Impairment of Corneal Epithelial Wound Healing in a TRPV1-Deficient Mouse

Abstract

Purpose.: To examine whether the absence or blockage of an ion channel receptor, transient receptor potential vanilloid subtype 1 (TRPV1), affects the healing of an epithelial injury using an experimental model of an epithelial defect in animal cornea.

Methods.: The expression of TRPV1 in the corneal epithelium was examined using immunohistochemistry in mice and rats. The migration of the corneal epithelium was examined in epithelium-debrided rat cornea in organ culture in the presence or absence of a TRPV1 agonist or its antagonist. Epithelial migration and cell proliferation following the debridement were examined in the cornea of a TRPV1-null mouse. Real-time RT-PCR was performed in samples of healing corneas to analyze the expression pattern of epithelial migration–related components (i.e., IL-6, substance P, and TGF-β1).

Results.: TRPV1 was detected mainly in the basal layer of mouse or rat corneal epithelium. Adding a TRPV1 receptor agonist to the culture medium enhanced epithelial healing in the rat cornea, and a TRPV1 antagonist retarded it in organ culture. The loss of TRPV1 did not affect the histology of the mouse cornea. In vivo analysis showed the loss of TRPV1-impaired re-epithelialization of the debrided area of the corneal epithelium by the suppression of both cell migration and proliferation. The lack of TRPV1 suppressed the expression of IL-6 and substance P but not of TGF-β1 in response to epithelial debridement in mice.

Conclusions.: TRPV1 signal is required for the upregulation of IL-6 and substance P and the healing of debrided corneal epithelium in mice.

Introduction

The epithelium serves as a barrier against external stimuli such as mechanical or chemical injury or infection by microorganisms. An epithelial defect in the cornea must be rapidly restored to avoid infection and further damage to the underlying basement membrane and stroma. The migration of the corneal epithelium is critical to the early phase of corneal epithelial healing, and the later proliferation activity of the cells is upregulated. ^1–4 Although various cytokines or growth factors and neurotransmitters are believed to orchestrate cell migration, TGF-β–activated p38 mitogen-activated protein kinase (MAPK) is one of the major signaling cascades involved in epithelial migration. ⁵  

Transient receptor potential (TRP) channels are polymodal receptors that are activated by multiple external and endogenous stimuli. ^6,7 The TRP superfamily is composed of 28 different genes that are subdivided into seven different subfamilies with variable cation permeability (TRPA, TRPC, TRPM, TRPML, TRPN, TRPP, and TRPV). ⁸ Among them, TRP vanilloid subtype 1 (TRPV1), the capsaicin receptor, is the prototype of the family members and is a nocioceptor. It elicits responses to a variety of noxious stimuli, including chemical irritants, inflammatory mediators, alterations in pH, moderate heat (≥43°C), and hypertonicity. ⁹ All these stimuli lead to nocioceptions and can evoke pain or pain-related behavior in animals. ^10–13 TRPV1 activation reportedly induces the release of tachykinin neuropeptides (e.g., substance P, neurokinin A, and calcitonin gene–related peptide) from the sensory nerves, inducing neurogenic inflammation. ^14,15 Although TRPV1 was originally found as a neuronal component, epidermal keratinocytes or mucosal epithelial cells also express TRPV1 to presumably exert a sensory function or modulation of the inflammatory response to external stimuli. ^16,17 By using cell culture experiments, we have shown that the TRPV1 channel is required for cell migration in association with calcium ion influx. ¹⁸ We have also demonstrated that TRPV1 is constitutively expressed in human, rabbit, and mouse corneal epithelium in vivo and that TRPV1 activation induces increased the proliferation and migration of SV40-immortalized human corneal epithelial cells through epidermal growth factor receptor transactivation, which leads to global MAPK pathway stimulation. ^19,20  

These findings indicate that the TRPV1 signal is involved in cell proliferation and migration of single epithelial cells. Along with this system in each epithelial cell, the behavior of in vivo corneal epithelium may also be modified by the neurotransmitters expressed in TRPV1-positive nerve fiber endings. The aim of the present study was to elucidate the role of TRPV1 in wound healing (cell migration and proliferation) in the corneal epithelium in vivo. The results demonstrated that the blockage of TRPV1 activation or the absence of TRPV1 suppressed the healing of an epithelial defect in the rat or mouse cornea in organ culture or in vivo in association with the suppression of the expression of substance P and IL-6. 

