Transplantation of human corneal limbal epithelial cell sheet harvested on synthesized carboxymethyl cellulose and dopamine in a limbal stem cell deficiency.

This study aimed to evaluate the efficacy and safety of transplantation with human corneal limbal epithelial (HCLE) cell sheets cultured on carboxymethyl cellulose (CMC)-dopamine (DA)-coated substrates and harvested via enzymatic digestion of CMC with cellulase in a rabbit animal model of limbal stem cell deficiency (LSCD). Synthesized CMC-DA was pretreated onto the surface of culture plates. Then, HCLE cells were cultured on precoated CMC-DA and HCLE cell sheets were harvested using cellulase-containing cell culture medium. HCLE cell sheets were evaluated using a live/dead assay, histological examination, and immunofluorescence staining. For in vivo assessment, HCLE cell sheets were transplanted in a rabbit model of LSCD for 2 weeks to determine the effectiveness of the repair. Primary culture of HCLE cells stained positively for p63, cytokeratin (CK)15, and CK12. HCLE cell sheets were generated with a well-preserved morphology and transparency ranging in size from 15 to 19 mm after cellulase-assisted cell sheet generation. HCLE cell sheets uniformly stained positively for human mitochondria, p63, CK15, CK12, CK3/2p, and zonula occludens (ZO)-1. HCLE cell sheet transplantation in a rabbit model of LSCD improved the corneal opacity and neovascularization scores. Transplanted HCLE cell sheets stained positively for p63 and CK12. Transplantation of HCLE cell sheets harvested on CMC-DA coating combined with cellulase is a safe and efficient procedure for corneal epithelial regeneration in a rabbit model of LSCD. This system could enable a promising strategy to regenerate corneal epithelium by transplantation in ocular surface disorders.

Correction: Safety and efficacy of tacrolimus-coated silicone plates as an alternative to mitomycin C in a rabbit model of conjunctival fibrosis.

[This corrects the article DOI: 10.1371/journal.pone.0219194.].

Safety and efficacy of tacrolimus-coated silicone plates as an alternative to mitomycin C in a rabbit model of conjunctival fibrosis.

PURPOSE: To find safer and more effective drugs than mitomycin C to prevent conjunctival fibrosis in a rabbit model. METHODS: Twenty-four rabbits were involved and randomly divided into four groups. Limbus-based peritomy was performed at the superior cornea, and normal saline (NS group), mitomycin C (MMC group), SR (SR group), or TC (TC group)-coated silicone plate was inserted at the sub-Tenon's space in each group. Conjunctival congestion was evaluated at 1 and 4 weeks postoperatively. At 4 weeks, the numbers of inflammatory cells, fibroblasts, myofibroblasts, blood vessels, and goblet cells were counted in the conjunctiva and Tenon's capsule around the silicone plate. RESULTS: At 4 weeks, conjunctival congestion was significantly less than that observed at 1 week in the SR and TC groups (p < 0.05), whereas the number of myofibroblasts was significantly lower in the MMC and TC groups (p < 0.05). The conjunctiva was significantly less congested in the TC group versus the other groups at 1 week and 4 weeks (p < 0.05). The TC group had the lowest number of inflammatory cells and MMC group had the lowest number of goblet cells among all groups (p < 0.05). CONCLUSIONS: The TC-coated silicone plate was more effective in inhibiting inflammation and fibrosis versus the MMC-coated silicone plate and was associated with fewer adverse effects in the rabbit model.

Human Conjunctival Epithelial Sheets Grown on Poly(Lactic-Co-Glycolic) Acid Membranes and Cocultured With Human Tenon's Fibroblasts for Corneal Repair.

Purpose: We determine the feasibility of human conjunctival epithelial cells (hCjECs) on poly(lactic-co-glycolic) acid (PLGA) membranes for corneal epithelium regeneration. hCjECs on PLGA or polyester (PET) membranes with or without coculture of human Tenon's fibroblasts (hTFs) were compared in vitro, and to determine whether epithelial sheets grown on PLGA membranes can repair injured rabbit corneal epithelium by transplantation for 2 weeks in vivo. Methods: Primary hCjECs were cultured on PLGA or the original PET membrane-based transwell inserts with or without coculture of hTFs on the floor of the culture plate. Cell behaviors, such as proliferation and differentiation, were compared. For in vivo assessment, the corneas of rabbits were burned, and PLGA-based epithelial sheets then were transplanted for 2 weeks before histologic staining was conducted and analyzed to determine the effectiveness of the repair. Results: Primary human epithelial cells on the PLGA membrane showed an increased proliferation when cocultured with fibroblasts, which was confirmed by CCK-8 analysis, and upregulation of Ki67, with the expression of the epithelial marker CK19. The stratified squamous cell marker MUC1 and conjunctival cell marker MUC5AC also were expressed in the epithelial sheet. The epithelial defect in the burned corneas was decreased in the PLGA-based epithelial sheet treatment group (6.1% ± 1.6% of the area) compared to that in the no-treatment group (30.5% ± 6.3%) 2 weeks postoperatively. Conclusions: We developed a coculture system using a human feeder cell layer and PLGA membrane-based transwell inserts to create human conjunctival epithelial sheets. This system represents a promising strategy to regenerate corneal epithelium by transplantation.

