Valproic Acid Induces Cutaneous Wound Healing In Vivo and Enhances Keratinocyte Motility

Background Cutaneous wound healing is a complex process involving several signaling pathways such as the Wnt and extracellular signal-regulated kinase (ERK) signaling pathways. Valproic acid (VPA) is a commonly used antiepileptic drug that acts on these signaling pathways; however, the effect of VPA on cutaneous wound healing is unknown. Methods and Findings We created full-thickness wounds on the backs of C3H mice and then applied VPA. After 7 d, we observed marked healing and reduced wound size in VPA-treated mice. In the neo-epidermis of the wounds, β-catenin and markers for keratinocyte terminal differentiation were increased after VPA treatment. In addition, α-smooth muscle actin (α-SMA), collagen I and collagen III in the wounds were significantly increased. VPA induced proliferation and suppressed apoptosis of cells in the wounds, as determined by Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining analyses, respectively. In vitro, VPA enhanced the motility of HaCaT keratinocytes by activating Wnt/β-catenin, ERK and phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling pathways. Conclusions VPA enhances cutaneous wound healing in a murine model and induces migration of HaCaT keratinocytes.

The ERK and PI3-kinase/Akt signaling pathways are involved in developmental processes, including skin development [19,20]. In addition, these two pathways are crucial for cutaneous wound healing [6,7]. Growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) accelerate keratinocyte migration and epithelialization in skin wound healing by activating the ERK and PI3-kinase/Akt signaling pathways [21,22]. Conversely, inhibition of these signaling pathways has been shown to impair corneal wound healing [23]. Taken together, these results indicate that the Wnt, ERK and PI3kinase/Akt signaling pathways may be useful therapeutic targets to enhance wound healing [7,13,24].
In this study, we investigated the effect of VPA on cutaneous wound healing in a murine model. The role of VPA in regulating wound healing was characterized by evaluating its effect on neoepidermis formation, fibroblast-myofibroblast transition, and cellular proliferation and apoptosis in the wounds. Finally, we assessed the effect of VPA on keratinocyte migration to confirm the role of VPA in wound healing in a human system.

Animals and in vivo Wound Healing Assay
Seven-week-old male C3H mice were purchased from Orient Bio Co. and allowed to adapt to their new environment for 1 week. All mouse experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine (09-013). To determine the effects of VPA on wound healing, 8week-old C3H mice, whose hair follicles are naturally synchronized in the telogen phase [32] and dermal depths are almost consistent [33], were anesthetized, backs were shaved by hair clipper, cleaned with ethanol, and full-thickness incision wounds were created. VPA (500 mM; Acros) was applied topically to the wounds daily (n = 10). Wound sizes were measured every other day on the assumption that wound depths in each animal are almost constant. The wounded skin tissue was also evaluated by immunohistochemical analysis.

Cell culture and in vitro Wound Healing Assay
HaCaT keratinocytes [34] were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% (v/ v) heat-inactivated fetal bovine serum (FBS, Gibco), 100 mg/ml penicillin (Gibco) and 100 mg/ml streptomycin (Gibco), and incubated in 5% (v/v) CO 2 at 37uC. For the in vitro wound healing assay, HaCaT cells (4610 5 cells/well) were seeded into 12-well plates in DMEM supplemented with 10% FBS and allowed to adhere overnight. The monolayers were then carefully scratched with sterile pipette tips and incubated with medium containing 2% serum with or without 100 mM VPA. After 24 h, the cells were washed once with cold phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 15 min at room temperature, and stained with 2% (w/v) crystal violet. The wound closure rate was measured using NIS-Elements imaging software (Nikon) (n = 3).

siRNA Preparation and Transfection
The human b-catenin small interfering RNA (siRNA) target sequences were 59-GAAACGGCTTTCAGTTGAG-39 and 59-AAACTACTGTGGACCACAAGC-39. The siRNAs were transfected into HaCaT cells using Lipofectamine Plus reagent (Invitrogen) at a final concentration of 100 nM. Transfected cells were grown for 24 h in 5% (v/v) CO 2 at 37uC. The cells were scratched and incubated for 24 h with medium containing 2% serum with or without 100 mM VPA.

