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Articles  |   July 2021
Effect of Vitamin D3 on Regulating Human Tenon's Fibroblasts Activity
Author Affiliations & Notes
  • Shuo Jia
    Department of Ophthalmology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
  • Fushun Chen
    Department of Ophthalmology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
  • Huogang Wang
    Department of Ophthalmology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
  • Gandhervin Kesavamoorthy
    Department of Ophthalmology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
  • Jimmy Shiu-Ming Lai
    Department of Ophthalmology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
  • Ian Yat-Hing Wong
    Department of Ophthalmology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
  • Kin Chiu
    Department of Ophthalmology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
  • Jonathan Cheuk-Hung Chan
    Department of Ophthalmology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong
  • Correspondence: Jonathan Cheuk-Hung Chan, Department of Ophthalmology, LKS Faculty of Medicine, University of Hong Kong, Room 301, Block B, Cyberport 4, 100 Cyberport Road, Hong Kong. e-mails: jonochan@hku.hk, jonnochan@yahoo.com 
Translational Vision Science & Technology July 2021, Vol.10, 7. doi:https://doi.org/10.1167/tvst.10.8.7
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      Shuo Jia, Fushun Chen, Huogang Wang, Gandhervin Kesavamoorthy, Jimmy Shiu-Ming Lai, Ian Yat-Hing Wong, Kin Chiu, Jonathan Cheuk-Hung Chan; Effect of Vitamin D3 on Regulating Human Tenon's Fibroblasts Activity. Trans. Vis. Sci. Tech. 2021;10(8):7. doi: https://doi.org/10.1167/tvst.10.8.7.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To study the in vitro effect of vitamin D3 on the healing response of human Tenon's fibroblasts (HTF) and its possible role in preventing excessive postoperative subconjunctival fibrosis.

Methods: Effect of vitamin D3 on cytotoxicity and cell survival of primary cultured HTF was measured by lactate dehydrogenase and PrestoBlue assays, respectively. Proliferation and migration of vitamin D3–treated HTF (D3-HTF) was determined by CyQUANT proliferation and scratch assay, respectively. The mRNA expression profiles of control-HTF and D3-HTF from six subjects (three with glaucoma and long-term use of topical medications, three with primary pterygium) were assessed by RNA sequencing analyses to identify potential biomarkers for the inhibitory effect on HTF by vitamin D3. Validation of these biomarkers and their potential pathways were performed by quantitative real-time polymerase chain reaction (qRT-PCR) detection.

Results: Pure monolayers of HTF from controls (retinal detachment or squint surgeries), pterygium, and glaucoma subjects were successfully prepared and passaged. Proliferation and migration of pterygium and glaucoma HTF were inhibited by vitamin D3 in a dose-dependent manner, and without cytotoxicity or decrease in cellular viability with concentrations up to 10 µM. The qRT-PCR results were consistent with the transcriptome analyses, vitamin D3 appears to enhance CYP24A1, SHE, KRT16 but suppresses CILP expression in HTF.

Conclusions: Vitamin D3 can inhibit the in vitro activity of HTF without compromising cellular survivability at concentration up to 10 µM. This has potential clinical application for improving the outcome of pterygium and filtering surgeries.

Translational Relevance: Vitamin D3 can suppress the in vitro proliferation, migration, and transdifferentiation of human Tenon's fibroblasts, without the cytotoxicity of mitomycin-C, the current standard antifibrotic agent in clinical use.

Introduction
Glaucoma is one of the leading causes for irreversible blindness,1 characterized by progressive damage to the optic nerve from an intraocular pressure (IOP) higher than its normal tolerance level.2 Although the tolerance level varies among individuals and the stage of disease, the aim in all cases is reduction of IOP or preventing its elevation.3 Lowering IOP by medications (usually as eye drops) is the first line of treatment in most clinical practices.4 When glaucoma progresses because the IOP remains high despite multiple medications, procedures involving lasers or incisional surgeries are often required to prevent blindness.5 Surgical procedures lower IOP by facilitating aqueous humor to exit the eye by decreasing the outflow resistance.6 Currently, the most common methods of lowering outflow resistance is by diverting aqueous out of the anterior chamber and into the subconjunctival space. Access to the subconjunctival space is by either surgically creating a new opening or inserting a stent-type implant into the anterior chamber.7 However, conjunctival wound healing after such drainage surgeries can sometimes be excessive, leading to subconjunctival fibrosis and scarring at the drainage site, with progressive reduction of aqueous outflow and surgical failure.8 Aside from surgery, long-term use of topical glaucoma medication can also activate human Tenon's fibroblasts (HTF).9 Excessive activation of HTF result in their proliferation and migration, synthesis of extracellular matrix components (ECM), and collagen contraction, leading to subconjunctival scarring.10 Various methods have been used for modulating postoperative wound scarring, but one that is free from serious adverse effect remains an elusive goal for subconjunctival aqueous drainage procedures.11 
Aside from its role in postoperative scarring after glaucoma surgery, HTF is also involved in the development of pterygium. Although pterygium excision is a relatively simple procedure, recurrences are common and more difficult to excise than primary pterygium in the initial surgery,12 so measures to mitigate proliferation of HTF after surgery are used to decrease the incidence of recurrent pterygium.13 
Antimetabolites such as 5-fluorouracil (5-FU) and mitomycin-C (MMC) are commonly used as antifibrotic agents to prevent or reduce postoperative fibrosis and scarring,14 but their use is limited by the cellular toxicity common to most antimetabolites, which manifests clinically as impaired wound healing and atrophic conjunctiva at the drainage site, with increased risk of bleb leak, hypotony, exposed implant, and infection.15 Therefore any new agents that can suppress fibrosis and scarring without concomitant cellular toxicity will greatly improve glaucoma surgery outcome.16 
Vitamin D3 (abbreviated as D3) is a multifunctional hormone that not only affects calcium homeostasis but plays an essential role in immune system regulation, as well as cell growth and survival.17 Many tissues in the eye are able to both activate and respond to D3,18 whereas D3 levels and genetic variations can influence the development of a wide range of ocular pathologies, such as myopia, age-related macular degeneration, diabetic retinopathy, uveitis, and glaucoma.18 Previous studies have shown that D3 may play a protective role in ocular health.19 This included inhibition of angiogenesis in transgenic murine retinoblastoma,20 inhibition of corneal neovascularization induced by sutures in murine eyes,21 inhibition of retinal neovascularization in mice model,22 prevention of retinal autoimmune disease and mitigation of uveitis,23 decreased retinal inflammation and levels of amyloid beta (Aβ) accumulation,24 as well as lower IOP in primates.25 
Because D3 appears to have a modulating effect on cutaneous scar formation,26 it may be potentially useful in ocular surgeries, although whether conjunctiva or Tenon tissues will respond similarly is currently unknown.27 As high doses of systemic D3 can result in hypercalcemia or anemia, our in vitro study will simulate the effect of D3 applied at the wound site and in direct contact with the HTF, similar to current application of 5-FU and MMC in glaucoma and pterygium surgeries. 
Materials and Methods
Primary Culture of Human Tenon's Fibroblasts
The study was conducted according to the tenets of the Declaration of Helsinki, and approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (IRB Reference Number: UW 19-366). All subjects were consecutive patients of Grantham Hospital, Hong Kong, scheduled for elective surgeries involving conjunctival dissection. We specifically recruited three types of subjects: (1) primary glaucoma patients undergoing trabeculectomy, with use of glaucoma eye drops longer than one year; (2) primary pterygium excision; (3) presumed normal without previous ocular surgery or long-term use of any eye drops (for example, eyes undergoing retinal detachment or squint surgeries). 
Details of the subjects and operated eyes, where Tenon's capsules were obtained, are shown in Table 1. None of the chosen operated eyes had previous surgery involving conjunctiva or Tenon's capsule. 
Table 1.
 