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing.

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Attenuation of corneal neovascularization by topical low-molecular-weight heparin-taurocholate 7 without bleeding complication.

AIM: To investigate the antiangiogenic effects and safety of topically administered low-molecular-weight heparin-taurocholate 7 (LHT7) on corneal neovascularization (CoNV). METHODS: Twenty-four Sprague-Dawley rats were randomly distributed into four groups of six rats each. The central corneas were cauterized using a silver/potassium nitrate solution. From 2d after cauterization, 12.5 mg/mL (low LHT7 group) or 25 mg/mL (high LHT7 group) LHT7 was topically administered three times daily; 12.5 mg/mL bevacizumab was topically administered as positive control (bevacizumab) group, with normal saline (NS) administered as negative control (NS group). The corneas were digitally photographed to calculate the CoNV percentage from the neovascularized corneal area at 1 and 2wk. RESULTS: The 4 study groups did not have different CoNV percentages at 1wk after injury (P>0.05). However, the low LHT, high LHT, and bevacizumab groups had significantly lower CoNV percentages than the NS group at 2wk (all P<0.05). No significant differences in CoNV percentage were found among the low LHT, high LHT, and bevacizumab groups (all P>0.05). All groups except the NS group had lower CoNV percentages at 2wk post-injury than the levels observed at 1wk (all P<0.05). CONCLUSION: Topically-administered LHT7 inhibited CoNV without complication after chemical cauterization in the rat.

Effect of nerve growth factor on the in vitro induction of apoptosis of human conjunctival epithelial cells by hyperosmolar stress.

PURPOSE: To evaluate the effects of nerve growth factor (NGF), which is activated during inflammatory episodes of ocular diseases, on the apoptotic response in cultured human primary conjunctival epithelial cells (pHCECs). METHODS: Levels of NGF transcripts and NGF protein in pHCEC grown in medium with normal osmolarity (307 mOsm/L) or hyperosmolar medium (350, 400, and 450 mOsm/L) were determined using RT-PCR or ELISA, respectively. To assess apoptosis, pHCEC were cultured in normal or 400 mOsm/L hyperosmolar medium with neutralizing anti-NGF antibody or recombinant human NGF for 6 hours before analysis by flow cytometry. Levels of Bcl-xL, Bax, phosphorylated JNK, and cleaved caspase-3 were detected using Western blotting. Levels of the inflammatory cytokine IL-6 was analyzed using ELISA. RESULTS: Hyperosmolar conditions increased NGF levels in cultured pHCECs. Hyperosmolarity and exposure to neutralizing anti-NGF antibody significantly increased the number of apoptotic cells. Addition of recombinant human NGF to 400 mOsm/L medium decreased the number of apoptotic cells, reduced the expression of phosphorylated JNK, Bax, and cleaved caspase-3 and increased the expression of Bcl-xL. Levels of IL-6 were increased by hyperosmotic stress but decreased by exposure to recombinant human NGF. CONCLUSIONS: Hyperosmolarity induces apoptosis of pHCECs by activating JNK signaling. Increased levels of NGF under hyperosmolar conditions may contribute, at least in part, to the reduced apoptosis of pHCECs and may be beneficial in recovering conjunctival damage due to chronic hyperosmolar stress.

Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing.

