Medical Lasers; Engineering, Basic Research, and Clinical Application 2017; 6(2): 67-76  
Effect of Photobiomodulation on Wound Healing of the Corneal Epithelium through Rho-GTPase
Yun-Hee Rhee1,2, Kyong Jin Cho2,3, Jin-Chul Ahn1,4, and Phil-Sang Chung1,2,5
1Beckman Laser Institute Korea, Dankook University, Cheonan, Korea, 2Laser Translational Clinical Trial Center, Dankook University Hospital, Cheonan, Korea, 3Department of Ophthalmology, College of Medicine, Dankook University, Cheonan, Korea, 4Department of Biomedical Science, College of Medicine, Dankook University, Cheonan, Korea, 5Department of Otolaryngology-Head and Neck Surgery, College of Medicine, Dankook University, Cheonan, Korea
Correspondence to: Phil-Sang Chung, Laser Translational Clinical Trial Center, Dankook University Hospital, 119 Dandae-ro, Cheonan 31116, Korea, Tel.: +82-41-550-3022, Fax: +82-41-559-7838, E-mail: pschung@dankook.ac.kr
Received: November 29, 2017; Revised: December 18, 2017; Accepted: December 18, 2017; Published online: December 30, 2017.
© Korean Society for Laser Medicine and Surgery. All rights reserved.

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Background and Objectives

In recent years, photobiomodulation (PBM) using low-level light has been applied to diverse clinical approaches because of its potential to elevate the cell metabolism or regulate various signaling pathways. This study evaluated the possibility of a short term effect of PBM on the wound healing of corneal epithelial cells. Rapid healing of the corneal epithelium and the return of an intact basement membrane can restore the eye’s normal mechanical barriers. The migration, proliferation, attachment, and cytoskeletal rearrangement play a critical role in the wound healing process of the corneal epithelium.

Materials and Methods

To determine which wavelength was most effective on corneal epithelium wound healing, light emitting diode (LED) arrays with wavelengths of 470, 530, 660, 740, and 850 nm were used. The proliferative effect was assessed using a MTT assay, cell cycle assay, and BrdU immunofluorescence (IF) staining, and the motility effect was examined using a wound healing assay after PBM. The cytoskeletal rearrangement effect of PBM was also evaluated by Western blot analysis and IF staining.

Results

PBM had no effect on cell proliferation; the cell cycle portion and BrdU were not changed after the PBM treatment, whereas cell survival was decreased at 470 nm. On the other hand, PBM at wavelengths greater than 660 nm affected migration. In particular, 740 nm was the most effective. The expression of Rho A and Rho C increased after PBM at wavelengths greater than 660 nm. The levels of cdc42 and mTORC2 expression were similar.

Conclusion

This study showed that PBM could increase the corneal epithelial cell migration capacity without cell proliferation in a short time via the activation of a part of Rho-GTPase pathways without the effect of the upstream signals. These findings may be used for the future development of PBM-based therapy for acute ocular surface diseases.

Keywords: Photobiomodulation, Corneal epithelium, Wound healing, Rho-GTPase
INTRODUCTION

The corneal epithelium is the outmost mechanical barrier of an eye in mammalian. Various corneal injuries result in corneal epithelial cell damage and break down. Although corneal epithelial cell had full vitalities, damages of corneal epithelium by fatal injuries such as alkali burn, ulcer, and surgical operation are difficult to repair and require transplantation in extreme cases. A repair system of injured corneal epithelium is delicate cross-talk of various signaling pathways for wound healing events such as proliferation, migration, adhesion, and differentiation of corneal epithelial cells.1,2 Rapid healing of the corneal epithelium and the return of an intact basement membrane can restore the eye’s normal mechanical barriers and prevent various epithelium-derived growth factors from leaking into the stroma.3,4

Meanwhile, the use of low levels of visible or near-infrared (NIR) light for reducing pain, inflammation, and edema, promoting healing of wounds, deeper tissues and nerves, and preventing tissue damage has been known. There are many studies on the use of light aiming to positively stimulate the healing process, but no report about corneal wound healing process. In this study, we hypothesized that photobiomodulation (PBM) using low-level light could restore the corneal epithelial damage faster than the typical corneal recovery time. Based on the corneal epithelial wound healing process, we investigated the effect of PBM on human corneal epithelial cells (HCE-T) using different wavelengths of light emitting diode (LED) array by evaluating of cell proliferation, migration, attachment and cytoskeletal rearrangement pathways.