Information of Sources for Tenon Tissues Used in This Study
Table 1.
 
Information of Sources for Tenon Tissues Used in This Study
Tenon's capsule sample, of approximately 1 to 2 mm by 1 to 2 mm in size, was obtained during surgery and transferred immediately into tubes containing Eagle's minimum essential medium (EMEM) growth medium (ATCC, Manassas, VA, USA), supplemented with 10% bovine calf serum, 7.5 mM L-glutamine, 5 ng/mL rh-FGF-basic, 5 µg/mL rh-insulin, 1 µg/mL hydrocortisone, 50 µg/mL ascorbic acid, 100 U/mL penicillin and 100 µg/mL streptomycin. After cutting into micro pieces under sterile conditions, the samples were air-dried and seeded in a CELLSTAR 6 well cell culture plate (Greiner Bio-One, Monroe, NC, USA). The HTF were maintained in the logarithmic growth phase in 5% CO2 humidified atmosphere at 37°C. A week after seeding, primary cultures were passaged at 60% confluence with 0.25% trypsin- ethylenediaminetetraacetic acid (EDTA; Invitrogen, Carlsbad, CA, USA) into Nunc EasYFlask Cell Culture Flasks 7 5cm2 with filter (Thermo Fisher Scientific, Waltham, MA, USA). For subsequent passaging, cells were dissociated using 0.25% trypsin-EDTA at 85%. The cultured HTF are then used in subsequent steps of the experiments as shown in Figure 1
Figure 1.
 
Experiment flow chart.
Figure 1.
 
Experiment flow chart.
Immunofluorescence Staining for Identification of HTF
For immunofluorescence, HTF were seeded at a concentration of 1 × 105 cells/mL on 4-well Nunc Chamber Slide System (Thermo Fisher Scientific) and incubated for one day to reach a subconfluent status. Cells were fixed in cold 4% paraformaldehyde (EM GRADE; Electron Microscopy Sciences, Hatfield, PA, USA) for 10 minutes, permeabilized in 0.05% Triton X-100 (Sigma-Aldrich Corp., St. Louis, MO, USA) in phosphate-buffered saline solution (PBS) for 15 minutes, blocked in 3% bovine serum albumin in PBS for one hour, and conjugated with primary antibody in 3% bovine serum albumin/PBS (Thermo Fisher Scientific) overnight (keep cells dark in a humidified chamber) at 4°C. After incubation with secondary antibody (1:500; Thermo Fisher Scientific) for one hour at room temperature, the upper chambers were removed. Before covering with 10 mm diameter round coverslip, one drop of ibidi Mounting Medium with DAPI (4ʹ, 6-diamidino-2-phenylindole) (ibidi USA, Fitchburg, WI, USA) was added on slides for mounting and cell nuclei staining and observed with a fluorescence microscope (Nikon ECLIPSE 80i; Nikon Inc., Melville, NY, USA) using SPOT Advanced software after 20 minutes. The antibodies used for identifying HTF in this assay are listed in Table 2
Table 2.
 
Monoclonal and Polyclonal Antibodies Used in This Study
Table 2.
 