Hematoxylin and Eosin Staining
Tissues were embedded in paraffin and cut into 4-mm sections. The sections were deparaffinized in three changes of xylene and rehydrated through a graded ethanol series. The sections were stained with hematoxylin for 5 min and with eosin for 1 min. The slides were then dehydrated through a graded alcohol series, cleared in xylene and mounted in Permount (Fisher Scientific). The hematoxylin and eosin (H&E)-stained slides were visualized using a bright-field optical microscope (Nikon TE-200U).

Apoptosis Assay
Tissue sections (4-mm thick) were deparaffinized and rehydrated. The slides were incubated in 20 mg/ml proteinase K for 15 min at room temperature, and washed twice with PBS for 5 min. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon).

Immunocytochemistry
Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed with PBS. For permeabilization, cells were treated with 0.1% Triton X-100 for 15 min at room temperature. After blocking with 5% BSA and 1% goat serum in PBS for 30 min at room temperature, the cells were incubated with mouse anti-b-catenin antibody (BD Transduction Laboratories; 1:100) or rabbit E-cadherin antibody (Cell Signaling Technology; 1:100) overnight at 4uC. The cells were rinsed with PBS and then incubated with Alexa Fluor 488conjugated goat anti-mouse antibody or anti-rabbit antibody (Molecular Probes; 1:400) for 1 h at room temperature, counterstained with DAPI (Boehringer Mannheim; 1:5000), and examined under confocal microscope, LSM510 META (Carl Zeiss).

Statistical Analysis
Statistical analyses were performed using unpaired two-tailed Student's t-test. Asterisks indicate the statistically significant differences with one asterisk being p,0.05 and two asterisks being p,0.005.

The Wnt/b-catenin Signaling Pathway is Activated during the Wound Healing Process
We investigated the activation status of the Wnt/b-catenin signaling pathway during the wound healing process in a murine model to characterize the role of this pathway in cutaneous wound healing. We created full-thickness wounds (diameter = 0.5 cm) on the backs of mice and monitored levels of b-catenin and wound healing markers. b-Catenin was highly expressed in epidermal keratinocytes and dermal fibroblasts at 7 and 10 d after wounding ( Figure 1A). The expression of keratin 14, a marker of undifferentiated keratinocytes, was also high in epidermis at 7 and 10 d post-wounding ( Figure 1A). The a-smooth muscle actin (a-SMA) was gradually increased in dermal fibroblasts and its level was maximal at 7 d post-wounding ( Figure 1A). Expression levels of collagen I and collagen III were also gradually increased in dermal fibroblasts of cutaneous wounds ( Figure 1A). In large cutaneous wounds (diameter = 1.5 cm), b-catenin and wound healing markers were also progressively increased during the wound healing process of mice ( Figure S1A). The high expressions of b-catenin and wound healing markers, especially at 7 and 10 d post-wounding, were also confirmed by western blot analyses ( Figure 1B). We founded that adult stem cell markers, Nestin and CD34 were expressed at 7 and 10 d after wounding (Figures 1C  and S1B).

VPA Promotes Cutaneous Wound Healing
Because of the relationship between Wnt/b-catenin signaling pathway and wound healing [4,5], we evaluated the effectiveness of VPA on cutaneous wound healing. With daily application of VPA to the wound area (diameter = 0.5 cm) of C3H mice, the wounds were markedly reduced in size and almost completely re-epithelialized after 7 d (Figures 2A and  2B). Histological analysis also showed restitution of normal tissue structure following application of VPA ( Figure 2C). VPA is known to activate the Wnt/b-catenin signaling pathway by inhibiting GSK3b [35,36]. To determine the effect of VPA on this pathway in our mouse model of wound healing, b-catenin expression was evaluated in VPA-treated wounds. We observed increased b-catenin expression in the wound neo-epidermis after a 7-d application of VPA ( Figures 3A and S2A). Both filaggrin and loricrin, markers of keratinocyte terminal differentiation involved in neo-epidermis formation, and keratin 14 were induced by VPA ( Figures 3A and S2A). Western blot analysis confirmed increased  Figures 3B and S3A).