MATERIALS AND METHODS

Chemicals

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), crystal violet, propidium iodide (PI), RIPA buffer, protease and phosphatase inhibitors were purchased from Sigma (Saint Louis, MO, USA). Protein DC kit was supplied by Bio-Rad (Hercules, CA, USA). BrdU, RhoA, RhoB, RhoC, phospho-Rac, Rac-123, cdc42, and mTOR were purchased from cell signaling technology (Beverley, MA, USA). β-actin was purchased from Sigma.

Cell

The human corneal epithelial cell line HCE-T was purchased from ATCC (Manassa, VA, USA) supplemented with 0.05 mg/ml bovine pituitary extract (BPE), 5 ng/ml epidermal growth factor (EGF), 500 ng/ml hydrocortisone in keratinocyte-serum free medium (Life Technologies, Grand Island, NY, USA). The cells were maintained at 37°C in a 5% CO2 humidified environment.

Photobiomodulation condition for low-level light therapy

The light sources were 470, 530, 660, 730, and 850 nm light emitting diode (WON Technology Co., Ltd, Daejeon, Korea). The elliptical fiber shape of the diode laser output had a diameter of 1.7 mm. The light device information is described in Table 1. The irradiance at the surface of the cell monolayer was measured by a power meter (Field-MAXII, Coherent Inc. Santa Clara, CA). We attached a supplementary data file of LED and the photobiomodulation parameter evaluation is provided in Table 1 and Fig. 1. The irradiance or power density was measured as 100 mW/cm2 and the operating mode was a continuous wave. The light dose measure for energy density or fluency was 30 J/cm2 with the duration of each treatment set.

Proliferation assay

Cells were inoculated into a 96-well, flat-bottomed microplate at a volume of 100 μl (2,000 cells) for a stationary culture and incubated overnight in growth medium to allow the cells to adhere the bottom of wells. Cells underwent by dual PBM treatment at 30 J/cm2 once and 10 J/cm2 three times then incubated for 24, 48 and 72 h in 5% CO2 at 37°C. After incubation 50 μl MTT solution (2 mg/ml) was added to each well. Four hours h after incubation in 5% CO2 at 37°C, media of each well was removed and 100 μl DMSO was added to dissolve violet blue crystals. The growth of cells was determined by measuring the absorbance at 570 nm using ELISA reader (TECAN, Männedorf, Switzerland).

Cell cycle analysis

To analyze cell cycle distribution at different stages in corneal epithelial cells with and without PBM, cell cycle analysis was performed by flow cytometry. Briefly, Cells were treated with PBM at 30 J/cm2 and incubated at 37°C and 5% CO2 for 24 h. Then the cells were collected by trypsinization and washed with PBS. Next, the cells were incubated with propidium iodide (50 μg/ml) (Sigma) for 15 minutes. The cell cycle distribution and sub-G1 DNA content were determined and analyzed by flow cytometry (Accuri C6, BD, CA, USA). The percentages of viable and dead were determined as 10,000 events per sample using an FL-2 filter and compared with control to study the efficacy of SFE. The histogram was prepared for showing a change in the percentage of cell numbers at different subpopulation.

Migration assay

Cells were seeded into 12-well plate and grown to confluence. The Wound was created by scraping confluent cell monolayers with a pipette tip. The cells were allowed to migrate for 8 h after 30 J/cm2 of PBM treatment. At 0 h and 8 h after scratching, the plate was stained with 0.5% crystal violet and wound images were taken under the inverted microscope to assess the ability of the cells to migrate into the wound area.

Western blot analysis

The expressions of Rho-GTPase signaling proteins were analyzed by Western blot technique. Cells were treated with same procedures of cell cycle assay described above, and the proteins were extracted in RIPA buffer (50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, and 0.1% sodium dodecyl sulfate with protease and phosphatase inhibitor cocktail (Sigma) and centrifuged at 15,000 rpm for 30 minutes at 4°C. The protein concentration was determined using the Bradford Protein Assay Reagent. Equivalent amounts of protein from each sample were loaded onto polyacrylamide gels and separated by electrophoresis. Then the proteins were transferred to PVDF membranes (Immuno-Blot PVDF; BioRad Laboratories, Hercules, CA, USA). Both electrophoresis and blotting were performed by using a PowerPac200 electrophoresis system (BioRad Laboratories). The membranes were blocked for 2 hours at room temperature in Tris-buffered saline containing 0.1% Tween-20 (TBST) and 3% BSA, and incubated overnight at 4°C with the primary antibody diluted with 3% BSA in TBST. The membranes were probed with horseradish peroxidase-conjugated anti-mouse IgG, anti-goat IgG or anti-rabbit IgG antibody for 2 hours. The protein band was developed by ECL Western Blotting detection reagents (GE Healthcare, Buckinghamshire, UK) and the pictures were taken and quantified by the image analyzer (Bio-Rad Laboratories).