Monoclonal and Polyclonal Antibodies Used in This Study
Drug Preparations
The 1α, 25-dihydroxyvitamin D3 powder 0.1 mg (Sigma-Aldrich Corp) was dissolved in 100 µL 96% ethanol (Sigma-Aldrich Corp) and 140 µL PBS to make 1mM D3 stock and stored in −80 °C. The same volume of 96% ethanol and PBS without 1α, 25-dihydroxyvitamin D3 powder was used as a vehicle control. Mitomycin-C (MMC) powder 10 mg (Calbiochem, San Diego, CA, USA) was dissolved in 1× PBS (Thermo Fisher Scientific) to make 2.5mM MMC stock and stored in −80 °C. The drugs were diluted with culture medium to achieve the desired concentrations for the experiments. 
Lactate Dehydrogenase Cytotoxic Analysis
Leakage of cytoplasmic lactate dehydrogenase (LDH) to the extracellular medium was measured with the Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific), with the presence of LDH in the medium representing damage to cell membrane. LDH activity is proportional to color intensity and is expressed as optical density. For the LDH assay, 104 HTF/mL were seeded in each well of 96-well black wall clear bottom plates (SPL Inc., Houston, TX, USA). Twenty-four hours after cell seeding, cells in triplicate wells were exposed to different concentrations of D3 (0.001 µM, 0.01 µM, 0.1 µM, 1 µM, 5 µM, 10 µM, 25 µM, 50 µM, and 100 µM), and MMC (0.025, 0.05, 0.075, 0.1, and 0.2 mg/mL) as positive control. In addition, there were wells with corresponding concentration levels of ethanol serving as vehicle control, and cell culture medium only to correct for background color (Supplementary Methods). Absorbance was measured at a wavelength of 490 nm and 680 nm using a multimodal microplate reader (SpectraMax iD5; Molecular Devices, San Jose, CA, USA). LDH activity was determined by subtracting the 680 nm (background signal from instrument) from the 490 nm absorbance value and presented as fold-change. 
PrestoBlue Cell Viability Assay
PrestoBlue Cell Viability Reagent (Invitrogen) was used for testing cell viability, based on the ability of viable cells, with active mitochondrial reductases of the electron transport chain, to convert resazurin dye (blue and nonfluorescent) to resorufin (red and highly fluorescent) (Supplementary Methods). The fluorescence is proportional to the number of viable cells and is quantified using absorbance measurements with a multimodal microplate reader (SpectraMax iD5, Molecular Devices) at 570 nm and normalized to 600 nm values. 
CyQUANT Cell Proliferation Assay
Cell proliferation assays were performed using the CyQUANT Cell Proliferation Assay kit (Invitrogen) (Supplementary Methods). The samples fluorescence was measured using a multimodal microplate reader (SpectraMax iD5, Molecular Devices) at 480 nm excitation and 520 nm emission. 
In Vitro Scratch Assay
Scratch assay was conducted to mimic surgical trauma and assess the subsequent migratory activity of pterygium and glaucoma HTF as part of the healing response. The HTF were grown on CELLSTAR six-well cell culture plate (Greiner Bio-One) at the bottom outer layer of which were previously marked a cross (with 25-gauge needle) for easy recognition. When the cells reached a confluence more than 90%, the culture medium was replaced by plain EMEM medium (without serum) overnight to synchronize the cell growth. Next, the cells were stimulated with fresh EMEM medium for the blank control group, 10 µM D3/EMEM for the treatment group, the same concentrated ethanol/EMEM for the vehicle control group, 0.2 mg/mL MMC/EMEM for the positive control group for one hour. Then, the cell monolayer was scraped with a sterilized 1000 µL pipette tip to generate a cell-free gap (scratch width) in each well. After washing with PBS three times, the cells were treated again with newly made culture medium for different groups. The gap of each well was imaged randomly under inverted phase-contrast microscope (Nikon ECLIPSE TE2000-S) using SPOT Advanced software. The migration distance was evaluated quantitatively in both pterygium and glaucoma HTF treated with 10 µM D3 and the vehicle control, at 0 and 24 hours, by calculating relative scratch width (normalized to the distance at 0 hour in each group). 
Western Blot Assay
After exposure to 10 µM D3/EMEM (treatment group) and the same concentration of ethanol/EMEM (vehicle control group), the cells were incubated in ice-cold RIPA Lysis Buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulphate, 1% sodium deoxycholate; 10% EDTA solution, 10% protease and phosphatase inhibitor cocktail; Abcam, Cambridge, MA, USA) for 30 minutes, then centrifuged at 13,200 rpm to obtain the supernatants, which were frozen at −80°C. Protein concentrations were determined with the Pierce BCA protein assay kit (Thermo Fisher Scientific). Protein samples were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes, which were then probed overnight at 4°C with primary antibodies against human α smooth muscle actin (α-SMA; ab5694, 1 µg/mL; Abcam), vimentin (EPR3776; ab92547, 1:5,000; Abcam), and GADPH (sc-47724, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubating with horseradish peroxidase–conjugated secondary antibodies (A16078, 1:20,000, Invitrogen; A6154, 1:20,000, Sigma-Aldrich Corp.) for two hours at room temperature. Labeled proteins were detected by enhanced chemiluminescence (no. 34579, Thermo Fisher Scientific). 
RNA Extraction and RNA Sequencing (RNA-Seq)
Total RNA in 10 µM D3 treated HTF, as well as its vehicle and positive control, was extracted with TRIzol Reagent (Invitrogen). The integrity and purity of RNA was assessed by analyzing 260/280 nm ratios (Ratio260/280) and 260/230 nm ratios (Ratio260/230) on a NanoDrop One/OneC and the RNA Integrity Number (RIN) using Agilent 4200 TapeStation (Agilent Technology, Tokyo, Japan). Life Invitrogen Qubit 3.0 Fluorometer was used for quantitation of RNA sample. To construct Illumina sequencing libraries, magnetic beads with oligo-dT were used to capture the mRNA with poly A structure. Subsequently, the first strand of cDNA was synthesized in the reverse transcriptase M-MuLV (from Moloney murine leukemia virus) system with fragmented mRNA as template and random oligonucleotides as primers. Next, RNA chains were degraded by ribonuclease H (RNase H), and the second strand of cDNA was synthesized from deoxyribonucleotide triphosphates (dNTPs) in the DNA polymerase I system. After purification of double-stranded cDNA and terminal repair, A tails were added and connected to sequencing joints. About 200bp of cDNA was purified by AMPure XP beads before polymerase chain reaction (PCR) amplification. Finally, the sequencing libraries were obtained by purifying the PCR products again using AMPure XP beads. After passing the library inspection (KAPA qPCR quantification and Agilent 4200 TapeStation detection), different libraries were pooled according to the requirements of effective concentration and target disembarkation data volume, then Illumina PE150 sequencing was performed. 
Post-Sequencing RNA-Seq Data Analysis
After obtaining raw data, the specific implementation process of biological information analysis was conducted (Supplementary Methods). Genes with adjusted P < 0.05 were considered significantly enriched by the differentially expressed genes. 
Validation Via Quantitative Real-Time PCR
Total RNA was harvested using RNA spin columns (9767; Takara Biotechnology Co., Kyoto, Japan). CDNA was synthesized by reverse transcriptase from total RNA with PrimeScript RT Master Mix (RR047A; Takara Biotechnology Co.). The expression of specific mRNAs was determined with StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the TB Green Premix Ex Taq (Tli RnaseH Plus) (RR420A; Takara Biotechnology Co., Kyoto, Japan) (Supplementary Methods). 
Statistics and Mathematical Analyses
Data analysis was performed using statistical software (GraphPad Prism Version 8.0.0; GraphPad, San Diego, CA, USA). All experiments were repeated at least three times. Values were expressed as mean ± standard error (SEM). For reporting purposes, all experimental replicates using multiple subjects’ samples were denoted using N = x, and the number of experimental repeats performed using each subject's sample will be denoted using n = y. The above data with more than two groups were analyzed with one‐way analysis of variance. For all analyses, P ≤ 0.05 was considered statistically significant. 
Results
HTF Characterization
After HTFs migrated from the initial tissue and passaged in vitro, the cells were grown in monolayer and exhibited a spindly, generally flat, elongated shape. As shown in Figure 2, the cultured cells from all Tenon samples were positive for the antibodies chosen to confirm their HTF identity; the anti-vimentin and anti-alpha smooth muscle actin antibodies staining showed green fluorescence in the cytoplasm, the anti-collagen I and the anti-fibroblast surface protein antibodies staining showed red fluorescence, and the DAPI showed oval-shaped, blue-stained nuclei. Interestingly, the expression of myofibroblast associate markers (α-SMA and FSP) were significantly higher in pterygium and glaucoma HTF compared with presumably normal HTF from eyes undergoing vitreoretinal or squint surgeries, indicating the differentiated state of the primary cultured HTF from eyes with primary pterygium or had used topical glaucoma medication for more than one year. 
Figure 2.
 