Methods

The experimental protocols and the use of experimental mice were approved by the DNA Recombination Experiment Committee and the Animal Care and Use Committee of Wakayama Medical University. They were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Organ Culture of Epithelium-Debrided Wistar Rat Corneas With an Agonist and/or an Antagonist of TRPV1 Receptor

To examine the role of TRPV1 channels in epithelial healing, we first performed an organ culture experiment as previously reported with a minor modification. ⁵ Under both general anesthesia and topical anesthesia, a round epithelial debridement (2.5 mm in diameter) was created in the corneas of 96 Wistar rats (Kiwa Laboratory Animals Co., Ltd., Kimino-cho, Japan). ⁵ The animals were killed without reawakening, and the eye globe was enucleated. The debrided eyes were then cultured in Eagle's medium supplemented with 2.0% fetal bovine serum with or without a TRPV1 receptor agonist (10 μM capsaicin; Sigma-Aldrich Corp., St. Louis, MO, USA) and/or antagonist (500 nM SB366791; Sigma-Aldrich Corp.). Closure of the epithelial defect was determined using fluorescein staining after 6, 12, 18, 24, 30, and 36 hours of culture. Four eyes were prepared and analyzed for each experimental condition at each time point. At various healing intervals, each cornea was photographed with green fluorescein staining, and the remaining defect of the epithelium was evaluated. The area of the fluorescein-stained epithelial defect was manually calculated from these photographs. The mean value of the vertical and horizontal diameter was obtained for determining the area by regarding the defect as a round shape. The data were analyzed using ANOVA. 

Healing of an Epithelial Defect in In Vivo Mouse Cornea

We then analyzed the in vivo wound healing of mouse corneal epithelium. Eight-week-old C57BL/6 wild-type (WT) mice (n = 16) (Japan SLC, Shizuoka, Japan) and TRPV1-null knockout (KO) mice (n = 16) of C57BL/6 background (Jackson Laboratories, Bar Harbor, ME, USA) were used. The mice were not littermates but were strictly aged matched. Under general anesthesia and topical anesthesia, a round epithelial debridement (2.0 mm in diameter) was created in the central cornea of the right eye as reported previously. ²¹ At various healing intervals of up to 36 hours, each cornea was photographed with green fluorescein staining, and the remaining defect of the epithelium was evaluated. The area of the fluorescein-stained epithelial defect was manually calculated from the photographs. The mean value of the vertical and horizontal diameters was obtained for determining the area by regarding the defect as a round shape. The data were analyzed using ANOVA. 

In another series of experiments, bromodeoxyuridine (BrdU) (120 μg/g of body weight; Sigma-Aldrich Corp.) was intraperitoneally administered to mice at 10 hours (n = 20), 22 hours (n = 20), and 34 hours (n = 20) after the debridement; the mice were killed 2 hours later by carbon dioxide asphyxiation and cervical dislocation as reported previously. ^5,22 The affected eyes at each time point were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 48 hours. Specimens were dehydrated through a graded ethanol series and embedded in paraffin. Sections were cut and processed for hydrogen chloride treatment and immunohistochemistry with anti-BrdU antibody (1:10 in PBS; Roche Applied Science, Mannheim, Germany) as reported previously. ^5,22 Four sections from one specimen (each 50 μm apart) were cut and immunostained for BrdU. The mean number of BrdU-labeled epithelial cells from the four sections derived from one central cornea represented the number of proliferating epithelial cells in one specimen. The data from the samples were analyzed using ANOVA. 

Gene Expression of Substance P, IL-6, and TGF-β1 in a Healing Cornea

Wild-type (n = 48) and KO (n = 48) mice were used. A round epithelial debridement (2.0 mm in diameter) was produced in the central cornea of the right eye as described above. At intervals of 6, 12, and 24 hours, each cornea was obtained and processed for total RNA extraction as reported previously. ²³ TaqMan real-time RT-PCR for mRNA of substance P, IL-6, and TGF-β1 was performed as reported previously using Applied Biosystems (Foster City, CA, USA) propriety gene expression assays (Δ/Δ threshold cycle analysis). ²³ All data were normalized for the endogenous expression of glyceraldehyde-3-phosphate dehydrogenase and were statistically analyzed using Tukey-Kramer test. The primers used are listed in the Table. 

