studies have reported that VEGF plays a critical role in wound healing through the induction of vascular permeability [32,36]. VEGF is produced in many cell types, including tumor cells and healthy cells, such as epidermal cells of the skin, including keratinocytes and fibroblasts [32,37,38]. A recent study has suggested that visfatin-induced proliferation of endothelial cells relies on the synthesis and secretion of VEGF [39], a key regulator of cell proliferation and angiogenesis. Moreover, visfatin has been shown to up-regulate the VEGF receptor 2, which mediates the angiogenic actions of VEGF [27,39]. Visfatin has been reported to regulate the expression of VEGF through activation of several pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt, the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38 signaling pathways [26,27,39–41]. Thus, we hypothesized that visfatin might induce wound healing through the induction of proliferation and migration. However, to date, there is poor evidence on the functions and direct effect of visfatin on wound healing in vitro and in vivo. In the present study, we aimed to evaluate the effect of visfatin on cell proliferation and migration.

2. Results

2.1. Visfatin Enhances the Proliferation and Migration of HDFs The proliferation and migration of skin dermal fibroblasts play an important role in wound repair. HDFs express numerous in response to epidermal growth factor (EGF) stimulus. We hypothesized that EGF-induced genes are directly or indirectly involved in the enhancement of cell proliferation and migration in wound repair. Thus, in this study, we performed a cDNA microarray to characterize the wound-related genes and investigate the role of these genes in wound healing in vitro and in vivo. According to cDNA microarray analysis, out of a total number of genes analyzed (36,079), expression of 1210 genes (3.35%) was up- or down-regulated more than 2-fold in EGF-stimulated HDFs, compared with the untreated control. As shown in Figure1A, as previous studies reported, we also found the up-regulated wound healing-related genes such as prostaglandin synthase 2 (PTGS2), intracellular adhesion molecule 1 (ICAM-1), matrix metalloproteinase 1 (MMP1), MMP3, 2 (HAS2), and chemokines including interleukin-1 (IL-1), chemokine (C-X-C motif) ligand-1 (CXCL-1), chemokine (C-C motif) ligand 2 (CCL2), CXCL-5, CCL11 and CXCL10 in EGF-stimulated HDFs. Notably, EGF markedly increased the expression of visfatin in a dose-dependent manner in HDFs (Figure1A,B). Based on several previous studies suggesting that visfatin promotes cell proliferation, migration and angiogenesis [26–29], we have selected a that visfatin could be closely related to wound healing. To further confirm the induction of visfatin expression, the expression levels of visfatin mRNA was determined by Q-PCR and RT-PCR. As shown in Figure1C,D, we found that HDFs induced the expression of visfatin in a dose- and time-dependent manner in response to EGF stimulus. Int. J. Mol. Sci. 2018, 19, 3642 3 of 15 Int. J. Mol. Sci. 2018, 19, x 3 of 16

We examinedWe examined the the effects effects of of visfatin visfatin on on thethe proliferation of of HDFs HDFs by by MTT MTT (3-(4,5-dimethylthiazol- (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium2-yl)-2,5-diphenyltetrazolium bromide) bromide) and and BrdU BrdU (5-Bromo-2 (5-Bromo-2′-deoxyuridine)0-deoxyuridine) cell cell proliferation proliferation assays, the importantassays, the steps important of wound steps of healing. wound As healing. shown As in shown Figure in1 FigureE, in cells 1E, in treated cells treated with 50 with ng/mL 50 ng/mL visfatin, the proliferationvisfatin, the proliferationof HDFs was of significantly HDFs was significantly increased by increased approximately by approximately 155.43 ± 7.94% 155.43 compared ± 7.94% to untreatedcompared control to untreated cells in MTT control assay. cells The in proliferationMTT assay. The ofvisfatin-stimulated proliferation of visfatin-stimulated HDFs was comparable HDFs was to that comparable to that of EGF-treated HDFs (146.77 ± 6.72%) at 50 ng/mL concentration. Furthermore, as of EGF-treated HDFs (146.77 ± 6.72%) at 50 ng/mL concentration. Furthermore, as shown in Figure1F, shown in Figure 1F, visfatin markedly increased cell proliferation in a dose-dependent manner, as visfatin markedly increased cell proliferation in a dose-dependent manner, as demonstrated by the BrdU demonstrated by the BrdU assay. The proliferation of visfatin-stimulated HDFs was comparable to assay.that The of proliferation EGF-stimulated of visfatin-stimulated HDFs (Figure 1F). HDFs Additionally, was comparable we observed to that thatof visfatin EGF-stimulated induced the HDFs (Figuremigration1F). Additionally, of HDFs in we a dose-dependent observed that visfatin manner induced(Figure 1G). the migrationA confluent of monolayer HDFs in a of dose-dependent HDFs was mannerscratched (Figure with1G). a A200-µL confluent pipette monolayer tip and HDFs of HDFs were was stimulated scratched for with24 h awith 200- variousµL pipette concentrations tip and HDFs wereof stimulated visfatin. HDFs for 24 actively h with various migrated concentrations into the scratched of visfatin. area at HDFs a concentration actively migrated of 50 ng/mL into thevisfatin, scratched area atwhich a concentration was comparable of 50 with ng/mL the migration visfatin, of which HDFs was upon comparable treatment with with 50 the ng/mL migration EGF. Thus, of HDFs EGF- upon treatmentinduced with up-regulation 50 ng/mL EGF. of visfatin Thus, suggests EGF-induced the possibility up-regulation that it of plays visfatin an important suggests therole possibility in wound that it playsrepair. an important role in wound repair.

A B C D

Visfatin Visfatin EGF (ng/mL) 0 1 10 50 9 9 EGF (ng/mL)-9h EGF (50 ng/mL) *

IL8 6 6 * 0 1 10 25 50 100 0 1 2 3 6 9 (h) Quantity CXCL1 *

mRNA Visfatin

CCL2 induction

of 3 PTGS2 (mRNA) 3 CSCL6 GAPDH Fold

1 Relative IL33 0 1 2 3 4 0 IcAM1 EGF (ng/mL) 0 1 10 50 EGF (ng/mL) 0 1 10 50 CXCL5 MMP1 CCL11 MTT assay BrdU assay IL6 E F

200 300 * * MIR29A * * ESM1 * * * * CYP7B1 * * 200 * * TFPI2 Control)

Control) 100

IL7R 100 vs

HAS2 vs

Proliferastion

Proliferation

IL1B (%

(% 1 C3 0 1 0 Visfatin EGF (ng/mL) 0 1 10 50 - - - - EGF (ng/mL) 0 1 10 50 - - - -

CXCL10 Visfatin (ng/mL) Visfatin (ng/mL) 0 1 10 50 IL13RA2 - - - - 0 1 10 50 - - - - MAP3K5 CYR61 Visfatin CXCL3 G EGF **