Primary culture cell verification. HTFs stained with Vimentin, collagen, fibroblast surface protein, α-smooth muscle actin and DAPI. (A) Pterygium HTF. (B) Normal HTF. (C) Glaucoma HTF (N = 3, n = 3). Scale bar: 25 µm
Figure 2.
 
Primary culture cell verification. HTFs stained with Vimentin, collagen, fibroblast surface protein, α-smooth muscle actin and DAPI. (A) Pterygium HTF. (B) Normal HTF. (C) Glaucoma HTF (N = 3, n = 3). Scale bar: 25 µm
Effect of Vitamin D3 on HTF Toxicity and Cell Viability
The amount of LDH in the culture media (Figs. 3A, 3B) was stable with D3 concentrations up to the maximum tested level of 100 µM, in contrast to the incremental changes (indicating increasing cytotoxicity) noted with increasing MMC concentrations beyond the lowest level (0.025 mg/mL) tested. Similar results were seen with PrestoBlue cell viability assay, where the absorbance values at 570 nm were stable until D3 concentration was increased to 100 µM, compared to the decremental changes (indicating decreasing cellular viability) with increasing MMC concentration beyond the lowest level (0.025 mg/mL) tested. With further testing of cellular toxicity and viability using D3 at 25 µM and 50 µM on both pterygium (Figs. 3C, 3E), and glaucoma HTF (Figs. 3D, 3F), we noted LDH release increases and HTF viability decreases with concentration above 25 µM. Note that 0.025 mg/mL MMC is about one-sixteenth to one-eighth of the concentration used clinically in pterygium and glaucoma surgeries. 
Figure 3.
 
Cytotoxicity and cell viability evaluation on pterygium HTF using the LDH and PrestoBlue assays, respectively, to compare (A) the Log 10 value of different concentrations of D3 (0.001 µM to 100 µM) with (B) mitomycin-C (75 µM to 598 µM) (N = 1, n = 3); effect of D3 (5 µM to 50 µM) on (C) pterygium HTF cytotoxicity and (D) glaucoma HTF cytotoxicity (N = 3 , n = 3); and effect of D3 (5 µM to 50 µM) on (E) pterygium HTF viability and (F) glaucoma HTF viability (N = 3 , n = 3). The arrows indicate clinically used MMC concentration of 0.1 and 0.2 mg/mL, equivalent to 299 and 598 µM. (**P ≤ 0.05; ****P ≤ 0.001)
Figure 3.
 
Cytotoxicity and cell viability evaluation on pterygium HTF using the LDH and PrestoBlue assays, respectively, to compare (A) the Log 10 value of different concentrations of D3 (0.001 µM to 100 µM) with (B) mitomycin-C (75 µM to 598 µM) (N = 1, n = 3); effect of D3 (5 µM to 50 µM) on (C) pterygium HTF cytotoxicity and (D) glaucoma HTF cytotoxicity (N = 3 , n = 3); and effect of D3 (5 µM to 50 µM) on (E) pterygium HTF viability and (F) glaucoma HTF viability (N = 3 , n = 3). The arrows indicate clinically used MMC concentration of 0.1 and 0.2 mg/mL, equivalent to 299 and 598 µM. (**P ≤ 0.05; ****P ≤ 0.001)
Vitamin D3 Inhibits HTF Proliferation, Migration, and Transdifferentiation to Myofibroblasts
Cell proliferation assessment by the CyQuant assay showed D3 treatment significantly decreased HTF proliferation after 24 hours, in a dose-dependent manner (Figs. 4A, 4B). 
Figure 4.
 
Inhibition of proliferation in (A) pterygium HTF and (B) glaucoma HTF seeded at 10,000 cells/well in a 96-well plate and grown for 24 hours in presence of ethanol or increasing concentrations of D3. Relative proliferation was measured by fluorescence determination of DNA content using the CyQuant Proliferation assay (N = 3, n = 3). Representative images of D3-induced inhibition of wound healing in (C) pterygium HTF and (D) glaucoma HTF as compared to the EMEM, ethanol (vehicle control), and MMC (positive control, 598 µM) at 0, 12, and 24 hours after scratch (Scale bar: 100 µm). Cell migration was evaluated quantitatively in (E) pterygium cases (N = 3, n = 4) and (F) glaucoma cases (N = 3, n = 3) respectively, between 10 µM D3-treated HTF and the vehicle control HTF at 0 and 24 hours by calculating relative wound closure percentage. Data are presented as the means ± SEM (***P < 0.001; ****P < 0.0001, compared with vehicle control group). (G) The expression of α-smooth muscle actin (α-SMA) was evaluated using Western blots.
Figure 4.
 
Inhibition of proliferation in (A) pterygium HTF and (B) glaucoma HTF seeded at 10,000 cells/well in a 96-well plate and grown for 24 hours in presence of ethanol or increasing concentrations of D3. Relative proliferation was measured by fluorescence determination of DNA content using the CyQuant Proliferation assay (N = 3, n = 3). Representative images of D3-induced inhibition of wound healing in (C) pterygium HTF and (D) glaucoma HTF as compared to the EMEM, ethanol (vehicle control), and MMC (positive control, 598 µM) at 0, 12, and 24 hours after scratch (Scale bar: 100 µm). Cell migration was evaluated quantitatively in (E) pterygium cases (N = 3, n = 4) and (F) glaucoma cases (N = 3, n = 3) respectively, between 10 µM D3-treated HTF and the vehicle control HTF at 0 and 24 hours by calculating relative wound closure percentage. Data are presented as the means ± SEM (***P < 0.001; ****P < 0.0001, compared with vehicle control group). (G) The expression of α-smooth muscle actin (α-SMA) was evaluated using Western blots.
In the scratch wound assays, cell migration was relatively fast in the control groups, with significant reduction of scratch width at 24 hours, in contrast to the D3 (10 µM) and MMC (0.2 mg/mL) treated groups, where it was not significantly different from 0 hour (immediately after scratching), as shown in Figures 4C and 4D, indicating suppression of cell migration. We also noted that the HTF started to float within the first hour in the MMC group, which was suggestive of cell death, although this was not observed in the blank, D3, or vehicle control groups. As shown in Figures 4E and 4F, simulated wound closure by HTF was significantly suppressed by 10 µM D3 treatment compared with the vehicle control. 
Because the accumulation of ECM proteins is an indicator of myofibroblast transdifferentiation, we evaluated the effect of D3 on expression of α-SMA, an ECM component, using Western blots. As shown in Figure 4G, D3 treatment strongly inhibited α-SMA expression in both pterygium and glaucoma HTF. 
The Molecular Pathology of HTF
To further investigate the molecular pathology of pterygium and glaucoma HTF, we compared the transcriptional profiles of pterygium (n = 4) and glaucoma (n = 4) versus normal (n = 1) conjunctival tissues by RNA-seq. Based on the RNA-seq data, both glaucoma and pterygium cases showed completely different expression patterns compared to that of the normal tissue (Figs. 5A, 5B). To explore the pathological process of glaucoma and pterygium HTF, we combined two differentially expressed genes (DEGs) by Venn diagram (Fig. 5C). Among these DEGs, 8642 transcripts both changed in the disease group against the normal. The top 20 genes with the most obvious expression changes based on adjusted P values are listed in Table 3 and Table 4
Figure 5.
 