Table

 

Gene Probes Used in m-RNA Detection

Gene	Probe
TGF-β1	Mm03024053_ml
IL-6	Mm01210732_gl
Substance P	Mm01166996_ml
Glyceraldehyde 3-phosphate dehydrogenase	Mm03302249_gl

Immunohistochemistry for TRPV1, Substance P, and IL-6

The epithelium in the central area (2 mm in diameter) of the cornea of the WT or KO mice (n = 10 of each genotype) was debrided and allowed to heal for 6, 12, 24, and 36 hours. An epithelial debridement (2.5 mm in diameter) was also produced in a cornea of adult Wistar rats (n = 6) and allowed to heal for 18 and 30 hours. The animals were killed, and the eyes were embedded in paraffin and then processed for histology. Paraffin sections (5 μm thick) of these mouse specimens, as well as the intact eyes of WT and KO mice, were deparaffinized, rehydrated, and subjected to immunohistochemistry for TRPV1, substance P, or IL-6 using rabbit polyclonal anti-TRPV1 antibody (1:500 in PBS; Neuromics, Edina, MN, USA), anti–substance P antibody (1:500 in PBS; Millipore, Temecula, CA, USA), or anti–IL-6 antibody (1:100 in PBS; Therma, Rockford, IL, USA), respectively, as reported previously. ⁵ Specimens from rats were processed for TRPV1 with the same antibody as above. Nuclei were counterstained with methyl green. 

Results

Protein Expression of TRPV1 in Mouse or Rat Corneal Epithelium

TRPV1 protein was detected in the basal layer of the uninjured epithelium of Wistar rats (Fig. 1A). The TRPV1 protein expression did not appear to be markedly altered in the healing corneal epithelium at 18 hours (Fig. 1B) and 30 hours (Fig. 1C) after the debridement in rats. TRPV1 protein was also detected mainly in the basal cell layer of uninjured mouse corneal epithelium (Fig. 1D). Compared with uninjured mouse epithelium, TRPV1 protein expression appeared less marked in the healing and migrating epithelium at 6 hours (Fig. 1E), 12 hours (Fig. 1F), and 24 hours (Fig. 1G) after the debridement. Regenerated mouse corneal epithelium recovered TRPV1 immunoreactivity in the basal cell layer (Fig. 1H). Negative control staining with omission of the primary antibody did not yield specific staining. 

Figure 1

 

Protein expression of TRPV1 in the corneal epithelium. (A) TRPV1 protein is detected in the basal layer of uninjured epithelium of Wistar rats. (B, C) TRPV1 expression did not appear to be markedly altered in the healing corneal epithelium at 18 hours (B) and 30 hours (C) after the debridement in rats. (D) TRPV1 protein is detected in the epithelium, particularly in the basal cell layer, of an uninjured mouse cornea. (E–G) Compared with the uninjured mouse epithelium shown in D, TRPV1 protein expression appeared less marked in healing, migrating epithelium at 6 hours (E), 12 hours (F), and 24 hours (G) after the debridement. Open arrows indicate the edge of the migrating epithelial sheets. Regenerated mouse epithelium recovered TRPV1 immunoreactivity in the basal cell layer (E) at 36 hours (H) after injury. Arrows indicate TRPV1 immunoreactivity in epithelium. epi, Epithelium. Scale bars: 20 μm.

Organ Culture Experiment

To exclude the involvement of the loss of TRPV1 from blood-derived inflammatory cells and trigeminal nerve components in epithelial healing, we first observed epithelial healing in an organ-cultured rat eye with a circular epithelial defect. A round epithelial defect in the rat cornea was re-epithelialized in organ culture during 36 hours. A TRPV1 receptor agonist (10 μM capsaicin) promoted the healing of the epithelial debridement; statistical significance was detected at 18 hours of culture. A TRPV1 antagonist (500 nM SB366791; Sigma-Aldrich Corp.) retarded epithelial defect closure; statistical significance was observed at 24 hours of culture (Figs. 2a, 2b). The presence of both the agonist and the antagonist did not alter epithelial healing (Figs. 2a, 2b). 