IL32 CON (50 ng/mL) (1 ng/mL) (10 ng/mL) (50 ng/mL) 60 ** 2.00 SLC22A4 * 1.33 TNIP1 0.67 MMP3 40 wound (%) 0.00 -0.67 -1.33 20 -2.00 area

1 Red: Actin (Rhodamine phalloidin) Relative 0

Blue: Nucleus (DAPI) EGF (ng/mL) - 50 - - - Visfatin (ng/mL) - - 1 10 50 FigureFigure 1. Visfatin 1. Visfatin stimulates stimulates the the proliferation proliferation and and migration migration of ofHDFs. HDFs. (A) Hierarchical (A) Hierarchical clustering clustering of of genesgenes that that werewere more more than than 2-fold 2-fold differentially differentially expressed expressed in cDNA in microarray. cDNA microarray. (B) HDFs were (B) HDFs seeded were seededinto into 6-well 6-well plates plates overnight overnight and then and cultured then cultured in a serum-free in a serum-free medium medium for further for 24 further h. Cells 24 were h. Cells weretreated treated with with EGF EGF or orphosphate-buffered phosphate-buffered saline saline (PBS) (PBS) at various at various concentrations concentrations and time and points. time The points. The expressionexpression of visfatin mRNA mRNA was was determined determined by by cDNA cDNA microarray, microarray, (C) ( CQ-PCR;) Q-PCR; * p < * p0.05<0.05 compared compared to theto saline-treated the saline-treated control control group. group. Values Values represent represent the the meanmean ± S.D.S.D. (n ( n= 6)= 6)and and (D) ( DRT-PCR.) RT-PCR. Data Data are are representative of three independent experiments. (E) HDFs were seeded in 96-well plates overnight representative of three independent experiments. (E) HDFs were seeded in 96-well plates overnight and cultured in a serum-free medium for further 24 h. Cells were then treated with visfatin, EGF, or and cultured in a serum-free medium for further 24 h. Cells were then treated with visfatin, EGF, PBS at the indicated concentrations for 24 h. The proliferation of HDFs was determined by MTT assay or PBS at the indicated concentrations for 24 h. The proliferation of HDFs was determined by MTT (E) and BrdU cell proliferation ELISA assays (F) as described in the Materials and Methods section. * assayp (

Several recent studies have suggested that visfatin might be closely related to the regulation of angiogenesis in various tumors, cell proliferation and migration [26–29]. Especially, VEGF plays Int. J. Mol. Sci. 2018, 19, 3642 4 of 15 a crucial role in cell proliferation and migration of keratinocytes and fibroblasts [32,37]. Here we examined the effects of visfatin on wound healing-related genes including collagens, MMPs, connective Int. J. Mol. Sci. 2018, 19, x 4 of 16 tissue growth factor (CTGF), EGF, fibroblast growth factors (FGFs), transforming growth factor beta (TGFβ), VEGFSeveral, VEGF recent receptor studies 1 have (VEGFR1 suggested), and that VEGFR2visfatin mightby be Q-PCR closely analysis.related to the We regulation foundthat of visfatin increasedangiogenesis the expression in various of VEGF tumors,and cell proliferationVEGFR2 (Figure and migration2A). However, [26–29]. Especially, visfatin VEGF did plays not a affect the expressioncrucial of other role genes in cell including proliferation MMPs, and migration FGFs, CTGF,of keratinocytes EGF and and TGF fibroblastsβ. Furthermore, [32,37]. Here we we confirmed examined the effects of visfatin on wound healing-related genes including collagens, MMPs, the effectsconnective of visfatin tissue on growth the factor expression (CTGF), EGF, of VEGF fibroblast in growth HDFs. factors HDFs (FGFs), were transforming cultured growth with various concentrationsfactor beta (0, 1,(TGFβ 10,), 25, VEGF 50,, VEGF and 100receptor ng/mL) 1 (VEGFR1 of visfatin), and VEGFR2 or EGF by Q-PCR (positive analysis. control) We found for different time periodsthat (0,visfatin 1, 2, increased 3, 6, and the 9expression h). The of mRNA VEGF and expression VEGFR2 (Figure of VEGF 2A). However, was determined visfatin did not by RT-PCR. Visfatin up-regulatedaffect the expression the expression of other genes of VEGFincluding gene MMPs, in aFGFs, dose- CTGF, and EGF time-dependent and TGFβ. Furthermore, manner we (Figure 2B). confirmed the effects of visfatin on the expression of VEGF in HDFs. HDFs were cultured with However,various visfatin concentrations did not affect (0, 1, the 10, expression 25, 50, and 100 of ng/mL) matrix of metalloproteinase visfatin or EGF (positive 2 (MMP2) control) for and MMP9, which alsodifferent play an time important periods (0, 1, role 2, 3, in 6, and the 9 wound h). The mRNA repair expression mechanism of VEGF (Figure was determined2B). Moreover, by RT- visfatin did not inducePCR. Visfatin the production up-regulated of the collagens expression in of mouse VEGF skingene in wound, a dose- comparedand time-dependent with the manner saline-treated skin wound(Figure as a 2B). control However, group visfatin (Figure did 2notC). affect In contrast, the expression EGF-treated of matrix skinmetalloproteinase wound as 2 a (MMP2) positive control and MMP9, which also play an important role in the wound repair mechanism (Figure 2B). Moreover, exhibited significantlyvisfatin did not induce increased the production production of collagens of collagens, in mouse comparedskin wound, compared with the with saline-treated the saline- mouse skin wound.treated To skin further wound determine as a control thegroup role (Figure of visfatin-induced 2C). In contrast, EGF-treated VEGF expressionskin wound as in a positive wound healing, HDFs werecontrol treated exhibited with significantly visfatin increased in the presence production ofof collagens, VEGF-neutralizing compared with antibody. the saline-treated As shown in Figure2D,mouse the visfatin-induced skin wound. To further proliferation determine ofthe HDFsrole of wasvisfatin-induced inhibited VEGF by VEGF-neutralizing expression in wound antibody healing, HDFs were treated with visfatin in the presence of VEGF-neutralizing antibody. As shown in a dose-dependentin Figure 2D, manner. the visfatin-induced The proliferation proliferation of VEGF-treated of HDFs was inhibited HDFs was by VEGF-neutralizing completely inhibited by VEGF-neutralizingantibody in antibody a dose-dependent (Figure 2 manner.D). Furthermore, The proliferation the VEGF-neutralizing of VEGF-treated HDFs antibody-treated was completely HDFs showed significantinhibited by inhibition VEGF-neutralizing of cell antibody migration (Figure (Figure 2D). Furthermore,2E). These the results VEGF-neutralizing suggest that antibody- visfatin plays a treated HDFs showed significant inhibition of cell migration (Figure 2E). These results suggest that critical role in wound healing through the induction of VEGF expression in HDFs. visfatin plays a critical role in wound healing through the induction of VEGF expression in HDFs. A COL4A1 COL5A1 MMP2 MMP9 CTGF COL1A1 COL3A1 N.S. N.S. N.S. N.S. 2 N.S. 2 2 N.S. 2 2 2 N.S. 2 Quantity

mRNA 1 1 1 1 1 1 1 of

1 0 1 0 1 0 1 0 1 0 1 0 1 0 Relative Visfatin 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 (ng/mL)

VEGF VEGFR1 VEGFR2 TGFb EGF FGF2 FGF7 FGF10 N.S. 6 * 2 2 * 2 N.S. 2 N.S. 2 N.S. 2 N.S. 2 N.S.