We reported the expression of phosphorylated HSP27 during epithelial wound healing in murine corneas (Jain et al., 2012) in July of 2012. This in vivo investigation demonstrated that the expression levels of phosphorylated HSP27 were greater in wounded corneal epithelial cells than in unwounded controls and that the localization of phosphorylated HSP27 was in the basal and superficial epithelia three days following corneal epithelial wounding. We suggested that phosphorylated HSP27 had a role in the early phase of corneal epithelial wound healing. The purpose of this study was to investigate the exact role of heat shock protein 27 (HSP27) phosphorylation for the wound healing of cultured human corneal epithelial cells (HCECs). HSP27-specific siRNAs and control-siRNAs, with no known homologous targets in HCECs, were created. The cultured HCECs were divided into two groups: Scrambled control-siRNA-transfected group vs. HSP27-specific siRNA-transfected group. The scratch-induced directional wounding assay, Western blotting, using antibodies against non-phosphorylated and phosphorylated HSP27, non-phosphorylated and phosphorylated Akt, and Bcl-2-associated X protein (Bax), immunofluorescence staining to determine the filament actin, flow cytometry to measure apoptosis, and proliferation assay were performed to determine the role of HSP27. Western blot assay showed that the expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding, compared with those in unwounded HCECs (all p < 0.05). Western blot assay also showed HSP27-specific siRNAs effectively blocked the expression of non-phosphorylated HSP27. The HSP27-specific siRNA-transfected group had more Bax expression, less phosphorylated Akt expression, and less non-phosphorylated and phosphorylated HSP27 expression (all p < 0.05). The scratch-induced directional wounding assay showed the HSP27-specific siRNA-transfected group with a less migrating cell number than the control-siRNA-transfected group (p < 0.05). Immunofluorescence staining showed that reorganization of actin cytoskeleton prominently decreased in the HSP27-specific siRNA-transfected group, compared with the control siRNA-tranfected group. Flow cytometry revealed that the HSP27-specific siRNA-transfected group had more HCEC apoptosis. Proliferation assay showed no difference between the two groups. In conclusion, the role of HSP27 in corneal epithelial wound healing can be epithelial cell apoptosis, as well as epithelial migration. HSP27 is involved in HCEC migration by the reorganization of actin cytoskeleton.

Topically administered bevacizumab had longer standing anti-angiogenic effect than subconjunctivally injected bevacizumab in rat corneal neovacularization.

AIM: To compare the effect of topically administered and subconjunctivally injected bevacizumab on experimental corneal neovascularization in rats for two weeks after treatment. METHODS: Twenty-eight Sprague-Dawley rats were divided into four groups of 7 animals. Each corneal center of right eye was cauterized with silver/potassium nitrate for 8s. After corneal burning, bevacizumab (12.5mg/mL) was topically administered three times per day (TB group) for two weeks or subconjunctivally injected on days 2 and 4 after cauterization (0.02mL; SB group). As negative controls, rats received 0.9% saline topically three times per day (TS group) or subconjunctivally on days 2 and 4 (0.02mL; SS group). Digital photographs of the cornea were taken 1 and 2 weeks after treatment and analyzed to determine the area of cornea covered by neovascularization as the percentage of corneal neovascularization. RESULTS: One week after treatment, the percentage of corneal neovascularization was significantly lower in the TB and SB groups than in the TS and SS groups (all P<0.05). Two weeks after treatment, the percentage of corneal neovascularization was significantly lower in the TB group than in the TS group (P<0.05). In all groups, the percentage of neovascularization was decreasing as time passed (all P<0.05). CONCLUSION: Topically administered bevacizumab has longer standing anti-angiogenic effect than subconjunctivally injected bevacizumab in corneal neovascularization following chemical injury in rats.

Subconjunctival injection of low-molecular-weight heparin-taurocholate 7 inhibits corneal neovascularization.

PURPOSE: To evaluate the effects of a subconjunctival injection of low-molecular-weight heparin-taurocholate 7 (LHT7) on corneal neovascularization (CoNV) in rats. METHODS: Twenty-four Sprague-Dawley rats were divided into 4 groups of 6 animals each. Corneal centers were cauterized by the application of a silver/potassium nitrate solution for 8 seconds. Either 0.02 or 0.04 mL of 25 mg/mL of LHT7 (low- and high-LHT7 groups, respectively) was subconjunctivally injected on days 2 and 4 after the cauterization was done; 0.02 mL of 25 mg/mL of bevacizumab was injected into rats in the positive control group, with normal saline (NS) being administered to a negative control group. Digital photographs of the cornea were taken 1 and 2 weeks later to calculate the percentage of CoNV using the area of the neovascularized cornea. To compare the differences in CoNV between weeks 1 and 2, the change in CoNV was calculated by subtracting the percentage of CoNV at 1 week from that at 2 weeks. RESULTS: The percentage of CoNV did not differ among the 4 groups either 1 or 2 weeks after the cauterization (P > 0.05). In all groups except the NS group, the percentage of CoNV significantly decreased at 2 weeks compared with that at 1 week (all P < 0.05). Moreover, the changes of CoNV in the high-LHT7 and bevacizumab groups significantly decreased compared with that in the NS group (all P < 0.05). Two corneal stromal hemorrhages occurred, 1 in each LHT7 group. CONCLUSIONS: Despite complications, including corneal stromal hemorrhage, subconjunctival injection of LHT7 attenuated CoNV after chemical cauterization.