Figure 2

 

(a) Effects of capsaicin (a TRPV1 receptor agonist) and SB366791 (a TRPV1 antagonist; Sigma-Aldrich Corp.) on wound healing of rat corneal epithelium defect in organ culture. Green fluorescein staining shows the remaining defect in each rat cornea. (b) Adding capsaicin significantly promoted defect closure at 18 hours of culture, and supplementation with SB366791 retarded it at 24 hours of culture. Error bar denotes SEM.

Loss of TRPV1 Retarding Re-epithelialization of the Debridement in In Vivo Corneal Epithelium

To examine the role of the TRPV1 receptor signal in the re-epithelialization of a debridement in the cornea in vivo, we evaluated the healing of the debridement in the central corneas of KO or WT mice. At 12, 18, 24, 30, and 36 hours after the debridement, the remaining defect in KO mice was larger than that in WT mice; this difference was not observed at 6 hours (Figs. 3a, 3b). The epithelial defect had resurfaced in all WT mice by 30 hours after the debridement, whereas even at 36 hours the defect had not completely resurfaced in the KO mice (Figs. 3a, 3b). 

Figure 3

 

In vivo healing of a defect in the corneal epithelium of WT and KO mice. (a) The remaining defect in the healing corneal epithelium is observed with green fluorescein staining. The defect resurfaced in a WT mouse at 30 hours after the debridement, whereas even at 36 hours after wounding the epithelial defect was not completely recovered in a KO mouse. (b) The area of the remaining defect was significantly larger in KO mice than in WT mice at 12, 18, 24, and 30 hours after the debridement. (c) Immunohistochemical detection of incorporated BrdU in healing corneal epithelium at 0 hours (A, B), 12 hours (C, D), 24 hours (E, F), and 36 hours (G, H) after the debridement in WT (A, C, E, G) and KO (B, D, F, H) mice. Scale bar: 20 μm. (d) The incidence of BrdU-labeled corneal epithelial cells was significantly lower in KO mice at 24 and 36 hours after the debridement. The incidence of BrdU-labeled cells in the healing KO epithelium at 36 hours was significantly less frequent compared with that in the WT epithelium at 24 hours after the debridement, although the size of the remaining epithelial defect was similar between WT mice at 24 hours and KO mice at 36 hours. Error bar denotes SEM.

With regard to cell proliferation activity in the healing epithelium, there was no difference between WT and KO mice at 12 hours after injury. However, BrdU-labeled cells were more frequently observed in WT healing epithelium compared with KO corneas at 24 and 36 hours after wounding (Figs. 3c, 3d). 

Gene Expression of IL-6, Substance P, and TGF-β1 in a Healing Cornea

To understand the mechanism of inhibition of epithelial cell migration through the loss of the TRPV1 channel, we examined if an absent TRPV1 could affect the expression levels of IL-6, TGF-β1, and substance P, which are all involved in the migration of the corneal epithelium or corneal epithelial wound healing. The expression of the mRNAs of these components was analyzed using real-time RT-PCR in the samples of epithelium-debrided corneas at intervals of 6, 12, and 24 hours. The results showed that at 12 hours after epithelial wounding the mRNA expression level of IL-6 was significantly suppressed by the loss of TRPV1 using Tukey-Kramer test (Fig. 4a). The expression level of TGF-β1 mRNA was not affected by the loss of the TRPV1 channel (Fig. 4b). The expression of substance P appeared lower in a KO cornea compared with a WT cornea after the debridement, although statistical significance was detected only at 12 hours (Fig. 4c). 

Figure 4

 

Expression pattern of IL-6, TGF-β1, and substance P in mouse corneas during healing after an epithelial defect. (a) The expression of IL-6 mRNA was significantly suppressed by the loss of TRPV1 at 12 hours after injury. (b) The expression of TGF-β1 mRNA was not significantly affected by the loss of TRPV1 at 6, 12, and 24 hours after injury. (c) The expression of substance P mRNA was significantly downregulated in a KO cornea at 12 hours after the debridement. Error bar denotes SEM.