Quantity * *

mRNA 4 * 1 1 1 1 1 1 1 of 2

1 1 1 1 1 1 1 1 Relative 0 0 0 0 0 0 0 0 Visfatin 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 (ng/mL)

Visfatin (ng/mL)-9h Visfatin (50 ng/mL) B C N.S. Saline EGF Visfatin 0 1 10 25 50 100 0 1 2 3 6 9

*

2 ßEp Ep Ep VEGF Saline

Red

ß ß ß 1.5 ß ß EGF MMP2 D Epi Tongue D Epi Tongue 1 Epi Tongue quantification Visfatin

D collagens MMP9 of 0.5 (Collagens) GT GT GT Picrosirius GAPDH 0 1 Relative Collagen # D 200 # E # * ** Visfatin (50ng/mL) + anti-VEGF 150 Visfatin CON (50ng/mL) 0.1 mg/mL 0.5 mg/mL 1 mg/mL

control) 100

vs

Proliferation 50 (%

0 1 Visfatin (ng/mL) 0 50 0 0 0 50 50 50 0 50 Anti-VEGF (mg/mL) 0 0 0.1 0.5 1 0.1 0.5 1 0 1 VEGF (ng/mL) 0 0 0 0 0 0 0 0 10 1 Figure 2. Visfatin-inducedFigure 2. Visfatin-induced VEGF VEGF expression expression is is involved involved in in wound wound healing healing in HDFs. in HDFs.(A) HDFs (A were) HDFs were seeded intoseeded 6-well into plates6-well plates and and then then cultured cultured in in a aserum-free serum-free medium medium for 24 h. forCells 24 were h. treated Cells with were treated visfatin at various concentrations and time points. Expression of various genes was determined by Q- with visfatinPCR at (A various,B) RT-PCR. concentrations * p < 0.05 compared and totime the saline-treated points. Expression control group. of Values various represent genes the was mean determined by Q-PCR (A,B) RT-PCR. * p < 0.05 compared to the saline-treated control group. Values represent

the mean ± S.D. (n = 6). N.S.: not significant. (C) The wound tissues of day 6 post-wound were stained with picrosirius red for the production of collagens (Magnification 100×, Scale bar, 200 µm). The black arrow indicates as follow: D (dermis), Ep (epidermis), Epi tongue (epithelial tongue), and GT (granulation tissue). The production of collagens was measured and graphed. * p < 0.05 compared to the saline-treated control group. Values represent the mean ± S.D. (n = 6). N.S.: not significant. Int. J. Mol. Sci. 2018, 19, 3642 5 of 15

(D) HDFs were seeded into 96-well plates overnight and then cultured in a serum-free medium for 24 h. Cells were treated with visfatin or PBS at the indicated concentrations for 24 h after pretreatment with 0.1, 0.5, or 1 µg/mL VEGF-neutralizing antibody. The proliferation of HDFs was determined by MTT assay. * p < 0.05 compared to the saline-treated control group. # p < 0.01 compared to the visfatin-treated group. ** p < 0.01 compared to the VEGF-treated control group. Values represent the mean ± S.D. (n = 6). (E) HDFs were scratched with a 200-µL tip and subsequently treated with visfatin at various concentrations for 24 h after treatment with 0.1, 0.5, or 1 µg/mL VEGF-neutralizing antibody. Subsequently, cells were fixed with 4% paraformaldehyde and stained with rhodamine phalloidin (red) and DAPI (blue) for actin and nucleus staining, respectively. Data are representative of three independent experiments. 2.3. Wound Healing Is Induced by Visfatin via ERK1/2 and JNK1/2 Activation in HDFs MAP kinase-signaling pathway has been shown to be involved in the proliferation and migration of skin keratinocytes and fibroblasts [42–44]. To investigate the effects of visfatin on the activation of MAP kinases in HDFs, cells were treated with visfatin and phosphorylation of each MAP kinase was determined by western blot analysis. As shown in Figure3A, visfatin rapidly phosphorylated ERK1/2 and c-Jun N-terminal kinase (JNK1/2) within 5 min. This phosphorylation reached a maximum 30 min after stimulation with visfatin and was maintained until 60 min. Visfatin-induced phosphorylation of JNK1/2 was in a time-dependent manner which was subsequently decreased (Figure3A). However, we observedInt. J.that Mol. Sci. visfatin 2018, 19, x did not affect phosphorylation of p38 MAP kinase in HDFs. 6 of 16

MTT assay BrdU assay Visfatin (50ng/mL) A B * C *

0 5 15 30 45 60 (min) 150 * 300 *

pp38 125 * 100 200 p38 Control)

Control) 75

pERK1/2

vs vs 50 100

Proliferation Proliferation (% (% ERK1/2 25

1 0 1 0 pJNK1/2 Visfatin (ng/mL) 0 50 0 50 0 50 0 50 Visfatin (ng/mL) 0 50 0 50 0 50 0 50

JNK1/2 U0126 - - + + - - - - U0126 - - + + - - - - SB203580 - - - - + + - - SB203580 - - - - + + - - Tubulin SP600125 ------+ + SP600125 ------+ +

Visfatin (50 ng/mL) D # CON - U0126 SB203580 SP600125 # (%) 60 ** # 50 area 0h 40 30 wound 20 24h 10