Immunofluorescence staining

Cells were seeded onto 11 mmΦ round coverslip in 24-well plate and grown to confluence. After 30 J/cm2 of PBM treatment, cells were incubated for 24 h, then fixed with chilled methanol. Fixed cells were blocked with 3% BSA and then incubated with primary antibodies (mTOR or BrdU) for overnight at 4°C in a humidified chamber. Cells were then incubated in a 1:100 dilution of Alexa488 or Alexa650 probed anti-rabbit IgG and observed using confocal laser scanning microscope (FV3000, Olympus, Tokyo, Japan). The green or red positive cells were counted under × 40 magnification.

Statistical analysis

The results are expressed as mean ± SD. Unpaired Student’s t-tests were used for comparisons between two means. The comparisons were performed by analysis of one way ANOVA, Tukey test (Graph Pad, Prism®, La Jolla, CA, USA). *p<0.1, **p<0.05 and ***p<0.0001 were considered statistically significant.

RESULTS

The PBM induced the migration of HCE-T corneal epithelial cells

To investigate whether PBM has an effect on the migration of corneal epithelial, a scratch wound healing assay was performed. As shown in Fig. 2, PBM treatment groups exhibited the increase of cell migration except for 470 nm irradiated group. Interestingly, both 740 nm and 850 nm showed the significant cell migration.

The PBM effect on proliferation of HCE-T corneal epithelial cells

To determine whether PBM-induced cell migration was due to cell proliferation, we performed the BrdU staining and cell proliferation assay. Since energy dose could affect the proliferation of cells, we performed the separated cell proliferation assay using 30 J/cm2 once and 10 J/cm2 three times. As shown in Fig. 3A and B, cell proliferative ratio was no significance between a control group and PBM group. The cell proliferative ratio with or without PBM was similar to that of control. In addition, there was no difference in the results of the cell proliferation between irradiated 30 J/cm2 at one time or 10 J/cm2 three times. The IF staining also showed the same results of cell proliferation (Fig. 3C). The BrdU positive cell number of a control group was no different from PBM treated group.

The PBM effect on cell cycle progression of HCE-T corneal epithelial cells

The cell cycle progression under PBM was evaluated via flow cytometry. A representative example depicting the effect of PBM on cell cycle phase distribution in the HCE-T cell line is shown in Fig. 4. At 24 h after PBM treatment, the percentage of cells in each phase were analyzed. There were no differences between control group and PBM treated group. Taken together, PBM had no effect on cell proliferation within 24 h whereas had an effect on migration.

The PBM effect on Rho-GTPase expression of HCE-T corneal epithelial cells

If so, we wondered what would have been possible for cell migration without proliferation. For cell movement, intracellular adherent and cellular structure should be remodeled. We investigated the expression marker of cytoskeletal rearrangements such as Rho family of small GTPase including Rho, Rac, and cdc42 acts as molecular switches to regulate processes such as cell migration, adhesion, proliferation and differentiation.5 As shown in Fig. 5, the expression of Rho A and Rho C was increased after PBM treatment at over 660 nm whereas the expression of RhoB and phospho-Rac was elevated only at 660 nm. The expression of cdc42 showed no difference.

The PBM effect on mTOR expression of HCE-T corneal epithelial cells

Next, we investigated the expression of mTORC2 after PBM for concerning the upstream of Rho/Rac activity.6 As shown in Fig. 6, the expression of mTOR was seemed to increase in PBM treatment group over 660 nm, but not significantly. However, the expression of mTORC2 has significantly decreased after PBM treatment at 470 and 530 nm.