Protein Expression of Substance P and IL-6 in Healing Mouse Cornea After the Debridement

Immunohistochemistry did not detect IL-6 protein in the healing cornea (data not shown), although real-time RT-PCR indicated that it was expressed in the tissue. Substance P protein was immunohistochemically detected in the posterior stroma of both WT and KO corneas (Figs. 5A, 5B). At 6 and 12 hours following the epithelial defect, the entire stroma was labeled for substance P in WT corneas (Figs. 5C, 5E). Marked substance P immunoreactivity was observed in the anterior stroma adjacent to the migrating epithelium in WT corneas (insets in Figs. 5C, 5E). At these time points, the KO anterior stroma stained more weakly for substance P compared with the WT anterior stroma (Figs. 5D, 5F). At 24 and 36 hours after injury, immunoreactivity for substance P was readily observed in the regenerated epithelial layer in WT mice (Figs. 5G, 5I); this was not seen in KO corneal epithelium (Figs. 5H, 5J). Negative control staining did not yield specific labeling (data not shown). 

Figure 5

 

Immunohistochemical detection of substance P in healing mouse corneas after the debridement. Substance P was detected in the posterior stroma of both WT (A) and KO (B) uninjured corneas. At 6 hours (C) and 12 hours (E) following an epithelial defect, the whole stroma (asterisk) was labeled for substance P in WT corneas. Marked substance P immunoreactivity was observed in the anterior stroma adjacent to the migrating epithelium in WT corneas at these time points (boxed areas). At these time points (D, F), the anterior stroma stained weakly for substance P in KO corneas compared with the anterior stroma in WT corneas. At 24 hours (G) and 36 hours (I) after injury, immunoreactivity for substance P was readily observed in the regenerated epithelial layer in WT mice (arrows) but not in KO corneal epithelium (H, J). The inset in each frame shows a higher-magnification photograph of the boxed area in each frame. Scale bar: 50 μm (10 μm in insets).

Discussion

The aim of the present study was to identify the role of the TRPV1 signal in wound healing of corneal epithelial defects. Using a scratch assay, our previous findings showed that blocking the TRPV1 signal retarded in vitro healing of a wound model. ^19,20 In the present study, we first showed that TRPV1 was detected in rat and mouse corneal epithelium. We also examined the effects of an agonist and antagonist of the TRPV1 receptor on the healing of rat corneal epithelium in organ culture under conditions that did not have neural function because sensory nerve endings express TRPV1 as well. The TRPV1 signal was positively involved in the healing of an epithelial defect in an organ-cultured rat cornea; the TRPV1 receptor agonist promoted, whereas the TRPV1 antagonist retarded, epithelial defect closure. Because the corneal epithelium expresses TRPV1, these findings may be attributable to the function of TRPV1 in epithelial cells. 

We then examined if these results were also true in vivo under a more complicated situation with possible inclusion of inflammatory cells and neuronal involvement. Trigeminal nerve sense is reportedly essential to the rapid healing of an epithelial defect in the corneal epithelium. ²⁴ TRPV1 protein expression was detected in the basal layer of rat corneal epithelium and did not appear to be affected by epithelial injury. On the other hand, TRPV1 immunoreactivity in the healing mouse corneal epithelium was reduced compared with that in an intact corneal epithelium. The exact reason for this difference remains unclear. Nevertheless, in vivo experiments have clearly shown that signaling via the TRPV1 channel receptor is required for wound healing of a defect in mouse corneal epithelium. The healing of an epithelial defect in the corneal epithelium mainly consists of two phases, namely, primary resurfacing of the defect with a migratory epithelium in the early phase and reestablishment of the squamous epithelial stratification through upregulation of cell proliferation in the later phase. ⁵ It is believed that corneal epithelial cells start to proliferate after the epithelial defect is covered with a monolayer of migrating epithelium. 

In the present study, a statistically significant delay in the resurfacing of the epithelial defect was detected at 12 hours, when there was no difference in cell proliferation in the healing epithelium between WT and KO mice. This indicated that the loss of TRPV1 certainly impaired the migration of healing corneal epithelium. Various growth factors, cytokines, and neuropeptides are involved in the migration of the corneal epithelium during wound healing. In order to understand the mechanism of inhibition of epithelial cell migration by the loss of TRPV1 function, we next examined if the absence of TRPV1 affected the expression levels of IL-6, substance P, and TGF-β1, which all are likely involved in cell migration of the corneal epithelium. The results showed that the loss of TRPV1 decreased the expression of IL-6 and substance P but not of TGF-β1; this may be included in the possible mechanisms for the impairment of epithelial healing in the KO cornea. 