1 Relative 0 Visfatin (ng/mL) 0 50 0 50 0 50 0 50 U0126 - - + + - - - - SB203580 - - - - + + - - SP600125 ------+ + Figure 3. VisfatinFigure 3. Visfatin enhances enhances the the proliferation proliferation and migration migration of HDFs of HDFs through through activation activation of ERK1/2 and of ERK1/2 and JNK1/2.JNK1/2. (A) (A HDFs) HDFs were were cultured cultured for for 24 h 24 and h then and treated then treatedwith 50 ng/mL with visfatin 50 ng/mL for 5, visfatin15, 30, 45 and for 5, 15, 30, 45 and 6060 min. min. Cells Cells were harvested harvested and andlysed lysed and cell and lysates cell were lysates analyzed were for analyzed the levels forof p38, the pp38, levels of p38, ERK1/2, pERK1/2, JNK1/2, and pJNK1/2 using the indicated antibodies; Tubulin was used as a loading pp38, ERK1/2, pERK1/2, JNK1/2, and pJNK1/2 using the indicated antibodies; Tubulin was used control. (B,C) Cells were treated with 50 ng/mL visfatin for 24 h after pretreatment with 10 µM U0126, as a loading20 µM control. SB203580, (B,C and) Cells 20 µM were SP600125 treated for 1 withh. The 50 growth ng/mL of HDFs visfatin was determined for 24 h after by MTT pretreatment (B) and with 10 µM U0126,BrdU 20proliferationµM SB203580, assays ( andC). * p 20 < 0.05µM compared SP600125 to forthe visfatin-treated 1 h. The growth group. of Values HDFs represent was determined the by MTT (B) andmean BrdU ± S.D. proliferation(n = 6). (D) HDFs assays were scratched (C). * p with< 0.05 a 200-µL compared tip and subsequently to the visfatin-treated treated with visfatin group. Values represent thefor 24mean h after± S.D. pretreatment (n = 6). ( withD) HDFs 10 µM were U0126, scratched 20 µM SB203580, with a 200- and µ 20L tip µM and SP600125 subsequently for 1 h. treated Subsequently, cells were fixed with 4% paraformaldehyde and stained with rhodamine phalloidin µ µ µ with visfatin(red) for and 24 DAPI h after (blue) pretreatment for actin and nucleus with10 staining,M U0126, respectively. 20 M** p SB203580, < 0.01 compared and to 20 theM control SP600125 for 1 h. Subsequently,group. # p < cells 0.01 compared were fixed to the with visfatin-treated 4% paraformaldehyde group. Values represent and stained the mean with ± S.D. rhodamine (n = 6). Data phalloidin (red) and DAPIare representative (blue) for of actin three andindependent nucleus experiments. staining, respectively. ** p < 0.01 compared to the control group. # p < 0.01 compared to the visfatin-treated group. Values represent the mean ± S.D. (n = 6). 2.4. Wound Healing Activity Induced by Visfatin in Keratinocytes Data are representative of three independent experiments. To further confirm the induction of visfatin expression by EGF treatment, the expression level of visfatin mRNA was determined in human keratinocytes by Q-PCR. As shown in Figure 4A, we found that keratinocytes in response to EGF stimulus induced the expression of visfatin in an EGF dose-dependent manner. Besides, we examined the effects of visfatin on the proliferation of keratinocytes by the BrdU cell proliferation assay. As shown in Figure 4B, the proliferation of keratinocytes was significantly increased by visfatin stimulation, compared to that of the untreated control cells. The proliferation of visfatin-stimulated keratinocytes was comparable to that of EGF- treated keratinocytes (Figure 4B). Also, we examined the effects of visfatin on the expression of the wound healing-related genes including collagens, MMPs, CTGF, EGF, FGFs, TGFβ, VEGF, VEGFR1 and VEGFR2 in human keratinocytes using Q-PCR. As shown in Figure 2A, we found that visfatin significantly increased the expression of VEGF and VEGFR2 (Figure 4C). However, visfatin did not affect the expression of other genes including MMPs, FGFs, CTGF, EGF, and TGFβ. Additionally, we observed that visfatin induced the migration of keratinocytes (Figure 4D). Keratinocytes actively migrated into the scratched area at a concentration of 50 ng/mL visfatin, which was comparable with the migration of keratinocytes upon treatment with 50 ng/mL EGF or 10 ng/mL VEGF. Importantly, VEGF-neutralizing antibody suppressed the visfatin-induced cell migration of keratinocytes (Figure

Int. J. Mol. Sci. 2018, 19, 3642 6 of 15

To further investigate the role of ERK1/2, p38, and JNK1/2 phosphorylation in visfatin-induced proliferationInt. J. Mol. Sci. and 2018 migration,, 19, x HDFs were treated with U0126, SB203580, and SP600125, which are 7 of specific 16 inhibitors of ERK1/2, p38, and JNK1/2, respectively. As shown in Figure3B–D, visfatin-induced 4D). Also, visfatin-induced migration of keratinocytes was completely inhibited by the specific proliferation and migration of HDFs were inhibited by the specific inhibitors of ERK1/2 and JNK1/2. inhibitors of p38, ERK1/2, and JNK1/2 (Figure 4D). Taken together, these results indicate that visfatin induces the proliferation and migration of HDFs via To investigate the effects of visfatin on the activation of MAP kinases in keratinocytes, cells were activationtreated of with ERK1/2 visfatin and and JNK1/2. phosphorylation of each MAP kinase was determined by western blot analysis. As shown in Figure 4E, visfatin rapidly induced phosphorylation of p38, ERK1/2 and 2.4. Wound Healing Activity Induced by Visfatin in Keratinocytes JNK1/2 within 5 or 15 min. This phosphorylation reached a maximum at 30 min after stimulation withTo further visfatin confirm (Figure the 4E). induction The phosphorylation of visfatin expression levels of by p38 EGF MAP treatment, kinase in the visfatin-treated expression level of visfatinkeratinocytes mRNA were was comparable determined to in those human of the keratinocytes visfatin-treated by Q-PCR.HDFs (Figure As shown 3A). To in Figurefurther4 A, we foundinvestigate that thekeratinocytes role of ERK1/2, in response p38, and to EGF JNK1/2 stimulus phosphorylation induced the in expression the visfatin-induced of visfatin cell in an EGFproliferation, dose-dependent keratinocytes manner. were Besides, treated we with examined U0126, SB203580, the effects and of SP600125, visfatin which on the are proliferation specific inhibitors of ERK1/2, p38, and JNK1/2, respectively. As shown in Figure 4F, the visfatin-induced of keratinocytes by the BrdU cell proliferation assay. As shown in Figure4B, the proliferation proliferation of keratinocytes was inhibited by the specific inhibitors of p38, ERK1/2 and JNK1/2 of keratinocytes was significantly increased by visfatin stimulation, compared to that of the (Figure 4F). Taken together, these results indicate that visfatin also induces the proliferation of untreatedkeratinocytes control via cells. activation The proliferationof p38, ERK1/2, of and visfatin-stimulated JNK1/2. keratinocytes was comparable to that of EGF-treated keratinocytes (Figure4B). Also, we examined the effects of visfatin on the expression2.5. Induction of the of Wound wound Healing healing-related by Visfatin In genesVivo including collagens, MMPs, CTGF, EGF, FGFs, TGFβ, VEGF, VEGFR1 and VEGFR2 in human keratinocytes using Q-PCR. As shown in Figure2A, To examine the therapeutic effects of visfatin on wound repair, 25 µL of 22% hydrogel containing we found1 µg visfatin that visfatinwas applied significantly to a 5-mm increasedwound on the back expression of BALB/c of mice VEGF once and a day VEGFR2 for 5 days. (Figure EGF4 C). However,(1 µg/wound) visfatin and did vehicle not affect alone the (saline) expression were used of otheras a positive genes includingand negative MMPs, control, FGFs, respectively. CTGF, EGF, andWound TGFβ. repair Additionally, was monitored we observed every day that for visfatin 10 days induced after injury the migrationand wound of closure keratinocytes was analyzed (Figure by4 D). KeratinocytesImageJ for actively the percentage migrated of into wound the scratched size (Figure area 5B). at Asa concentration shown in Figure of 50 5A, ng/mL in the visfatin, hydrogel which wascontaining comparable visfatin with theor EGF-treated migration ofmice keratinocytes the wound size upon was treatment decreased with significantly 50 ng/mL from EGF day or 2 10 to ng/mL 10 VEGF.after Importantly, injury, compared VEGF-neutralizing to those treated antibody with vehicle suppressed alone. Ten the days visfatin-induced after injury, the cell wounds migration of of keratinocytesvisfatin-treated (Figure mice4D). were Also, healed visfatin-induced approximately migration 100% compared of keratinocytes to those wounded was completely and left (Figure inhibited by the5B). specific inhibitors of p38, ERK1/2, and JNK1/2 (Figure4D).