DISCUSION

There is evidence that multiple mammalian cell types can respond to low-level light irradiation. For wound healing type studies, these cells are likely to be endothelial cells,7 fibroblasts,8 keratinocytes,9 and possibly some classes of leukocytes.10

Corneal epithelial repair involves a multifaceted series of events with specific physiologic functions. At this phase, metabolic activity was increased and the cellular structure was reorganized for allowing the epithelial cells migration over the wound surface.4 Additionally, intercellular adherens and gap junctions are also lost, desmosomes are remodeled, and structural proteins and actin filaments are assembled in preparation for cellular migration.1 The epithelial healing process is referred to as the migration phase, in which cells begin to move and cover the epithelial defect. This begins with the flattening of cells at the wound edge into a monolayer, and the formation of lamellipodia and filopodia that aid in cellular movement.2 Focal contacts of migrating cells to the provisional extracellular matrix, and subsequent contraction of the cytoskeletal actin filaments, allow the layer of cells to slide together as a sheet, eventually fully covering the wound bed.11 It is important to note that throughout the latent and migration phases, there is no mitotic activity of the cells in or around the area of the epithelial defect; these initial processes are completely independent of cellular proliferation. Based on this initial wound healing process of corneal epithelial cells, we hypothesized that PBM could induce cell motility in short time. Wound closure is usually accomplished within 2 to 4 days following a corneal injury, the entire epithelial healing process typically requires weeks after to be fully restored. In this study, we observed that PBM regulate cell migration but not proliferation in corneal epithelial cells. We evaluate the cell proliferation using BrdU assay, MTT assay (Fig. 3), and cell cycle progression assay (Fig. 4), however, PBM could not elevate the corneal epithelial cell proliferation. Thus, we focused the Rho-GTPase activity after PBM treatment. The Rho family of small GTPases, including Rho, Rac, and Cdc42, are small monomeric G proteins that cycle between an inactive GDP-bound form and an active GTP-bound form and regulate actin cytoskeleton, cell migration, and proliferation.12,13 Rho regulates actin polymerization, resulting in the formation of stress fibers and the assembly of focal adhesion complex.14 Rho has been implicated in cell migration, actin organization, focal adhesion formation, as well as adherents and gap junction assembly in the corneal epithelium.15,16 Rho kinases (ROCKs) were initially characterized for their roles in mediating the formation of RhoA-induced stress fibers and focal adhesions through their effects on the phosphorylation of myosin light chain.17 In the cornea, ROCKs have been suggested to be involved in epithelial differentiation, 18 cell cycle progression,11 cell-cell adhesion,15 endothelial barrier integrity,19 stromal cell phenotype conversion,20 cytoskeleton reorganization, contractility,21 and cell-matrix interaction.22 Thus, we investigated the Rho-GTPase activity by PBM and found that the expression of RhoA and RhoC was significantly increased after PBM treatment. However, the expression Cdc 42 after PBM was not changed (Fig. 5). The precise mechanism of Rho-GTPase during corneal epithelial cells should be more investigated, however, we suggested that the Rho-GTPase had a critical role during corneal epithelial wound healing but not all Rho-GTPase factors are involved. Next, we investigated whether Rho-GTPase activation by PBM treatment was downstream of mechanistic target of rapamycin catalytic domain 2 (mTORC2). Although both mTORC1 and mTORC2 are central mediators of growth factor responses and cellular metabolism, mTORC1 is uniquely activated by environmental cues such as adequate nutrients (amino acids), energy (ATP/AMP), and oxygen availability, which results in activation of pathways leading to cellular growth (protein, DNA, and lipid synthesis) and inhibition of autophagy. Conversely, mTORC1-mediated cellular growth is inhibited by cellular stresses such as DNA damage, low energy states, and hypoxia. mTORC2, by comparison, is more specifically activated by growth factor signaling, and facilitates cell survival and cytoskeletal reorganization to promote cell migration and adhesion.6,23 In addition, the Rho-GTPase family has been known to the target of mTORC2 kinases.6 Unlike what we expected, the expression of mTORC2 was significantly decreased after PBM treatment at 470 and 530 nm, and not increased by PBM treatment over 660 nm (Fig. 6). This results suggested that under 530 nm of wavelength inhibited the cell migration via mTORC2 regulation, nevertheless over NIR wavelength did not increase or affect the expression of mTORC2.

In summary, the current study revealed that PBM could increase corneal epithelial cell migration capacity in short time. The activation of Rho-GTPase pathways was involved in this process. In addition, mTORC2, the upstream of Rho-GTPase was not changed by PBM treatment. These results may be used for future development of PBM-based therapy for acute ocular surface diseases.