(A) Hierarchical clustering of all DEGs between the normal and the pterygium. (B) Hierarchical clustering of all DEGs between the normal and the glaucoma. Expression values are Z score transformed. Samples were clustered using complete linkage and Euclidean distance. (C) Venn diagram of DEGs that both changed (P < 0.05) in the pterygium and glaucoma in comparison to that of the normal.
Figure 5.
 
(A) Hierarchical clustering of all DEGs between the normal and the pterygium. (B) Hierarchical clustering of all DEGs between the normal and the glaucoma. Expression values are Z score transformed. Samples were clustered using complete linkage and Euclidean distance. (C) Venn diagram of DEGs that both changed (P < 0.05) in the pterygium and glaucoma in comparison to that of the normal.
Table 3.
 
Top 20 DEGs of HTFs Versus Normal
Table 3.
 
Top 20 DEGs of HTFs Versus Normal
Table 4.
 
Top 20 DEGs for MMC and VD3 Versus Vehicle
Table 4.
 
Top 20 DEGs for MMC and VD3 Versus Vehicle
Vitamin D3 Enhances CYP24A1, SHE, KRT16 But Suppresses CILP Expression in HTF
To further investigate the mechanism of effect by D3 on HTF, we performed RNA-seq analysis for pterygium, glaucoma, and normal HTF. This consists of normal blank control (normal_bl; n = 1), pterygium blank control (ptery_bl_1; n = 1), glaucoma blank control (glau_bl_1; n = 1), pterygium MMC positive control (ptery_MMC_1; n =1), glaucoma MMC positive control (glau_MMC_1; n = 1), pterygium vehicle control (ptery_veh_1, ptery_veh_2, ptery_veh_3; n = 3), glaucoma vehicle control (glau_veh_1, glau_veh_2, glau_veh_3; n = 3), pterygium treated with D3 (ptery_vD3_1, ptery_vD3_2, ptery_vD3_3; n = 3), and glaucoma treated with D3 (glau_vD3_1, glau_vD3_2, glau_vD3_3; n = 3). To eliminate any outliers, both principal-component analysis and hierarchical clustering were applied to segregate the transcriptomic profiles of vehicle (veh) from D3-treated (D3) pterygium and glaucoma HTF, leaving seven samples demonstrating the greatest difference between the vehicle and D3 group (i.e., glau_veh_1, glau_veh_2, ptery_veh_2, ptery_veh_3, glau_vD3_2, ptery_vD3_2, ptery_vD3_3) (Figs. 6A, 6B). Differential expression analysis of these samples revealed 4251 DEGs in the D3-treated group compared with the vehicle group. The top 10 genes were displayed in volcano plot (Fig. 6C). Two of the upregulated genes, CYP24A1 and KRT16, were selected for PCR verification, which appeared consistent with the RNA-seq data (Figs. 6D, 6E). 
Figure 6.
 
(A) Principal-component analysis (PCA) plot of n = 4251 genes expressed in the D3-treated group (n = 3) and the vehicle group (veh) (n = 4). (B) Hierarchical clustering of all DEGs between the D3 and the vehicle groups. Expression values are Z score transformed. Samples were clustered using complete linkage and Euclidean distance. (C) Volcano plots for DEGs between the D3-treated and the vehicle groups. As observed in the figure, the upregulated and downregulated genes were marked as a dot, those significantly up-regulated genes were highlighted in red, the down-regulated genes were highlighted in blue, and the nonsignificant genes were labeled as gray dots. In the figure, the gray lines indicate the marginal lines separating DEGs from non-DEGs, with the horizontal lines denoting the P value threshold (P ≤ 0.05). The top 10 upregulated and downregulated genes were showed in graph: pterygium (D) and glaucoma (E) treated with 10 µM D3 or vehicle for 24 hours. Then mRNA expressions of CYP24A1, KRT16 in pterygium and glaucoma were measured by qPCR and normalized to GAPDH (n = 3). The vehicle and D3-treated groups consist of primary cultured pterygium or glaucoma HTF that are not treated or treated with specific concentration of D3, respectively. All bar graphs are expressed as mean ± SEM (***P < 0.001; ****P < 0.0001). (F) GO analysis of DEGs between the D3-treated and the vehicle groups. The graph displays the classification term enrichment status and term hierarchy. The color scale shows the P value cutoff levels for each biological process. (G) KEGG analysis of DEGs between the D3-treated and the vehicle groups. The color scale shows the P value, and the dot size shows the related DEGs.
Figure 6.
 
(A) Principal-component analysis (PCA) plot of n = 4251 genes expressed in the D3-treated group (n = 3) and the vehicle group (veh) (n = 4). (B) Hierarchical clustering of all DEGs between the D3 and the vehicle groups. Expression values are Z score transformed. Samples were clustered using complete linkage and Euclidean distance. (C) Volcano plots for DEGs between the D3-treated and the vehicle groups. As observed in the figure, the upregulated and downregulated genes were marked as a dot, those significantly up-regulated genes were highlighted in red, the down-regulated genes were highlighted in blue, and the nonsignificant genes were labeled as gray dots. In the figure, the gray lines indicate the marginal lines separating DEGs from non-DEGs, with the horizontal lines denoting the P value threshold (P ≤ 0.05). The top 10 upregulated and downregulated genes were showed in graph: pterygium (D) and glaucoma (E) treated with 10 µM D3 or vehicle for 24 hours. Then mRNA expressions of CYP24A1, KRT16 in pterygium and glaucoma were measured by qPCR and normalized to GAPDH (n = 3). The vehicle and D3-treated groups consist of primary cultured pterygium or glaucoma HTF that are not treated or treated with specific concentration of D3, respectively. All bar graphs are expressed as mean ± SEM (***P < 0.001; ****P < 0.0001). (F) GO analysis of DEGs between the D3-treated and the vehicle groups. The graph displays the classification term enrichment status and term hierarchy. The color scale shows the P value cutoff levels for each biological process. (G) KEGG analysis of DEGs between the D3-treated and the vehicle groups. The color scale shows the P value, and the dot size shows the related DEGs.
To gain further insight into the various targets and their associated pathways, we performed pathway enrichment analysis of the differently expressed sequences' target genes based on the GO/KEGG database. By subjecting the differentially expressed genes from the group of D3 and vehicle control in both glaucoma and pterygium HTF, we observed numerous enriched gene sets. During the GO enrichment analysis, the genes were mainly enriched in extracellular matrix organization, extracellular structure organization and homophilic cell adhesion via plasma membrane adhesion molecules (Fig. 6F). We further investigated the functional implications of these differentially expressed genes of D3 treatment by KEGG pathway analysis. A number of the differentially expressed genes were enriched in four KEGG pathways, including calcium signaling pathway, retrograde endocannabinoid signaling, inflammatory mediator regulation of TRP channels and AGE-RAGE signaling pathway in diabetic complications (P < 0.05) (Fig. 6G). Because MMC is commonly used as an antifibrotic agent during pterygium and glaucoma surgery, we compared the effect of D3 and MMC on HTF by analyzing their DEGs by Venn diagram. As shown in Figure 7A, a total of 533 DEGs were obtained from comparison between D3/MMC and the vehicle group, and these genes reached the threshold P value <0.05. Among these DEGs, CYP24A1 showed changes in both D3 and MMC treated group. In the pterygium group, it showed that KCNE4, GDPD5, TDRD10, SHE, HSPG2, CILP, CLMN, SULT1C2, MLXIPL changed in both D3 and MMC group, while IL11, GDF15, ATF3, STC1, ADAMTS15, HBEGF, PMAIP1 were found to change in the glaucoma group. Among these genes, we selected SHE and CILP to conduct PCR verification using pterygium HTF (Fig. 7B). The real-time PCR results showed that D3 could significantly upregulate SHE and downregulate CILP in pterygium HTF. 
Figure 7.
 