A C 4 Visfatin VEGF VEGFR1 VEGFR2 MMP2 MMP9

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Red: Actin (Rhodamine phalloidin) ** 100 Visfatin (50ng/mL) BrdU assay (%) ** E F * # ** # 0 5 15 30 45 60 (min) *

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EGF 50 Control) pERK1/2 ------50 Visfatin (ng/mL) vs 50 50 50 50 50 - - - - ERK1/2 Proliferastion U0126 (% - - - + - - - - - 0 1 SB203580 - - - - + - - - - pJNK1/2 Visfatin (ng/mL) 0 50 0 50 0 50 0 50 SP600125 - - - - - + - - - U0126 JNK1/2 - - + + - - - - VEGF(ng/mL) ------10 - SB203580 + + Annti-VEGF(ng/mL) ------1 - 1 Tubulin SP600125 ------+ + FigureFigure 4. Visfatin 4. Visfatin enhances enhances the the proliferation proliferation and migration of of human human keratinocytes keratinocytes through through activation activation of ERK1/2of ERK1/2 and and JNK1/2. JNK1/2. (A) Human keratinocytes were seeded into 6-well plates overnight and then cultured in a serum-free medium for 24 h. Cells were treated with EGF at various concentrations or

Int. J. Mol. Sci. 2018, 19, 3642 7 of 15

(A) Human keratinocytes were seeded into 6-well plates overnight and then cultured in a serum-free medium for 24 h. Cells were treated with EGF at various concentrations or PBS as a negative control. The expression of visfatin mRNA was determined by Q-PCR. * p < 0.05 compared to the saline-treated control group. Values represent the mean ± S.D. (n = 6). Data are representative of at least three independent experiments. (B) Human keratinocytes were treated with visfatin, or EGF at the indicated concentrations for 24 h. The proliferation of keratinocytes was determined by BrdU cell proliferation assay. * p < 0.05 compared to the saline-treated control group. Values represent the mean ± S.D. (n = 6). (C) Human keratinocytes were treated with visfatin at various concentrations or PBS as a negative control. Expression of various genes was determined by Q-PCR. * p < 0.05 compared to the saline-treated control group. Values represent the mean ± S.D. (n = 6). N.S.: not significant. (D) Human keratinocytes were scratched with a 200-µL tip and treated with visfatin, EGF, or VEGF at the indicated concentration after pretreatment with 10 µM U0126, 20 µM SB203580, or 20 µM SP600125 for 1 h. Also, cells were treated with 50 ng/mL visfatin for 24 h after treatment with 1 µg/mL VEGF-neutralizing antibody. Subsequently, cells were fixed with 4% paraformaldehyde for 10 min and stained with rhodamine phalloidin (red) for actin. # p < 0.01 compared to the control- or VEGF-treated group. ** p < 0.01 compared to the visfatin-treated control group. Values represent the mean ± S.D. (n = 3). (E) Human keratinocytes were treated with 50 ng/mL visfatin for 5, 15, 30, 45, and 60 min. Cells were harvested and lysed and cell lysates were analyzed for the levels of p38, pp38, ERK1/2, pERK1/2, JNK1/2, and pJNK1/2 using the indicated antibodies. Tubulin was used as a loading control. (F) Human keratinocytes were treated with 50 ng/mL visfatin for 24 h after pretreatment with 10 µM U0126, 20 µM SB203580, and 20 µM SP600125 for 1 h. The cell growth of keratinocytes was determined by BrdU proliferation assay. * p < 0.05 compared to the visfatin-treated control group. Values represent the mean ± S.D. (n = 6). Data are representative of three independent experiments.

To investigate the effects of visfatin on the activation of MAP kinases in keratinocytes, cells were treated with visfatin and phosphorylation of each MAP kinase was determined by western blot analysis. As shown in Figure4E, visfatin rapidly induced phosphorylation of p38, ERK1/2 and JNK1/2 within 5 or 15 min. This phosphorylation reached a maximum at 30 min after stimulation with visfatin (Figure4E). The phosphorylation levels of p38 MAP kinase in visfatin-treated keratinocytes were comparable to those of the visfatin-treated HDFs (Figure3A). To further investigate the role of ERK1/2, p38, and JNK1/2 phosphorylation in the visfatin-induced cell proliferation, keratinocytes were treated with U0126, SB203580, and SP600125, which are specific inhibitors of ERK1/2, p38, and JNK1/2, respectively. As shown in Figure4F, the visfatin-induced proliferation of keratinocytes was inhibited by the specific inhibitors of p38, ERK1/2 and JNK1/2 (Figure4F). Taken together, these results indicate that visfatin also induces the proliferation of keratinocytes via activation of p38, ERK1/2, and JNK1/2.

2.5. Induction of Wound Healing by Visfatin In Vivo To examine the therapeutic effects of visfatin on wound repair, 25 µL of 22% hydrogel containing 1 µg visfatin was applied to a 5-mm wound on the back of BALB/c mice once a day for 5 days. EGF (1 µg/wound) and vehicle alone (saline) were used as a positive and negative control, respectively. Wound repair was monitored every day for 10 days after injury and wound closure was analyzed by ImageJ for the percentage of wound size (Figure5B). As shown in Figure5A, in the hydrogel containing visfatin or EGF-treated mice the wound size was decreased significantly from day 2 to 10 after injury, compared to those treated with vehicle alone. Ten days after injury, the wounds of visfatin-treated mice were healed approximately 100% compared to those wounded and left (Figure5B). Next, the wound tissues were collected at day 6 after wound puncture and the histological analysis of the wound tissues were performed using hematoxylin and eosin (H&E) staining (Figure5C). We found that visfatin-treated group increased the length of epithelial tongues and epidermis around the wound edge (Figure5C). These results demonstrated that the visfatin-treated group restored the skin wound tissues, as similar to the EGF-treated group (Figure5C). We further confirmed the Int. J. Mol. Sci. 2018, 19, 3642 8 of 15 mechanism by which visfatin activates ERK and JNK pathway in vivo. As shown in Figure5D, the induction of phosphorylation of ERK and JNK was confirmed by the immunofluorescence staining of the tissue sections, compared to the saline-treated group (Figure5D). The induction of ERK and JNK phosphorylation was comparable to that of the EGF-treated group (Figure5D). Moreover, we demonstrated that visfatin increased the VEGF expression in the wound tissue section, compared to the saline-treated group. These results indicate that visfatin promotes wound healing in vivo. TheInt. J. therapeutic Mol. Sci. 2018 effect, 19, x of visfatin on wound repair was comparable to that of EGF treatment. 9 of 16

