Hyperforin/HP-β-Cyclodextrin Enhances Mechanosensitive Ca2+ Signaling in HaCaT Keratinocytes and in Atopic Skin Ex Vivo Which Accelerates Wound Healing

Cutaneous wound healing is accelerated by mechanical stretching, and treatment with hyperforin, a major component of a traditional herbal medicine and a known TRPC6 activator, further enhances the acceleration. We recently revealed that this was due to the enhancement of ATP-Ca2+ signaling in keratinocytes by hyperforin treatment. However, the low aqueous solubility and easy photodegradation impede the topical application of hyperforin for therapeutic purposes. We designed a compound hydroxypropyl-β-cyclodextrin- (HP-β-CD-) tetracapped hyperforin, which had increased aqueous solubility and improved photoprotection. We assessed the physiological effects of hyperforin/HP-β-CD on wound healing in HaCaT keratinocytes using live imaging to observe the ATP release and the intracellular Ca2+ increase. In response to stretching (20%), ATP was released only from the foremost cells at the wound edge; it then diffused to the cells behind the wound edge and activated the P2Y receptors, which caused propagating Ca2+ waves via TRPC6. This process might facilitate wound closure, because the Ca2+ response and wound healing were inhibited in parallel by various inhibitors of ATP-Ca2+ signaling. We also applied hyperforin/HP-β-CD on an ex vivo skin model of atopic dermatitis and found that hyperforin/HP-β-CD treatment for 24 h improved the stretch-induced Ca2+ responses and oscillations which failed in atopic skin.


Introduction
Epidermal keratinocytes are located at the surface of the skin and are exposed to various environmental stimuli including mechanical and physical stimuli and are susceptible to these stimuli. During the wound healing process, these exogenous stimuli and the endogenous stimuli, such as the tension and traction forces generated between the migration of the foremost cells and the cells that are located behind them, may affect the rate of wound closure. Our earlier study demonstrated that mechanical stretching facilitated wound closure in bovine aortic endothelial cells [1]. We recently reported that wound healing in HaCaT keratinocytes was accelerated by stretching and treatment with hyperforin, which is a major component of a traditional herbal medicine and which is known to be a TRPC6 activator, further accelerated wound closure [2]. We revealed that the facilitation of wound closure by mechanical stretching and hyperforin occurs due to the release of ATP via mechanosensitive hemichannels at the wound edge and the P2Y receptor-mediated Ca 2+ influx via TRPC6 in the cells located behind the wound edge using realtime ATP luminescence imaging and Ca 2+ fluorescence measurement [2]. The influx of Ca 2+ through TRPC6 channels was also reported to be essential for wound healing in vivo in TRPC6 knockout mice [3].
Hyperforin is a major active constituent of St. John's wort (Hypericum perforatum L.) extract, which is widely used in traditional herbal medicines, to promote wound healing [4][5][6][7][8][9]. The use of hyperforin-rich cream as a topical medication for atopic dermatitis was recently reported [10][11][12][13][14]. In spite of its potential therapeutic activities, the extreme sensitivity of hyperforin to photodegradation has impeded its topical application. The complexation of St. John's wort extract with -and -cyclodextrin (CD) was reported to enhance the photoprotection and solubility of hyperforin in aqueous solutions [15][16][17]. In the present study, we aimed to develop a novel formation of encapsulated hyperforin with hydroxypropyl--cyclodextrin (HP--CD) to improve its aqueous solubility and photostability, because HP--CD has been shown to possess the highest solubility not only in water but also in ethanol among several of the CD compounds that are commonly used. We also assessed the effects of the compound on the wound healing and ATP-Ca 2+ signaling in HaCaT keratinocytes.
Atopic dermatitis is a chronic inflammatory skin disease that develops due to various factors that are associated with epidermal barrier dysfunction [18]. It is known that the Ca 2+ gradient in the epidermis is necessary for maintaining the barrier function; however, the Ca 2+ dynamics of atopic skin remain to be elucidated. We herein measured the stretchinduced Ca 2+ responses ex vivo in atopic skin using a confocal microscope. We found that the Ca 2+ responses were impaired in the atopic epidermis and that the responses recovered after the application of hyperforin/HP--CD. The data suggested that hyperforin/HP--CD is a potent targeted therapeutic agent that can be used to promote epidermal wound healing and treat atopic dermatitis.