(A) Venn diagram showing DEGs that are both changed in vitamin D3 (vD3)– and MMC-treated, compared to normal, tissues. (B) Pterygium HTF were treated with 10 µM vD3 or vehicle for 24 hours, then mRNA expression of SHE, CILP were measured by qPCR and normalize to GAPDH (n = 3). All bar graphs are expressed as mean ± SEM. (*P < 0.5; ***P < 0.001; ****P < 0.0001).
Figure 7.
 
(A) Venn diagram showing DEGs that are both changed in vitamin D3 (vD3)– and MMC-treated, compared to normal, tissues. (B) Pterygium HTF were treated with 10 µM vD3 or vehicle for 24 hours, then mRNA expression of SHE, CILP were measured by qPCR and normalize to GAPDH (n = 3). All bar graphs are expressed as mean ± SEM. (*P < 0.5; ***P < 0.001; ****P < 0.0001).
Discussion
The outcome of ocular procedures like glaucoma filtration surgery (trabeculectomy) and pterygium excision are closely dependent on the postoperative response of subconjunctival Tenon's fibroblasts.28 Therefore the inhibition of excessive proliferation and migration of HTF continues to be a key goal for improving surgical success.29 However, the use of current antifibrotic agents such as MMC and 5-FU involves off-label use of cytotoxic agents with potentially serious complications including bleb leak, blebitis, and endophthalmitis after trabeculectomy, and corneoscleral melt after pterygium excision.30,31 
Our study showed that D3 had a regulatory effect on the activity of subconjunctival fibroblasts, similar to their previously reported effect on cutaneous fibroblasts. At concentration up to 25 µM, D3 also appears safer than MMC, currently the most commonly used antifibrotic agent, even at concentration well below clinical usage. However, D3 appears to be cytotoxic at concentration above 25 µM. Our CyQuant proliferation assays on glaucoma and pterygium HTF treated with D3 for 24 hours showed that it could suppress HTF proliferation in a dose-dependent manner. We believe that the optimal concentration of D3 is around 10 to 25 µM, with regard to cellular safety and inhibitory effect on HTF proliferation. In our Western blot analysis of ECM components, α-SMA expression was reduced after D3 treatment, indicating suppressed myofibroblast transdifferentiation. In 2007, Albert et al. also demonstrated that calcitriol (1α,25(OH)2D3) concentrations ≥50 µM can significantly decrease endothelial cell viability in retinal capillaries.32 A limitation of our study was that MMC and D3 concentrations may not be strictly maintained during the entire experiment (24 hours), because of their degradable properties when exposed to light or relatively high temperature. 
For our mRNA profile analysis, although we imported data from 17 samples, we only performed further analysis for seven samples (i.e., glau_veh_1, glau_veh_2, ptery_veh_2, ptery_veh_3, glau_vD3_2, ptery_vD3_2, ptery_vD3_3), because these clustered the best to display the difference between the vehicle and D3 group. Our qPCR result was consistent with that of RNA-seq. 
In 2016, Srikuea et al.33 reported administration of 1α,25(OH)2D3 at a supra-physiological dose can decrease satellite cell differentiation, delay regenerative muscle fiber formation, and increase muscular fibrosis. The 1α,25(OH)2D3 has been reported to activate intracellular signaling molecules such as protein kinases A and C, phosphatidylinositol 3-kinase, phospholipase C, as well as open calcium channels in addition to transcriptional activation.34 The concentration of 1α,25(OH)2D3 is regulated by the action of the vitamin D3-catabolizing enzyme, encoded by the CYP24A1 gene, to convert it to the inactive metabolite, calcitroic acid.35 As one of the major enzymes regulating D3, the upregulation of CYP24A1 in D3 treated HTF confirms the presence of local metabolism of 1α,25(OH)2D3. 
SHE (Src Homology 2 Domain Containing E) belongs to the Src homology 2 (SH2) domain family. SHE is reported to be associated with diseases such as blepharoconjunctivitis.36 It is now known that the human genome encodes ∼120 SH2 domains that are dispersed in more than 110 proteins.37 These include protein or lipid kinases, protein phosphatases, small GTPases, cytoskeleton regulators, and adaptor/scaffolding proteins and other regulators of signal transduction.38 For example, Src homology 2 domain‐containing inositol 5ʹ‐phosphatase is a negative regulator of the phosphatidylinositol 3‐kinase/protein kinase B (PI3K/Akt) signaling.39 In 2017, Li et al. suggests that decreased Src homology 2 domain‐containing inositol 5ʹ‐phosphatase mediates high glucose‐induced TGF‐β1 upregulation and ECM deposition through activation of the PI3K/Akt pathway in renal tubular cells.40 PI (3,4,5) P3 initiates a variety of signaling cascades by interacting with pleckstrin homology (PH) domain‐containing proteins, most notably the serine kinase Akt.41 The activity of the PI3K/Akt signaling axis is multifunctional and is involved in renal ECM accumulation and interstitial fibrosis.40 The upregulation of SHE after treatment with D3 may suggest suppression of TGF‐β1 induced subconjunctival fibrosis via PI3K/Akt signaling axis. 
Keratin 16 (K16), a type I cytokeratin encoded by the KRT16 gene, when paired with keratin 6 in epithelial tissues, was found to maintain collective cell adhesion and integrity by increasing cell-cell and cell-matrix contact and decreasing cell migration and directionality during skin wounding.42 In 2010, Nima Aden et al. confirmed that in systemic sclerosis, epidermal keratinocytes have a delayed differentiation and expression of K16.43 In the same year, Trost et al. investigated on the influence of excess K16 on the migratory behavior of HaCaT keratinocytes in vitro and found that cells overexpressing K16 displayed a 90% reduction in migration potential compared to mock-transfected immortalized human keratinocytes.44 In our study, D3 treatment significantly upregulates KRT16 gene expression, and possibly leading to enhanced cellular adhesion and migration suppression. 
Cartilage intermediate layer protein (CILP) is a key regulator of cardiac remodeling and heart failure.45 In 2018, Zhang et al. reported that CILP-1 alleviates pressure overload-induced cardiac fibrosis via interfering with TGF-β1 signaling and concluded that CILP-1 is a novel ECM protein possessing antifibrotic ability in pressure overload-induced fibrotic remodeling.45 They demonstrated that C-terminal CILP-1 increased Akt phosphorylation, promoted the interaction between Akt and Smad3, and suppressed Smad3 phosphorylation.45 The antifibrotic effect of CILP-1 is attributed to it interfering with TGF-β1 signaling through its N- and C- terminal fragments.46 Blockade of PI3K-Akt pathway attenuated the inhibitory effect of C-CILP-1 on TGF-β1-induced Smad3 activation.45 The downregulation of CILP may indicate lower fibrotic remodeling. 
Apart from the qPCR validation results, our mRNA profiles revealed some candidate genes like IGFBP-5, ADAM33 and KRT14 (Table 3.), which have been reported to play vital roles in fibrosis or ECM reconstruction.4749 In addition, vitamin D3 could significantly downregulated chemokine CXCL1, retinol metabolic related gene ADH4 (Fig. 6C). It appears that further investigation of the possible regulatory effect of these candidate genes is warranted. Both RNA-seq and qPCR data suggest that D3 affects not only scar tissue hyperplasia but also the inflammatory response after tissue injury. We hope to validate these effects of D3 in a future, in vivo, filtration surgery animal model, which will allow us to further study the effect of different mode of administration (subconjunctival injection or topical application), frequency of administration (intraoperative or perioperative), and its effect on adjacent tissues (for example, corneal and conjunctival epithelium). 
Conclusions
This study showed for the first time that D3 appears capable of suppressing HTF proliferation, migration, and transdifferentiation, and these effects are mediated by its ability to promote the expression of the CYP24A1, KRT16, and SHE genes, while suppressing CILP gene expression. Clinically, D3 may have an important role in preventing subconjunctival fibrosis after surgery involving conjunctiva and Tenon's capsule and possibly improving their postoperative outcome, especially for lowering recurrences after pterygium excision and maintaining aqueous filtration after trabeculectomies. 
Acknowledgments
Supported by Seed Fund for Basic Research, University Research Committee, the University of Hong Kong. 
Disclosure: S. Jia, None; F. Chen, None; H. Wang, None; G. Kesavamoorthy, None; J.S.M. Lai, None; I.Y.H. Wong, None; K. Chiu, None; J.C.H. Chan, None 
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Figure 1.
 