Saline EGF Visfatin Saline EGF Visfatin

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Day 0 2 4 6 8 10 VEGF FigureFigure 5. 5.Treatment Treatment with with visfatin visfatin accelerates accelerates wound wound healing healingin in vivo vivo.(. A(A)) Representative Representative photographs photographs ofof wounds wounds from from mice mice treated treated with with visfatin, visfatin, EGF, EGF, and and control control saline. saline. The The hydrogel hydrogel containing containing visfatin visfatin (1(1µ µg/wound)g/wound) and EGF (1 µg/wound)µg/wound) were were applied applied every every 24 24 h hfor for 5 5days. days. Digital Digital images images indicate indicate the thestatus status of wound of wound healing healing during during the the 10 10days days of ofthe the wound wound repair repair process. process. (B ()B The) The wound wound closure closure in incontrol control saline-, saline-, visfatin-, visfatin-, or or EGF-treated EGF-treated mice mice was was measured measured digitally by counting counting pixels pixels at at the the indicatedindicated days daysafter after surgicalsurgical incisionincision andand thethe wound closure was was expressed expressed in in terms terms of of percentage. percentage. * *pp << 0.05 0.05 compared compared toto the the saline-treated saline-treated control control group. group. Values Values represent represent the mean the mean ± S.D.± (nS.D. = 6). ( n(C=) For 6). (Cthe) For histological the histological analysis, analysis, the H&E-stained the H&E-stained wound wound tissues tissues of day of 6 day post-wound 6 post-wound were were analyzed analyzed and andgraphed graphed for forepithelial epithelial tongue tongue (Magnification (Magnification 100×, 100 Scale×, Scale bar, bar, 200 200 µm).µm). The The black black arrow arrow indicates indicates as asfollow: follow: D D(dermis), (dermis), EpEp (epidermis), (epidermis), Epi Epi tongue tongue (epithelial (epithelial tongue), tongue), and andGT (granulation GT (granulation tissue). tissue). * p < *0.05p < 0.05compared compared to the to saline-treated the saline-treated control control group. group. Values Values represent represent the mean the ± mean S.D. (±n =S.D. 6). N.S.: (n = not 6). N.S.:significant. not significant. (D) For ( theD) For immunofluorescence the immunofluorescence staining, staining, tissue tissuesections sections of day of 6 day post-wound 6 post-wound were weretreated treated with with fluorescence-conjugated fluorescence-conjugated antibodies antibodies against against ERK, ERK, JNK, JNK, or or VEGF and and photographed photographed (Magnification(Magnification× ×200,200, Scale Scale bar, bar, 200 200 µm).µm). * p *< p0.05< 0.05compared compared to the to saline-treated the saline-treated control control group. group.Values Valuesrepresent represent the mean the mean± S.D. ±(n S.D.= 6). ((nE=) Proposed 6). (E) Proposed scheme scheme of the therapeutic of the therapeutic action of action visfatin of visfatin in wound in woundhealing. healing. 3. Discussion 3. Discussion The clinical applications of growth factors, including epidermal growth factor (EGF) which is The clinical applications of growth factors, including epidermal growth factor (EGF) which is commercially available and basic fibroblast growth factor (bFGF) have been rapidly developed in commercially available and basic fibroblast growth factor (bFGF) have been rapidly developed in wound healing. The topical applications of these growth factors have been proven to be effective in wound healing in China [45]. However, the mechanism by which EGF promotes wound healing is still unclear. Thus, identification of the EGF-induced genes might be helpful in understanding the process of wound healing. In this study, we have identified the wound healing-related gene, visfatin, by cDNA microarray analysis in EGF-stimulated human dermal fibroblasts (HDFs) and then examined the effects of visfatin on wound healing in vitro and in vivo. Many studies have reported that visfatin is secreted from peripheral-blood monocytes, lymphocytes, dendritic cells, macrophages, adipose tissues and expressed in various malignant tumors [13,15,17–24,46]. Additionally, visfatin was shown to be expressed in fibroblasts or macrophages in the synovial

Int. J. Mol. Sci. 2018, 19, 3642 9 of 15 wound healing. The topical applications of these growth factors have been proven to be effective in wound healing in China [45]. However, the mechanism by which EGF promotes wound healing is still unclear. Thus, identification of the EGF-induced genes might be helpful in understanding the process of wound healing. In this study, we have identified the wound healing-related gene, visfatin, by cDNA microarray analysis in EGF-stimulated human dermal fibroblasts (HDFs) and then examined the effects of visfatin on wound healing in vitro and in vivo. Many studies have reported that visfatin is secreted from peripheral-blood monocytes, lymphocytes, dendritic cells, macrophages, adipose tissues and expressed in various malignant tumors [13,15,17–24,46]. Additionally, visfatin was shown to be expressed in fibroblasts or macrophages in the synovial tissues with rheumatoid arthritis and psoriatic skin [47,48]. The function of visfatin has been examined in atherosclerotic diseases and various types of tumors, such as glioblastoma, malignant astrocytomas, breast and, prostate cancer [20,49–53]. Visfatin is also known to function as a growth factor causing proliferation and differentiation. Moreover, visfatin has been shown to promote vascular smooth muscle cell maturation and is well characterized for insulin mimetic effect [17,54]. However, there is no evidence to date on therapeutic effects of visfatin in wound repair. In this study, we have demonstrated the role of visfatin and its underlying mechanism in wound healing. EGF significantly induced the expression of visfatin in a dose- and time-dependent manner in HDFs (Figure1A–D). Thus, we hypothesized that visfatin could be involved in the wound healing process. As we expected, human recombinant visfatin significantly induced the migration and proliferation of HDFs (Figure1E–G). However, the mechanism by which visfatin promotes wound repair is still unclear. The recent study demonstrated that visfatin induced the proliferation of endothelial cells by induction of VEGF production [27,39]. Visfatin was also reported to induce the expressions of MMP-2 and MMP-9 [27,39]. As shown in Figure2, visfatin induced the expression of VEGF. However, MMP2 and MMP9 expression remained unchanged upon visfatin treatment (Figure2A,B). We expect that this discrepancy depends on cell types used for the experiments. Also, we demonstrated that visfatin enhanced the expression of VEGF in wound tissue section (Figure5D). Recent studies have reported that visfatin increases VEGF expression through activation of PI3K/Akt, ERK1/2, and p38 MAPK signaling pathways [26,27,39–41]. Consistent with these reports, we found that visfatin activated the phosphorylation of ERK1/2 and JNK1/2 MAPK in HDFs (Figure3A). Furthermore, in the histological analysis, we found that visfatin increased the length of epithelial tongues and epidermis around the wound edge, as similar to the EGF-treated group (Figure5C). As shown in Figure5D, visfatin induced the phosphorylation of ERK and JNK in tissue sections, compared to the saline-treated group (Figure5D). In addition, visfatin-induced proliferation and migration of HDFs were significantly suppressed by specific inhibitors of ERK1/2 and JNK1/2, that is, U0126 and SP600125, respectively (Figure3B–D). We also found that visfatin improved wound healing by enhancing the proliferation and migration through the induction of VEGF via the MAPK pathway in keratinocytes, as similar to the HDFs model (Figure4). Therefore, we demonstrated that visfatin induced the proliferation and migration of cells through ERK1/2 and JNK1/2 signaling pathways. As shown in Figure5E, we suggested that visfatin promotes wound repair by enhancing VEGF expression via phosphorylation of the MAP ERK1/2 and JNK1/2. The results of the present study have several critical clinical implications. First, EGF, a typical growth factor, significantly increased the expression of visfatin which was shown to play an important role in wound healing in a time- and dose-dependent manner in HDFs. Thus, we suggest that visfatin may be used as a novel therapeutic agent for wound repair along with other growth factors, such as EGF and bFGFs. Second, undefined receptors of visfatin should be demonstrated to improve the healing potential of visfatin in promoting wound healing. These data indicate that visfatin may become a potential therapeutic agent to improve tissue repair through triggering the proliferation and migration of HDFs and human keratinocytes via induction of VEGF expression. However, further studies on visfatin expression and activity are needed to develop a novel therapeutic agent for tissue repair. Int. J. Mol. Sci. 2018, 19, 3642 10 of 15