The Preparation of Hydrophilic and Stable
Hyperforin/HP--CD. Solutions of 4.66 × 10 −4 M hyperforin (in 1 mL methanol) and 1.86 × 10 −3 M HP--CD (in 1 mL ethanol) were mixed and then stirred for 30 min. The solvents were then removed in vacuo with a centrifugal evaporator (0.1 Mpa, 2800 rpm, 90 min, WKN-PV-1200, Wakenyaku, Kyoto, Japan) at ambient temperature. The obtained white solid was dissolved in Milli-Q water by ultrasonication for at least 10 min. The resulting aqueous solution of hyperforin/HP--CD was syringe-filtered with a 0.20 m pore size and kept in a freezer until use. All of the procedures were performed under light-shielded conditions. Stoichiometry of the reaction between hyperforin and HP--CD was spectroscopically determined. The concentration of hyperforin in these studies was 4.66 × 10 −4 M whereas the HP--CD concentration was used in the range of 0-8.0 equivalents. The UV spectra of hyperforin were recorded using a UV/VIS scanning spectrophotometer (Gene Spec III, Hitachi Naka Instruments, Hitachinaka, Japan). The changes in the absorbance of hyperforin following the addition of various concentrations of the HP--CD complexing agent were measured at max 281 ± 7 nm.

The Analysis of Irradiated Hyperforin Solution by HPLC.
An irradiation test was performed using a 6-watt LED light bulb (total luminous flux 480 lm, color temperature: 6700 K, Panasonic, Osaka, Japan) that was placed 14 cm above the samples. Irradiation was conducted in a dark room under temperature control (25 ∘ C). Aliquots of 40 L were taken every 30 min for the analysis. All of the quantitative measurements were conducted using a Hitachi LaChrom Elite HPLC system (Hitachi High-Technologies, Tokyo, Japan) equipped with a quaternary pump (L-2130), an autosampler (L-2200), a column oven (L-2300), and a diode array detector (DAD/L-2450). Separation was performed using a TSKgel ODS-100Z reversed phase column (4.6 mm × 250 mm, 5 m, Tosho, Tokyo, Japan) with a mobile phase composed of acetonitrilewater-methanol-trifluoroacetic acid (72 : 18 : 10 : 0.5, v/v/v/v). The flow rate was 1.6 mL/min. The UV detector was set at 270 nm. Curve fitting was performed using Excel (MS Office 2013) to minimize the 2 value.
The physiological experiments were performed as described previously [2]. A brief explanation follows.

Cell Stretch Experiments and Wound Closure
Assay. The cells were cultured in a stretch chamber molded out of Silpot 184 W/C silicone elastomers (Dow Corning Toray, Tokyo, Japan). A chamber with cultured cells was attached to a stretching machine (NS-600W or ST-600W, STREX, Osaka, Japan) mounted on the stage of an inverted microscope (IX-70, Olympus, Tokyo, Japan) for intracellular Ca 2+ imaging or an upright microscope (BX51WI, Olympus) for extracellular ATP imaging. HaCaT cells were seeded on collagen-coated silicone stretch chambers or 15 mm round glass coverslips at 3 × 10 5 cells/cm 2 and grown to confluence. A narrow cellfree gap (about 250 m) was created in a fully confluent monolayer by removing a silicone strip that was attached to the bottom of the stretch chamber during cell seeding. The wound closure process was monitored every 3 h after making the scratch using an inverted microscope (IX-70 Olympus) with a 4x (UPlanFL N, 0.13) objective. The wound closure speed was defined as the percentage of the wound closure area, which was calculated from the ratio of the final migrated area to the initial cell-free area. The stretch-induced release of ATP was measured in real-time using the imaging system, as described previously [20]. Briefly, the luciferin-luciferase ATP bioluminescence was detected using a high-sensitivity camera system simultaneously with infrared DIC imaging to monitor exact cell locations and extension during stretching. At 3 h after making a scratch, the cells in the stretching chamber were attached to the stretching device on the stage of an upright microscope (BX51WI, Olympus) with a 4x objective (340 Fluor XL, 0.28) and the medium was replaced with DME/F12 medium (2.0 mM Ca 2+ and 10 mM HEPES, pH 7.4) containing highsensitivity luciferin-luciferase solution (60315; Lucifer HS Set, Kikkoman Biochemifa, Tokyo, Japan). Images were acquired using the MetaMorph software with a stream acquisition mode (exposure time 100 ms).