Experiment flow chart.
Figure 1.
 
Experiment flow chart.
Figure 2.
 
Primary culture cell verification. HTFs stained with Vimentin, collagen, fibroblast surface protein, α-smooth muscle actin and DAPI. (A) Pterygium HTF. (B) Normal HTF. (C) Glaucoma HTF (N = 3, n = 3). Scale bar: 25 µm
Figure 2.
 
Primary culture cell verification. HTFs stained with Vimentin, collagen, fibroblast surface protein, α-smooth muscle actin and DAPI. (A) Pterygium HTF. (B) Normal HTF. (C) Glaucoma HTF (N = 3, n = 3). Scale bar: 25 µm
Figure 3.
 
Cytotoxicity and cell viability evaluation on pterygium HTF using the LDH and PrestoBlue assays, respectively, to compare (A) the Log 10 value of different concentrations of D3 (0.001 µM to 100 µM) with (B) mitomycin-C (75 µM to 598 µM) (N = 1, n = 3); effect of D3 (5 µM to 50 µM) on (C) pterygium HTF cytotoxicity and (D) glaucoma HTF cytotoxicity (N = 3 , n = 3); and effect of D3 (5 µM to 50 µM) on (E) pterygium HTF viability and (F) glaucoma HTF viability (N = 3 , n = 3). The arrows indicate clinically used MMC concentration of 0.1 and 0.2 mg/mL, equivalent to 299 and 598 µM. (**P ≤ 0.05; ****P ≤ 0.001)
Figure 3.
 
Cytotoxicity and cell viability evaluation on pterygium HTF using the LDH and PrestoBlue assays, respectively, to compare (A) the Log 10 value of different concentrations of D3 (0.001 µM to 100 µM) with (B) mitomycin-C (75 µM to 598 µM) (N = 1, n = 3); effect of D3 (5 µM to 50 µM) on (C) pterygium HTF cytotoxicity and (D) glaucoma HTF cytotoxicity (N = 3 , n = 3); and effect of D3 (5 µM to 50 µM) on (E) pterygium HTF viability and (F) glaucoma HTF viability (N = 3 , n = 3). The arrows indicate clinically used MMC concentration of 0.1 and 0.2 mg/mL, equivalent to 299 and 598 µM. (**P ≤ 0.05; ****P ≤ 0.001)
Figure 4.
 
Inhibition of proliferation in (A) pterygium HTF and (B) glaucoma HTF seeded at 10,000 cells/well in a 96-well plate and grown for 24 hours in presence of ethanol or increasing concentrations of D3. Relative proliferation was measured by fluorescence determination of DNA content using the CyQuant Proliferation assay (N = 3, n = 3). Representative images of D3-induced inhibition of wound healing in (C) pterygium HTF and (D) glaucoma HTF as compared to the EMEM, ethanol (vehicle control), and MMC (positive control, 598 µM) at 0, 12, and 24 hours after scratch (Scale bar: 100 µm). Cell migration was evaluated quantitatively in (E) pterygium cases (N = 3, n = 4) and (F) glaucoma cases (N = 3, n = 3) respectively, between 10 µM D3-treated HTF and the vehicle control HTF at 0 and 24 hours by calculating relative wound closure percentage. Data are presented as the means ± SEM (***P < 0.001; ****P < 0.0001, compared with vehicle control group). (G) The expression of α-smooth muscle actin (α-SMA) was evaluated using Western blots.
Figure 4.
 