VPA Induces Fibroblast-to-myofibroblast Transition
Fibroblast-to-myofibroblast transition is an important step in cutaneous wound healing [37]. Protein levels of a-SMA, collagen I, and collagen III, markers of myofibroblast differentiation, were significantly increased by treatment of VPA onto the wounds, as shown by Western blot and immunohistochemical analysis ( Figures 3B, 3C, S2B and S3B).

VPA Induces Cell Proliferation and Inhibits Apoptosis in the Wound
The increase in cell proliferation and decrease in apoptosis are important phenomena in early wound healing [38]. We stained cells for Ki67 to detect cell proliferation and performed the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to evaluate apoptosis in VPA-treated wounds. Our results showed that VPA stimulated cell proliferation and suppressed apoptosis in the wound ( Figure 3D). Moreover, Ki67 was expressed in a linear pattern in the epidermal layer of VPAtreated wounds, but was expressed in a scattered pattern in the vehicle-treated wounds ( Figure 3D). Caspase 8, mediator of apoptosis during wound healing [39], was significantly decreased by application of VPA onto the wounds ( Figure 3B). In contrast, protein levels of Mcl-1, an anti-apoptotic protein, and PCNA, a proliferation marker, were increased in VPA-treated wounds ( Figure 3B). HaCaT keratinocytes were used to determine the effect of VPA on human keratinocyte migration. Cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, streptomycin (100 mg/ml), and penicillin (100 mg/ml) in 5% CO 2 at 37uC. After scratch wounding with sterile pipette tips, HaCaT keratinocytes were incubated with medium containing 2% serum with or without 100 mM VPA for 24 h. (A) Cells treated with or without 100 mM VPA were stained with crystal violet for 24 h (first row panel; original magnification 640). Immunocytochemical analyses of phalloidin, b-catenin, or E-cadherin in control and VPA treated HaCaT cells (second, third, and fourth row panel; original magnification 6400). (B) The relative wound closure rate of HaCat cells treated with or without 100 mM VPA. The wound closure rate was measured using NIS-Elements imaging software. Asterisks denote the significant differences between control and test groups as measured by t-test with one asterisk being p,0.05 (n = 3). (C) Western blot analysis of b-catenin, Ecadherin, p-ERK, p-Akt, or a-tubulin in control and VPA-treated HaCaT cells. (D) HaCaT cells were transfected with 100 nM b-catenin siRNA before VPA treatment. The wound closure rate was measured using NIS-Elements imaging software. Asterisks indicate the statistically significant differences as measured by t-test with one asterisk being p,0.05 and two asterisks being p,0.005 (n = 3). (E) 10 mM U0126 or LY294002 was pre-treated for 1 h before VPA treatment, and the wound closure rate was measured after 24-h VPA treatment. One asterisk means p,0.05 and asterisks being p,0.005 (n = 3). doi:10.1371/journal.pone.0048791.g006 VPA enhances cutaneous healing in large wounds.
To investigative the effect of VPA on cutaneous wounds of different sizes, we created large cutaneous wounds (diameter = 1.5 cm) on the dorsal skin of C3H mice and topically applied VPA to the wound. VPA accelerated healing of large cutaneous wounds, as determined by measurements of wound diameters ( Figure S4), and the wound size was markedly reduced in VPAtreated wounds after 10 d ( Figure 4A). Histological analysis also showed that the distance between the wound edges were decreased by VPA ( Figure 4B). Levels of b-catenin, filaggrin, loricrin, keratin 14, and PCNA in the neo-epidermis of VPA-treated wounds were significantly increased ( Figure 4C). In addition, a-SMA, collagen I, and collagen III were significantly increased by VPA ( Figure 4D). Western blot analyses also confirmed that b-catenin, filaggrin, loricrin, keratin 14, a-SMA, and collagen I were increased after VPA treatment ( Figure 4E).