4. Materials and Methods

4.1. Mice and Cell Culture BALB/c mice were purchased from Orient Bio, Inc. (Seoul, Korea). All experiments were ethically performed under the guidelines of the Korea University Institutional Animal Care and Use Committee (Seoul, Korea; approval no. KUIACUC-2015-244, 2017-109). Human dermal fibroblasts (HDFs) and human keratinocytes (HaCaT cells) were purchased from Biosolution (Seoul, Korea) with passage number 2. HDFs and HaCaT cells were cultured in the mixture (1:3) of F-12K and DMEM or DMEM with 10% fetal bovine serum and 100 U/mL penicillin and 0.1 mg/mL streptomycin (Life Technologies, ◦ Carlsbad, CA, USA) at 5% CO2, 37 C incubator, respectively.

4.2. Reagents and Antibody Antibodies for JNK1/2 (sc-571), phosphor-JNK1/2 (sc-6254), ERK1/2 (sc-153), phosphor-ERK1/2 (sc-7383), p38 (sc-535), Tubulin (sc-69969), and VEGF (sc-152) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for goat anti-rabbit IgG Alexa fluor 488 (A-11034) and goat anti-mouse IgG Alexa fluor 488 (A-11029) secondary antibody were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies for phospho-p38 (#9216), Rabbit-HRP (#7074) and Mouse-HRP (#7076) were purchased from Cell Signaling (Danvers, MA, USA). Recombinant human visfatin (10990-H20B) was purchased from Sino Biological (Beijing, China). Pluronic®F-127 (P2443) for the preparation of hydrogel (22%) to delivered EGF or visfatin was purchased from Sigma Aldrich (Kenilworth, Merck, Germany).

4.3. cDNA Microarray Fragmented and Labeled single strand-DNA (ss-DNA) was prepared according to the standard Affymetrix protocol from 500 ng total RNA (GeneChip® WT PLUS Reagent Kit Manual, Carlsbad, CA, USA). Following fragmentation, 3.5 µg of ss-DNA were hybridized for 16 h at 45 ◦C with 60 rpm on GeneChip® Mouse Gene 2.0 ST Array (Carlsbad, CA, USA). GeneChips were washed and stained on the Affymetrix Fluidics Station 450 (Carlsbad, CA, USA). GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G (Carlsbad, CA, USA). To normalize the results, the data were analyzed with Robust Multichip Analysis (RMA) using Affymetrix default analysis settings and global scaling. The trimmed mean target intensity of each array was arbitrarily set to 100. The values of normalized and log-transformed intensity were then analyzed using GeneSpring GX 13.1.1 (Agilent Technologies, Santa Clara, CA, USA). Fold change filters included the requirement that the genes be present in at least 200% of controls for up-regulated genes (2-fold up) and lower than 50% of controls for down-regulated genes (2-fold down).

4.4. MTT and BrdU Proliferation Assays In Vitro HDFs and HaCaT cells were seeded and pre-cultured into 96-well plates at a density of 5 × 103 cells/well for 24 h. After cells were cultured in serum-free media for 16 h and then cells were stimulated with various concentration of visfatin or EGF as a positive control in serum-free medium. After 24 h stimulation, the cells were treated with 20 µL of 100 µg/mL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution for 2 h and then dissolved in 150 µL of DMSO and then determined absorbance at 560 nm. For BrdU incorporation assay, the 5-bromo-20-deoxy-uridine (BrdU) cell proliferation ELISA kit (colorimetric) was purchased from Roche (11647229001, Basel, Switzerland) and the assay was performed according to the manufacturer’s instructions. In brief, after 24 h stimulation with visfatin or EGF, cells were treated with BrdU for 3 h. BrdU-integrated DNA was determined at 490 nm and quantified according to the relative absorbance. Int. J. Mol. Sci. 2018, 19, 3642 11 of 15

4.5. Migration Assay In Vitro HDFs and HaCaT cells were seeded and pre-cultured into 12-well plates at a density of 8 × 104 cells/well for 24 h and then cells were cultured with serum-free media for 16 h. HDFs were scratched with the 200-µL tip. Cells were treated with a various concentration of visfatin or EGF for 24 h. Following stimulation, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then permeabilized with 0.1% Triton X-100 in PBS for 5 min. Next, the cells were stained with rhodamine phalloidin for 2 h. Nuclei were stained with DAPI. Fluorescence images were acquired with a fluorescence microscope (Olympus IX 71, Shinjuku, Tokyo, Japan).