ACKNOWLEDGEMENTS

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI15C1524) and was a part of the project titled “Development of marine material based near infrared fluorophore complex and diagnostic imaging instruments”, funded by the Ministry of Oceans and Fisheries, Korea.

Figures
Fig. 1. The figure of light emitting diode (LED) array. We used light diode array to evaluate PBM effect on corneal epithelium. A LED array was 8 cm × 12 cm which was manufactured by WON technology Co., Ltd. The elliptical fiber shape of the diode laser output had a diameter of 1.7 mm. The light device information is described in . The irradiance at the surface of the cell monolayer was measured by a power meter.
Fig. 2. The effect of PBM on cell migration of HCE-T cells. Cells were seeded into 12-well plate and grown to confluence. The wound was created by scraping confluent cell monolayers with a pipette tip. The cells were allowed to migrate for 8 h after 30 J/cm2 of PBM treatment. At 0 h and 8 h after scratching, the plate was stained with 0.5% crystal violet and wound images were taken under the inverted microscope to assess the ability of the cells to migrate into the wound area. The graphs are shown as the mean ± SD of three independent experiments. **p<0.05, ***p<0.001.
Fig. 3. The effect of PBM on cell proliferation of HCE-T cells. (A, B) Cells were seeded onto 96-well at a density of 2,000 cells/well and incubated overnight in a growth medium. Cells underwent by dual PBM treatment at 30 J/cm2 (A) once and 10 J/cm2 three times (B) then incubated for 24, 48 and 72 h in 5% CO2 at 37°C. After incubation, 2 mg/ml of MTT solution was added to each well then was replaced by 100 μl DMSO after 4 h. The growth of cells was determined by measuring the absorbance at 570 nm. (C) Cells were seeded onto 11 mmΦ round coverslip in 24-well plate and were underwent PBM treatment at 30 J/cm2. After 24 h, cells were fixed by chilled methanol. Fixed cells were blocked with 3% BSA and then incubated with BrdU (1:100) for overnight at 4°C in a humidified chamber. Cells were then incubated in a 1:100 dilution of Alexa650 probed anti-rabbit IgG and observed using confocal laser scanning microscope. The red positive cells were counted under × 40 magnification. Every assay was performed 3 times and the results are expressed as mean ± SD. One-way ANOVA (Tukey test) were used for comparisons between two means.
Fig. 4. The effect of PBM on cell cycle progression of HCE-T cells. Cells were treated with PBM at 30 J/cm2 and incubated at 37°C and 5% CO2 for 24 h. Cells were collected by trypsinization and were incubated with propidium iodide for 15 minutes. The cell cycle distribution and sub-G1 DNA content were determined and analyzed by flow cytometry. The percentages of viable and dead were determined as 10,000 events per sample using an FL-2 filter and compared with control. Every assay was performed 3 times and the results are expressed as mean ± SD. One-way ANOVA (Tukey test) were used for comparisons between two means.
Fig. 5. The effect of PBM on Rho-GTPase activity of HCE-T cells. Cells were treated with same procedures of cell cycle assay described above, and the proteins were extracted in RIPA buffer. Equivalent amounts of protein were analyzed by Western blotting. The expression of Rho-GTPase was taken and quantified by the image analyzer. Every assay was performed 3 times and the results are expressed as mean ± SD. One-way ANOVA (Tukey test) were used for comparisons between two means. *p<0.1, **p<0.05, ***p<0.001.
Fig. 6. The effect of PBM on mTOR expression of HCE-T cells. Cells were seeded onto 11 mmΦ round coverslip in 24-well plate and were underwent PBM treatment at 30 J/cm2. After 24 h, cells were fixed by chilled methanol. Fixed cells were blocked with 3% BSA and then incubated with mTOR (1:100) for overnight at 4°C in a humidified chamber. Cells were then incubated in a 1:100 dilution of Alexa488 probed anti-rabbit IgG and observed using confocal laser scanning microscope. The green positive cells were counted under × 40 magnification. Every assay was performed 3 times and the results are expressed as mean ± SD. One-way ANOVA (Tukey test) were used for comparisons between two means. **p<0.05, ***p<0.001.
Tables
Table 1

Photobiomodulation (PBM) by low level light condition

Irradiation parameters (nm)470, 530, 660, 740, 850
Treated surface diameter (mm)35
Treated area (cm2)9.61
Power input (mW/cm2)5.2
Irradiation time (sec)600
Total energy (J)30
Total energy density (J/cm2)3.12
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