VPA Induces the Expression of Stem Cell Markers in the Wound
Multipotent adult stem cells contribute to cutaneous wound healing [40][41][42]. To investigative the effect of VPA on stem cells in the wounds, the expression levels of stem cell markers, Nestin and CD34 were determined by immunohistochemical anlaysis. VPA induced the expression of Nestin and CD34 in dermis of small wounds after 7 d (Figures 5A and S5A). Nestin-or CD34expressing cells were also increased in large wounds after a 10d application of VPA ( Figures 5B and S5B).

VPA Enhances Motility of HaCaT Keratinocytes by Activating Wnt/b-catenin, ERK and PI3-kinase/Akt Signaling Pathways
To determine whether VPA can influence keratinocyte migration in a human system, we evaluated the effect of VPA on HaCaT keratinocyte migration after scratch wounding. After 24 h, VPA enhanced narrowing of the scratch wound in HaCaT keratinocytes ( Figures 6A and 6B), showing a maximum enhancement at the concentration of 100 mM ( Figure S6). VPA-treated cells exhibited more stress fibers and a thicker cortical network than control cells, as demonstrated by phalloidin staining ( Figure 6A). b-Catenin level was increased by 24-h VPA treatement, but Ecadherin level was decreased ( Figures 6A and 6C). The effect of Wnt/b-catenin signaling pathway on VPA-induced keratinocyte migration was analyzed by using b-catenin siRNA transfection. VPA increased nuclear translocation of b-catenin, but this VPA effect was significantly reduced by b-catenin knockdown, as shown by immunocytochemical and western blot analyses ( Figures S7A  and S7C). The VPA-induced keratinocyte migration was blocked ( Figures 6D and S8) and decrease of E-cadherin level by VPA treatment was recovered by b-catenin siRNA (Figures S7B and  S7C).
The ERK and PI3-kinase/Akt signaling pathways are known to be involved in keratinocyte migration [21,22]. Therefore, we next investigated the involvement of ERK and PI3-kinase/Akt signaling pathways in VPA-induced keratinocyte migration. Western and immunocytochemical analysis showed that the activities of ERK and Akt were significantly increased by 1-h VPA treatment (Figures 6C and S9). Such activations were specifically abolished by pre-treatment with U0126 or LY294002, selective inhibitors for MEK and PI3-kinase, respectively ( Figure S9). After 24 h, the VPA-induced keratinocyte migration was also blocked by U0126 or LY294002 (Figures 6E and S10).