The
Knockdown of TRPC6 by shRNA. TRPC6 shRNA plasmids that coexpressed RFP (TF308626; OriGene Technologies, Rockville, MD) were used. The shTRPC6 targeting sequence was 59-AAGCAGGACATCTCAAGT-CTCCGCTATGA-39. A scrambled noneffective plasmid with the same nucleotide content was used as a negative control. Each shRNA at a concentration of 45 nM was transfected into HaCaT cells using Lipofectamine reagent, according to the manufacturer's instructions. 2+ Imaging of the Epidermis. Biopsies were taken from the outer forearm of a volunteer with atopic skin. Written informed consent was obtained from the volunteer. The study was approved by the Pixy Central Institute Ethics Committee, 2009. The sample of skin tissue was placed in PBS prior to treatment with dispase. After overnight digestion with 500 U/mL dispase in serum-free F12/DME with or without hyperforin/HP--CD at 4 ∘ C, the epidermis was peeled off from the dermis with forceps. The detached pieces of epidermis were fixed with intradermal needles on an elastic silicone chamber and were further incubated at 37 ∘ C in a humidified 5% CO 2 atmosphere for 12 h. Epidermis tissue was loaded with 1 M Fluo-8 AM using 0.2% of Cremophor EL in culture media for 1 h at 37 ∘ C. After washing away the dye with DME/F12 containing 2.0 mM Ca 2+ , the chamber containing the cells was attached to a pulse-motor-driven stretching machine (NS-600W or ST-600W, STREX) mounted on the stage of an inverted laser scanning confocal microscope (LSM510 with a 10x lens, Carl Zeiss, Jena, Germany). Time-lapse Fluo-8 fluorescence and Nomarski differential interference contrast images were acquired at 1 s intervals. The Ca 2+ imaging experiments were performed at room temperature (24±2 ∘ C).

Preparation of Stable Hydrophilic Hyperforin Encapsulated in HP--CD and Its Effect on Wound
Closure. To improve the photostability and aqueous solubility of hyperforin, it was molecularly encapsulated in cyclodextrin. Hyperforin was complexed with hydroxypropyl--cyclodextrin (HP--CD) at different molar ratios using the solvent evaporation method. The hyperforin/HP--CD complexes were investigated with UV/Vis spectroscopy in aqueous solution. The molar ratio method was used to determine the stoichiometry of the inclusion complex formed by hyperforin and HP--CD. ΔA, the difference in the absorbance of hyperforin with and without HP--CD, was plotted against the molar ratio of HP--CD to hyperforin at 280 nm (Figure 1(a)). The curve for hyperforin/HP--CD showed an inflexion point at a ratio of 1 : 4, suggesting that the inclusion complex formed HP--CD tetracapped hyperforin at hemiterpene terminal moieties (Figure 1(b)). Next, we checked the light stability of a 1 : 4 complex of hyperforin/HP--CD using HPLC. We previously demonstrated that the wound closure of keratinocytes was accelerated by stretching and that hyperforin treatment further enhanced the effect [2] (Figure 1(d)). In the present study, we examined the effect of hyperforin/HP--CD on wound closure. A confluent monolayer of HaCaT cells cultured on silicone membrane was linearly scratched to create a cell-free gap of ∼250 m width, and the wound was allowed to heal under various conditions. Figure 1(d) shows representative wound closing waves occurred due to the release of ATP from the leading cells and its diffusion to the surrounding cells behind the edge, as shown in Figure 2(b) (Movie S2). The stretch applied parallel to the linear gap had essentially the same effect on ATP-Ca 2+ signaling and wound healing in HaCaT cells [2]. One notable advantage of hyperforin/HP--CD was that the effects on the stretch-induced ATP and Ca 2+ signaling were more reproducible than those obtained simple hyperforin. This may be attributed to the improvement of photostability and the aqueous solubility of hyperforin/HP--CD.