Lower energy to make a corneal flap with a 60 kHz femtosecond laser reduces flap inflammation and corneal stromal cell death but weakens flap adhesion.

PURPOSE: To compare corneal flaps created in rabbits with a 60 kHz femtosecond (FS) laser using different levels of raster energy and to measure early inflammation, corneal stromal cell death, and late postoperative adhesion strength. METHODS: Sixty rabbits were divided into three groups of 20 each. A flap 110 µm thick and 9.0 mm in diameter was made in one eye of each rabbit at raster energies of 0.7 µJ, 1.1 µJ, and 2.4 µJ. Histopathological evaluation for inflammation and apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed at 4 and 24 hours after flap creation. The adhesion strength of the flaps was measured with a tension meter at 1 and 3 months. RESULTS: Twenty four hours after flap creation, the 2.4 µJ group had more inflammatory and CD11b-positive cells than the 0.7 and 1.1 µJ groups. The number of TUNEL-positive cells increased with raster energy at 4 and 24 hours. The grams of force (gf) needed to detach the flaps at 3 months was significantly higher in 2.4 µJ group (170 gf) than in 0.7 µJ group (97.5 gf) and 1.1 µJ group (100 gf, p = 0.03). CONCLUSIONS: Using raster energy lower than 1.1 µJ to make a flap with a 60 kHz FS laser decreases inflammatory cell infiltration and corneal stromal cell death in the central cornea but may result in a weaker flap than using higher raster energy (2.4 µJ).

Bevacizumab and rapamycin can decrease corneal opacity and apoptotic keratocyte number following photorefractive keratectomy.

PURPOSE: We investigated the effects of bevacizumab and rapamycin on central corneal opacity and apoptotic keratocyte number after photorefractive keratectomy (PRK) followed by ultraviolet B (UV-B) irradiation. METHODS: A total of 60 right eyes of Sprague-Dawley rats in four groups (n = 15 each) underwent PRK ablation to 80 µm with a 3-mm zone. Sponges soaked with 0.02% mitomycin C (MMC), 2.5% bevacizumab, 0.01% rapamycin, and balanced saline solution were applied for 2 minutes to these eyes in the MMC, bevacizumab, rapamycin, and control groups, respectively. At 3 weeks after PRK, all right eyes were exposed to 100 mJ/cm(2) UV-B irradiation. Biomicroscopy was used to determine the amount of haze, and TUNEL staining for apoptosis and histology were performed at 3, 6, and 12 weeks. RESULTS: Contrary to the results at 3 weeks, central corneal haze, and apoptotic keratocyte and keratocyte number decreased significantly in the MMC, bevacizumab, and rapamycin groups compared to the control group, and the keratocyte number was lower in the MMC group than the bevacizumab and rapamycin groups at 6 weeks (all P < 0.05). At 12 weeks, the apoptotic keratocyte number was lower in the MMC, bevacizumab, and rapamycin groups than the control group, and the keratocyte number was significantly lower in the MMC than the rapamycin and control groups (all P < 0.05). CONCLUSIONS: Intraoperative bevacizumab and rapamycin administration decreases central corneal haze and apoptotic keratocyte number after PRK. Bevacizumab and rapamycin may be safe alternatives to MMC during refractive surgery to prevent postoperative corneal opacity less affecting the keratocyte number.

TC1 (C8orf4) enhances the Wnt/beta-catenin pathway by relieving antagonistic activity of Chibby.

The Wnt/beta-catenin pathway has been implicated in human cancers. Here, we show that TC1 (C8orf4), a small protein present in vertebrates, functions as a positive regulator of the pathway. TC1 interacts with Chibby (Cby) and thereby enhances the signaling pathway by relieving the antagonistic function of Cby on the beta-catenin-mediated transcription. Upon coexpression in mammalian cells, TC1 redistributes from nucleolus to nuclear speckles, where it colocalizes with Cby. TC1 up-regulates the expression of beta-catenin target genes that are implicated in invasiveness and aggressive behavior of cancers, such as metalloproteinases, laminin gamma2, and others. Our data indicate that TC1 is a novel upstream regulator of the Wnt/beta-catenin pathway that enhances aggressive behavior of cancers.