Interleukin 6 reportedly accelerates the migration of the corneal epithelium in organ culture and in vivo. ^25,26 In a previous study, ²⁰ we observed that the stimulation of TRPV1 by a specific agonist upregulated the expression of IL-6 in SV40-immortalized cultured corneal epithelial cells, along with the enhancement of cell migration. The reduction of IL-6 expression in a KO healing cornea may be attributable to impaired in vivo or in situ epithelial migration. 

Substance P is a neuropeptide of an undecapeptide that functions as a neuromodulator of the tachykinin neuropeptide family. ²⁷ It exhibits a variety of biological activities, including the stimulation of cell growth and migration of epithelial cells in culture. ²⁷ The expression of substance P was significantly more prominent in WT healing corneas compared with KO corneas at certain time points as revealed by both real-time RT-PCR and immunohistochemistry. The expression of substance P is reportedly mediated by the TRPV1 receptor signal, ^14,15 which may explain the mechanism for the suppression of substance P expression in the healing cornea of a KO mouse compared with that in a WT cornea. The effects of substance P on corneal epithelial cells have been well investigated by researchers. A research group with Nishida reported that substance P did not promote the migration of the corneal epithelium but did augment the accelerating effects of insulin-like growth factor 1 on the migration of rabbit corneal epithelium in organ culture. ^28–30 Compared with healthy subjects, the concentration of substance P in tear fluid is reportedly low in patients with neurotrophic keratitis, suggesting the possible contribution of substance P to the maintenance of the integrity of the corneal epithelium. ³¹ Further investigation will be needed to determine if the suppression of substance P expression in healing KO mouse cornea is involved in the impairment of cell migration. 

Our previous data demonstrated that blocking TRPV1 suppressed the proliferation of a cultured corneal epithelial cell line, ²⁰ which may explain the current in vivo phenomenon. Cell proliferation in the healing epithelium as detected by BrdU immunohistochemistry was less active in KO mouse cornea compared with WT cornea at 24 and 36 hours after the debridement. Cell migration is associated with the cessation of cell proliferation in a healing corneal epithelium. ⁵ Nevertheless, the incidence of BrdU-labeled epithelial cells in a WT cornea at 24 hours after injury was significantly more common compared with the incidence in a KO cornea at 36 hours, although the size of the remaining defect was similar between each genotype of mouse. Garcia-Hirschfeld et al. ³² and another group ³³ reported that the addition of substance P to the culture medium promoted the proliferation of corneal epithelial cells and another epithelial cell type in vitro. Interleukin 6 reportedly promoted the proliferation of keratinocytes but not of corneal epithelial cells, ^34,35 although both cell lines were derived from stratified squamous epithelium. The suppression of these two components by the loss of TRPV1 may be a mechanism for the reduction of cell proliferation in KO healing epithelium. 

We previously reported that TGF-β1–activated p38 MAPK was one of the most important signaling cascades involved in cell migration. ⁵ However, in the present study the loss of TRPV1 did not affect the expression level of TGF-β1 in the healing cornea after the debridement. In a previous study, ²³ we reported that the absence of TRPV1 suppressed TGF-β1 mRNA expression levels in a whole-tissue sample of healing mouse cornea after alkali burn. On the other hand, the ablation of the TRPV1 gene markedly enhanced fibrosis in the myocardium after myocardial infarction, possibly via the stimulation of the TGF-β1–Smad2 signaling pathway in mice. ³⁶ The effects of the loss of TRPV1 on the expression of TGF-β1 may differ among tissues. 

In conclusion, the TRPV1 signal is required for the upregulation of IL-6 and substance P and the healing (migration and proliferation) of debrided corneal epithelium in mice. Although capsaicin (the prototype TRPV1 agonist) caused severe irritation when topically applied, nonirritating TRPV1 agonists may be applicable for the treatment of refractory corneal epithelial disorders. 

Acknowledgments

Supported by Ministry of Education, Science, Sports, and Culture of Japan Grants C40433362 (TS), C21592241 (YO), C25462759 (OY), C19592036 (SS), and EY04795 (PSR). 

Presented as an abstract at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, United States, May 2009, and May 2012. 

Disclosure: T. Sumioka, None; Y. Okada, None; P.S. Reinach, None; K. Shirai, None; M. Miyajima, None; O. Yamanaka, None; S. Saika, None 

Footnotes

 TS and YO contributed equally to the work presented here and should therefore be regarded as equivalent authors.