The Pharmacological Analysis of the Stretch-Induced Ca 2+ Responses and Wound Closure in Hyperforin/HP--CD-Treated Cells.
To analyze the characteristics of the stretchinduced Ca 2+ response, the effects of various inhibitors on the Ca 2+ response were evaluated in hyperforin/HP--CDtreated HaCaT cells. The time course of the intracellular Ca 2+ response induced by a 20% stretch was measured at different distances (0-240 m) from the scar (Figure 3(a), control; hyperforin/HP--CD-treated cells). At 0 m (wound edge), the Ca 2+ response was evoked immediately after stretching and it was prolonged by several min. When the distance from the edge was increased, a longer time lag was found before the onset of the activity; however, the amplitudes of the plateau phase were nearly the same. These results were consistent with the idea that Ca 2+ waves caused by the simple diffusion of ATP released from the leading cells at wound edge and the activation of P2Y in the surrounding cells behind the wound edge. When Gd 3+ (10 M), an inhibitor of the stretch-activated channel, was applied, the Ca 2+ response in the peak was reduced and the rate of decay was obviously faster, especially at the distant regions (Figure 3(b)). The Ca 2+ responses were similarly measured under various conditions and inhibitors and were evaluated by the peak response in an averaged trace of the responses at different distances (Figure 3(c)). The suppression observed in Ca 2+ -free medium, in hyperforin/HP--CDuntreated cells and in shTRPC6-treated cells, suggested the involvement of the influx of Ca 2+ via TRPC6. The inhibition by the treatments with suramin (P2-receptor antagonist, 100 M), apyrase (ATP-hydrolyzing enzyme, 20 U/mL), and CBX (hemichannel blocker, 100 M) suggested the contribution of ATP signaling in this process. The reduction by each treatment with U73122 (PLC inhibitor, 10 M) and diC8-PIP2 (a water-soluble PIP 2 analog that suppresses the activity of PLC by competing with PIP 2 , 10 M) suggested that the P2Y receptor-Gq-PLC-DAG-mediated signaling cascade was involved in the activation of TRPC6. These results were the same as those obtained by treatment with hyperforin (nonencapsulate) and stretch stimulation [2]. This suggests the involvement of the release of ATP via hemichannels in the leading cells and that the activation of P2Y in the cells behind the wound edge prolonged the influx of Ca 2+ via TRPC6 through the Gq-PLC-DAG cascade.

The Effects of Hyperforin/HP--CD Treatment on the Ca 2+
Responses in the Ex Vivo Skin of Atopic Dermatitis. Next, we assessed the effects of hyperforin/HP--CD treatment and stretch mechanical stimulation on an ex vivo epidermis of atopic dermatitis. The epidermis, which was detached from the dermis after overnight treatment with dispase, was loaded with Fluo-8AM and observed with a laser confocal microscope. Normal skin exhibited frequent spontaneous Ca 2+ oscillations and a large Ca 2+ response to stretch stimulation (1 s single) and subsequent Ca 2+ waves with the long-lasting elevation of Ca 2+ (Figure 4(a), Movie S3). In contrast, the epidermis of atopic dermatitis showed few oscillations and only a small response to stretching without any waves (Figure 4(b), Movie S4). In contrast, atopic skin that had been treated with hyperforin/HP--CD for 24 h exhibited autonomous Ca 2+ oscillation, and a transient longlasting increase in Ca 2+ and more frequent Ca 2+ oscillations following stretch stimulation (Figure 4(c), Movie S5). The application of hyperforin/HP--CD-treatment to atopic skin for 24 h led to the recovery of the mechanosensitive ATP-Ca 2+ signaling, which was dysfunctional in the untreated atopic epidermis.