4.6. Wound Healing Assay In Vivo

Seven-week-old mice (n = 6) were anesthetized with 65% N2, 30% O2 and 5% isoflurane and then maintained with 2% isoflurane during wound puncture. Dorsal hair was shaved and wiped with 70% ethanol before wound puncture (5 mm diameter). The wounds were topically treated with saline, 1 µg of EGF, or 1 µg of visfatin in hydrogel per wound at once a day for 5 days. The wound closure was monitored by taking the picture at the same height with fixed height stand. Measurement of the wound area in the images was analyzed using by the Image J program (NIH, Bethesda, MD, USA). Wound repair was calculated using the percentage of wound size as follows [55]:

(Wound Area on Day 0) − (Wound Area on Day post − wounding) Wound closure (%) = × 100 Wound Area on Day 0

4.7. Histologic Analysis of Wounds and Immunofluorescence Microscopy Histologic analysis was performed on digital images using the ImageJ program. Cross-sections (5 µM) of paraffin-embedded skin tissues of day 6 post-wound were stained with hematoxylin and eosin (H&E) or picrosirius red for the extent of re-epithelialization and collagen expression, respectively. Measurement of the collagen in the images was analyzed using by the ImageJ program. The length of the epithelial tongue was determined as the distance between the margin of the wound and the end of the epithelial using by ImageJ program. The relative epithelial tongue was calculated compared with the saline-treated group. For immunofluorescence microscopy, immunofluorescence detection of phospho-ERK, phosphor-JNK, and VEGF proteins was performed using rabbit anti-VEGF, anti-mouse pERK, anti-mouse pJNK (Santa Cruz Biotechnology; Dallas, TX, USA), goat anti-rabbit IgG Alexa fluor 488 (A-11034), and goat anti-mouse IgG Alexa fluor 488 (A-11029) secondary antibody (Invitrogen, Carlsbad, CA, USA) in the paraffin section of mouse skin tissue of day 6 post-wound. Stained sections were observed under fluorescence microscopy systems (100×, Olympus IX 71, Shinjuku, Tokyo, Japan). The fluorescence was analyzed and quantified using by the ImageJ program.

4.8. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Real-Time Quantitative PCR (Q-PCR) Analysis HDFs (1 × 105 cells/well) were treated with various concentrations of visfatin for the different time and then total RNA was extracted with RiboEX total RNA kit (GeneAll Biotechnology, Seoul, Korea). Total RNA (1 µg) was reverse-transcribed into cDNA by reverse transcriptase. The cDNAs were amplified with specific primers by PCR using GenieTM 32 Thermal Block (Bioneer, Daejeon, Korea). Specific primer sequences were as follow: hGAPDH, 50-ACA TCA AGA AGG TGG TGA AG-30 (sense) and 50-ATT CAA GAG AGT AGG GAG GG-30 (anti-sense); hvisfatin, 50-AGG GTT ACA AGT TGC TGC CA-30 (sense) and 50-CAA AAT TCC CTG CTG GCG TC-30 (anti-sense); hVEGF, 50-CCT TGC CTT GCT GCT CTA CC-30 (sense) and 50-CCT ATG TGC TGG CCT TGG TG-30 (anti-sense). The PCR products were separated on 1.5% agarose gels and stained with Staining Star (Dyenbio, Seongnam, Kyeongki, Korea). Quantitative-PCR analyses were performed with Fast SYBRTM Green Master Mix (Applied BiosystemsTM, Waltham, MA, USA). The reactions of 6.25 µL fast SYBRTM Green Master Mix, 1 µL of cDNA, 0.5 µM primers in a final volume of 12.5 µL were analyzed in an optical 96-well Int. J. Mol. Sci. 2018, 19, 3642 12 of 15 fast clear reaction plate (Applied BiosystemsTM, Waltham, MA, USA). Reactions were amplified and using human GAPDH as an endogenous control. The primer sequences are listed in quantified by using a StepOnePlus Real-Time PCR System and the manufacturer’s corresponding software (StepOnePlus Software v2.3; Life Technologies, Waltham, MA, USA). The relative quantities of mRNAs were determined by using the comparative Ct method and were normalized Table1.

Table 1. Primer sequences used for Q-PCR.

Gene Forward (50-30) Reverse (50-30) hCOL1A1 CATGACCGAGACGTGTGGAA GGCAGTTCTTGGTCTCGTCA hCOL3A1 TGTTCCAGGAGCTAAAGGCG CTCCTGGGATGCCATTTGGT hCOL4A1 TTTTGTGATGCACACCAGCG AGTAATTGCAGGTCCCACGG hCOL5A1 AGACATGGGCATCAAGGGTG CCGAGTTTTCCCTTCTCCCC hMMP2 CCCTGTGTCTTCCCCTTCAC GTAGTTGGCTGTGGTCG hMMP9 GGACAAGCTCTTCGGCTTCT TCGCTGGTACAGGTCGAGTA hFGF2 AAAAACGGGGGCTTCTTCCT AGCCAGGTAACGGTTAGCAC hFGF7 TGGATCCTGCCAACTTTGCT TTCTTGTGTGTCGCTCAGGG hFGF10 GGAGCTACAATCACCT ACGGGCAGTTCTCCTTCTTG hEGF AGTCCGTGACTTGCAAGAGG CCTCTTCTTCCCTAGCCCCT hTGFβ TGGTGGAAACCCACAACGAA GAGCAACACGGGTTCAGGTA hCTGF GTTTGGCCCAGACCCAACTA GGCTCTGCTTCTCTAGCCTG hVEGF CTTGCCTTGCTGCTCTACCT GCAGTAGCTGCGCTGATAGA hVisfatin TCGGTTCTGGTGGAGGTTTG TTGGGATCAGCAACTGGGTC hVEGFR1 GGGGGAAGCAGCCCATAAAT GCCAGTGTGGTTTGCTTGAG hVEGFR2 CGGTCAACAAAGTCGGGAGA CAGTGCACCACAAAGACACG hGAPDH TCCTGCACCACCAACTGTT GTCCACTGTCTTCTGGGTGG

4.9. Western Blot Analysis Whole cell lysates were separated on 10% SDS–PAGE gels and transferred to polyvinylidene difluoride membranes (PVDF). The membrane was blocked with 5% skim milk (Neogen Co., Lansing, MI, USA) and then incubated with a 1:1000 diluent of a primary antibody in TBS with 5% BSA. The membrane was washed 5 times in TBS-T and then incubated with 1:5000 diluent of the corresponding secondary antibodies HRP-conjugated IgG antibody (Cell Signaling Technology, Danvers, MA, USA) in TBS with 5% skim milk. Target proteins were visualized by using the ECL detection system (Amersham ECL Prime Western blotting Detection Regent, GE Healthcare, Chicago, IL, USA).

4.10. Statistical Analysis All experiments were repeated at least three independent experiments. Data in bar graphs were shown as the mean ± standard deviation (S.D.). Statistical analysis between the control and the treated groups were assessed using a two-tailed Student’s t-test, Normality of distribution was assessed by the Shapiro-Wilk test and comparisons more than two conditions (k > 2) were determined using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test, using SPSS 22 statistics software (IBM). * p < 0.05, ** p < 0.01 and # p < 0.01 were considered statistically significant.

Author Contributions: B.-C.L. conceived and designed experiments. B.-C.L., J.S. and A.L., performed experiments. B.-C.L. and T.S.K. wrote the manuscript. B.-C.L., D.C. and T.S.K. analyzed data. T.S.K. was responsible for overall study design and supervised the project. Funding: This research was supported by the Creative Materials Discovery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science ICT and Future Planning (grant no. 2016M3D1A1021387). Conflicts of Interest: The authors declare no conflict of interest. Int. J. Mol. Sci. 2018, 19, 3642 13 of 15

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