Discussion
VPA is an antiseizure drug commonly prescribed for epilepsy and used as a mood stabilizer for bipolar disorder over the last several decades. Previous studies have shown that VPA exerts differential effect on cell proliferation and migration. For example, VPA suppresses breast cancer cell migration by specifically targeting HDAC2 and down-regulating survivin [43]. VPA also inhibits proliferation and migration of prostate cancer cells by hyperacetylation of histone H3 and H4 [44]. However, VPA enhances mesenchymal stem cell migration by inhibiting GSK3b and increasing protein levels of matrix metalloproteinase-9 (MMP-9) [45]. VPA also stimulates the proliferation of hematopoietic stem cells by inhibiting GSK3b and up-regulating HOXB4, a target of Wnt/b-catenin signaling pathway [46]. Thus, VPA regulates proliferation and migration in a cell-specific or tissuespecific manner. In this study, we found that VPA induced proliferation and suppressed apoptosis in wounds and enhanced motility of HaCaT keratinocytes. The finding that VPA increases keratinocyte proliferation is consistent with the results of our previous studies reporting that VPA induces hair regeneration [47].
VPA has been shown to activate Wnt/b-catenin signaling pathway by inhibiting GSK3b [35,36]. We found that VPA increased markers of keratinocyte terminal differentiation, which are regulated by Wnt/b-catenin signaling pathway [48,49] in the wound neo-epidermis. Wnt/b-catenin signaling pathway is also important for regulation of cell proliferation and motility in the wounds [13,14]. Although hypertrophic scars and keloids are the result of dysregulated Wnt/b-catenin signaling pathway [50], we believed that the moderate regulation of Wnt/b-catenin signaling pathway in the normal cutaneous wounds or other types of wounds is important for enhancing wound healing.
During early wound healing, cell proliferation is controlled by a decrease in apoptosis [13]. VPA has been shown to activate the ERK and PI3-kinase/Akt signaling pathways [51,52]. Our results indicate that VPA may regulate proliferation in early wound healing via the ERK and PI3-kinase/Akt signaling pathways.
We found that VPA induced the expression of stem cell markers, Nestin and CD34. Nestin-expressing interfollicular blood vessel network contributes to skin wound healing [41]. It is also known that CD34 positive cell transplantation improves wound healing by increasing neo-vascularization [42]. The induction of Nestin and CD34 by VPA may be important in the enhancement of efficacy of stem cell therapy in cutaneous wounds.
Taken together, our results suggest that VPA induces cutaneous wound healing in murine model and enhances HaCaT keratinocyte migration. VPA induced complete re-epithelialization in wounds of mice at an earlier time than other agents, although the direct comparison of the effectiveness of the healing between VPA and those agents are difficult [53,54]. In terms of safety, drugs used for cuatenous wound healing such as growth factors and small molecules often result in potential side effects [55]. However, VPA, which has been used for several decades to safely treat neurological disorders [25], can potentially be applied without significant side effects. Therefore, VPA may be useful for the development of therapies to enhance wound healing. Figure S1 b-catenin status, wound healing and stem cell markers during the healing process in large wounds. Full-thickness wounds (diameter = 1.5 cm) were generated on the backs of 8-week-old C3H mice. Wounded tissues were excised from CH3 mice at 1, 4, 7, and 10 d post-wounding, and subjected to H&E staining and immunohistochemical analyses. (A) H&E staining (first row panels) (original magnification 6100) and immunohistochemical staining for b-catenin, keratin 14, a-SMA, collagen I, and collagen III (other row panels) in the wounds (original magnification 6200). EP, epidermis. (B) Immunohistochemical analysis of Nestin or CD34 in the wounds at 1, 4, 7, and 10 d post-wounding (original magnification 6635). (TIF) Figure S2 Effects of VPA on levels of b-catenin, wound healing markers in small wounds. The wounded skin of 8-week-old male C3H mice was treated daily with 500 mM VPA for 7 days. Wounded skin was fixed in paraformaldehyde overnight. (A) Immunohistochemistry was performed with b-catenin, filaggrin, loricrin, or keratin 14 antibodies in the neo-epidermis of control and VPA-treated wounds. The protein levels were quantified using TissueQuest analysis software. Asterisks denote the significant differences between control and test groups as measured by t-test with one asterisk being p,0.05, two asterisks being p,0.005 (n = 5). (TIF) Figure S3 Effects of VPA on levels of b-catenin, wound healing markers in small wounds. The relative protein expression was calculated as the ratio of each protein level to a-tubulin level. The software used for the quantification was Multi-Gauge V 3.0 (Fujifilm). Asterisks denote the significant differences between control and test groups as measured by t-test with two asterisks being p,0.005 (n = 5). (TIF) Figure S4 Quantitative data for effects of VPA on cutaneous wound healing in large wounds. A full-thickness skin excision (diameter = 1.5 cm) was made on the backs of 8-week-old C3H mice, and 500 mM VPA was topically applied to the wounds daily ( Figure 4A). Wound sizes were measured at 1, 3, 5, 7, and 9 d after wounding. Asterisks denote the significant differences between control and test groups as measured by t-test with two asterisks being p,0.005 (n = 10). (TIF) Figure S5 Effects of VPA on the expression of stem cell markers in wounds (Low magnification images). A full-thickness skin excision (diameter = 0.5 cm or 1.5 cm) was made on the backs of 8-week-old C3H mice, and 500 mM VPA was topically applied to the wounds daily. The relative wound closure rate was measured using NIS-Elements imaging software. Asterisks denote the significant differences between control and test groups as measured by t-test with one asterisk being p,0.05 and two asterisks being p,0.