Discussion
The topical application of hyperforin, which is a traditional folk remedy, has anti-inflammatory, antioxidative, antibacterial, antinociceptive, and wound healing effects. Recently, accumulating evidence indicates that hyperforin facilitates the keratinocyte differentiation caused by the uptake of Ca 2+ through TRPC6 [11]. Our previous studies showed that hyperforin-treated HaCaT keratinocytes could accelerate wound closure in conjunction with exogenous and endogenous mechanical stretching through the facilitation of the ATP-Ca 2+ signaling cascade [2]. The impact of hyperforin on mechanosensitivity remains unclear, but hyperforin certainly amplifies ATP-Ca 2+ signaling and facilitates reepithelialization during wound healing. However, due to the photoinstability of hyperforin, daylight initiates its facile oxidative degradation [21]. Prenyl side chains (hemiterpene moieties) containing conjugated double bonds are generally prone to oxidation. In order to enhance the stability of hyperforin and exert its topical therapeutic potential, hyperforin was encapsulated by forming a supramolecular complexation with HP--CD. The solubility of HP--CD is highest in ethanol as well as water among the CD compounds, -CD, -CD, methylated--CD, sulfobutyl ethyl--CD, -CD, and HP--CD. This amphipathic property was a major advantage when making the inclusion complex with hydrophobic hyperforin. The molar ratio method indicated that the optimal ratio of the hyperforin/HP--CD complex was 1 : 4. This meant the formation of HP--CD-tetracapped hyperforin, where the hyperforin was encapped with HP--CD at each hemiterpene moiety [22] as shown in Figure 1(b). The novel inclusion complex showed obvious photostability in comparison to hyperforin (Figure 1(c)). The curve fitting of the decay time course of hyperforin/HP--CD indicated the existence of a large nondecayed component that corresponded to photostable hyperforin. This modification can contribute to both pharmaceutical application and topical medication.
We assessed the effects of hyperforin/HP--CD on wound healing and ATP-Ca 2+ signaling in keratinocytes. Hyperforin/HP--CD enhanced the acceleration of wound closure by stretching with a similar efficiency to hyperforin (Figure 1(d)). In hyperforin/HP--CD-treated keratinocytes, stretching induced a conspicuous increase in the Ca 2+ levels in the leading cells facing the wound edge and the Ca 2+ waves slowly propagated to the cells behind the wound edge (Figure 2(a)). These propagating Ca 2+ waves were entirely due to the release of ATP from the leading cells (Figure 2(b)). The pathway of ATP release was CBX sensitive (Figure 3(c)) and presumably pannexin hemichannels from our previous study [2]. The migrating cells at the wound edge represented morphological changes that were similar to those observed at the epithelial-to-mesenchymal transition and might be more susceptible to endogenous and exogenous mechanical stress [2,23]. The increase in Ca 2+ in the cells behind the wound edge was dependent on the influx of Ca 2+ via the TRPC6 channels, which were activated by the activation of P2Y through the Gq-PLC-DAG-mediated signaling cascade (Figure 3(c)) [2] and lasted for a relatively long period. The concentration and duration of the Ca 2+ increase were dependent on the distance from the wound edge, making a Ca 2+ gradient from the leading cells to the following cells. This Ca 2+ gradient may be essential for organized wound healing, including cell migration, molecular relocation, and gene expression. The cell traction of the cells located behind the edge by migrating leading cells is also an important mechanical cue for wound healing that is controlled by Ca 2+ dependent cell-cell interaction molecules such as Ecadherin. This Ca 2+ signaling is enhanced by treatment with hyperforin/HP--CD.
Interestingly, the reagents that blocked the increase in Ca 2+ also suppressed the acceleration of wound closure in response to stretching in hyperforin/HP--CD-treated cells (Figure 3(d)). Thus, the hyperforin/HP--CD complex shows a similar efficiency to hyperforin in inducing mechanosensitive ATP-Ca 2+ signaling and wound closure in keratinocytes. In fact, hyperforin/HP--CD seems to be superior due to the reproducibility of the data concerning stretch-induced ATP and Ca 2+ signaling, which may be attributed to the photostability and aqueous solubility of hyperforin/HP--CD.
In our experimental design, HaCaT keratinocytes were cultured under low extracellular Ca 2+ conditions (0.07 mM), which mimicked the extracellular Ca 2+ environment for barrier-perturbed epidermis, such as the environment that would result from skin stripping or the use of surfactants. Atopic dermatitis is also a skin barrier dysfunction. Topical medication of hyperforin-rich St. John's wort cream has been shown to be effective in patients with atopic dermatitis [10][11][12][13][14]. The analysis of the laser scanning microscopy images has shown that the hyperforin-rich cream reduces the skin surface dryness and improves the moisture level of the stratum corneum [14]. However, the mechanism underlying the improvement of symptoms in atopic skin remains unclear. Our present study is the first to demonstrate how the Ca 2+ dynamics of atopic skin behave under mechanical environments such as wound healing and reepithelialization. We observed the Ca 2+ dynamics induced by the stretching of epidermis ex vivo using a confocal microscope (Figure 4). In atopic epidermis, there was a remarkable decrease in the Ca 2+ responses and oscillations induced by stretching (Figure 4(b)). Treatment with hyperforin/HP--CD for 24 h restored the Ca 2+ responses and oscillations, even in atopic skin (Figure 4(c)). These results suggest that the pathogenesis of atopic dermatitis is related to ATP-Ca 2+ signaling and that hyperforin/HP--CD may have therapeutic application in the treatment of atopic dermatitis.

Conclusions
Cutaneous wound healing is accelerated by mechanical stress both exogenously and endogenously, and treatment with hyperforin enhances the acceleration through the facilitation of ATP-Ca 2+ signaling in keratinocytes. We succeeded in making HP--CD-tetracapped hyperforin (hyperforin/HP--CD), which possessed increased aqueous solubility and improved photoprotection. Treatment with hyperforin/HP--CD enhanced the mechanically induced ATP-Ca 2+ signaling and accelerated wound closure in HaCaT keratinocytes with equal (or greater) efficacy to hyperforin. We also applied hyperforin/HP--CD on atopic skin ex vivo and found that hyperforin/HP--CD treatment for 24 h improved the stretch-induced Ca 2+ responses and oscillations, which reduced in atopic skin. The data suggest that hyperforin/HP--CD is a potent targeted therapeutic agent that can be used to promote epidermal wound healing and to treat atopic dermatitis.