Inhibition of proliferation in (A) pterygium HTF and (B) glaucoma HTF seeded at 10,000 cells/well in a 96-well plate and grown for 24 hours in presence of ethanol or increasing concentrations of D3. Relative proliferation was measured by fluorescence determination of DNA content using the CyQuant Proliferation assay (N = 3, n = 3). Representative images of D3-induced inhibition of wound healing in (C) pterygium HTF and (D) glaucoma HTF as compared to the EMEM, ethanol (vehicle control), and MMC (positive control, 598 µM) at 0, 12, and 24 hours after scratch (Scale bar: 100 µm). Cell migration was evaluated quantitatively in (E) pterygium cases (N = 3, n = 4) and (F) glaucoma cases (N = 3, n = 3) respectively, between 10 µM D3-treated HTF and the vehicle control HTF at 0 and 24 hours by calculating relative wound closure percentage. Data are presented as the means ± SEM (***P < 0.001; ****P < 0.0001, compared with vehicle control group). (G) The expression of α-smooth muscle actin (α-SMA) was evaluated using Western blots.
Figure 5.
 
(A) Hierarchical clustering of all DEGs between the normal and the pterygium. (B) Hierarchical clustering of all DEGs between the normal and the glaucoma. Expression values are Z score transformed. Samples were clustered using complete linkage and Euclidean distance. (C) Venn diagram of DEGs that both changed (P < 0.05) in the pterygium and glaucoma in comparison to that of the normal.
Figure 5.
 
(A) Hierarchical clustering of all DEGs between the normal and the pterygium. (B) Hierarchical clustering of all DEGs between the normal and the glaucoma. Expression values are Z score transformed. Samples were clustered using complete linkage and Euclidean distance. (C) Venn diagram of DEGs that both changed (P < 0.05) in the pterygium and glaucoma in comparison to that of the normal.
Figure 6.
 
(A) Principal-component analysis (PCA) plot of n = 4251 genes expressed in the D3-treated group (n = 3) and the vehicle group (veh) (n = 4). (B) Hierarchical clustering of all DEGs between the D3 and the vehicle groups. Expression values are Z score transformed. Samples were clustered using complete linkage and Euclidean distance. (C) Volcano plots for DEGs between the D3-treated and the vehicle groups. As observed in the figure, the upregulated and downregulated genes were marked as a dot, those significantly up-regulated genes were highlighted in red, the down-regulated genes were highlighted in blue, and the nonsignificant genes were labeled as gray dots. In the figure, the gray lines indicate the marginal lines separating DEGs from non-DEGs, with the horizontal lines denoting the P value threshold (P ≤ 0.05). The top 10 upregulated and downregulated genes were showed in graph: pterygium (D) and glaucoma (E) treated with 10 µM D3 or vehicle for 24 hours. Then mRNA expressions of CYP24A1, KRT16 in pterygium and glaucoma were measured by qPCR and normalized to GAPDH (n = 3). The vehicle and D3-treated groups consist of primary cultured pterygium or glaucoma HTF that are not treated or treated with specific concentration of D3, respectively. All bar graphs are expressed as mean ± SEM (***P < 0.001; ****P < 0.0001). (F) GO analysis of DEGs between the D3-treated and the vehicle groups. The graph displays the classification term enrichment status and term hierarchy. The color scale shows the P value cutoff levels for each biological process. (G) KEGG analysis of DEGs between the D3-treated and the vehicle groups. The color scale shows the P value, and the dot size shows the related DEGs.
Figure 6.
 
(A) Principal-component analysis (PCA) plot of n = 4251 genes expressed in the D3-treated group (n = 3) and the vehicle group (veh) (n = 4). (B) Hierarchical clustering of all DEGs between the D3 and the vehicle groups. Expression values are Z score transformed. Samples were clustered using complete linkage and Euclidean distance. (C) Volcano plots for DEGs between the D3-treated and the vehicle groups. As observed in the figure, the upregulated and downregulated genes were marked as a dot, those significantly up-regulated genes were highlighted in red, the down-regulated genes were highlighted in blue, and the nonsignificant genes were labeled as gray dots. In the figure, the gray lines indicate the marginal lines separating DEGs from non-DEGs, with the horizontal lines denoting the P value threshold (P ≤ 0.05). The top 10 upregulated and downregulated genes were showed in graph: pterygium (D) and glaucoma (E) treated with 10 µM D3 or vehicle for 24 hours. Then mRNA expressions of CYP24A1, KRT16 in pterygium and glaucoma were measured by qPCR and normalized to GAPDH (n = 3). The vehicle and D3-treated groups consist of primary cultured pterygium or glaucoma HTF that are not treated or treated with specific concentration of D3, respectively. All bar graphs are expressed as mean ± SEM (***P < 0.001; ****P < 0.0001). (F) GO analysis of DEGs between the D3-treated and the vehicle groups. The graph displays the classification term enrichment status and term hierarchy. The color scale shows the P value cutoff levels for each biological process. (G) KEGG analysis of DEGs between the D3-treated and the vehicle groups. The color scale shows the P value, and the dot size shows the related DEGs.
Figure 7.
 
(A) Venn diagram showing DEGs that are both changed in vitamin D3 (vD3)– and MMC-treated, compared to normal, tissues. (B) Pterygium HTF were treated with 10 µM vD3 or vehicle for 24 hours, then mRNA expression of SHE, CILP were measured by qPCR and normalize to GAPDH (n = 3). All bar graphs are expressed as mean ± SEM. (*P < 0.5; ***P < 0.001; ****P < 0.0001).
Figure 7.
 
(A) Venn diagram showing DEGs that are both changed in vitamin D3 (vD3)– and MMC-treated, compared to normal, tissues. (B) Pterygium HTF were treated with 10 µM vD3 or vehicle for 24 hours, then mRNA expression of SHE, CILP were measured by qPCR and normalize to GAPDH (n = 3). All bar graphs are expressed as mean ± SEM. (*P < 0.5; ***P < 0.001; ****P < 0.0001).
Table 1.
 
Information of Sources for Tenon Tissues Used in This Study
Table 1.
 
Information of Sources for Tenon Tissues Used in This Study
Table 2.
 
Monoclonal and Polyclonal Antibodies Used in This Study
Table 2.
 
Monoclonal and Polyclonal Antibodies Used in This Study
Table 3.
 
Top 20 DEGs of HTFs Versus Normal
Table 3.
 
Top 20 DEGs of HTFs Versus Normal
Table 4.
 
Top 20 DEGs for MMC and VD3 Versus Vehicle
Table 4.
 
Top 20 DEGs for MMC and VD3 Versus Vehicle
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