Corneal Epithelial Proliferation Research Articles

The Role of SLIT3-ROBO4 Signaling in Endoplasmic Reticulum Stress-Induced Delayed Corneal Epithelial and Nerve Regeneration.

In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration. Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells. Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment. ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.

The therapeutic potential of chondroitin sulfate in Aspergillus fumigatus keratitis

PurposeTo explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsThe nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators. ResultsCS at 400 μg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1β, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells. ConclusionCS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.

Current and future therapies for persistent corneal epithelial defects and neurotrophic keratopathy.

The corneal epithelium is a crucial barrier against pathogens, and when disrupted in the setting of certain underlying risk factors such as neurotrophic keratopathy (NK), may result in persistent epithelial defects (PEDs) of the cornea. Management is challenging and may require a variety of different approaches ranging from conservative medical therapy to surgical intervention. The purpose of this review is to provide an update on current and potential future therapeutic options for PEDs and NK. Recent research has yielded promising results for numerous novel therapies aimed at treating PEDs. Many of these attempt to stimulate healing at the cellular level, via signaling of corneal epithelial differentiation, migration, and proliferation. Considerable advances have also been made regarding medical and surgical promotion of corneal re-innervation and restoration of corneal sensitivity to directly address the underlying NK condition. Together with the current well established therapeutic options available for PEDs and NK, growing research on newer alternatives suggest increasing potential for both more effective and more convenient therapies for these difficult situations.

Vitamin D receptor and metabolite effects on corneal epithelial cell gap junction proteins

Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)2D3 and 24R,25(OH)2D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR). The purpose of this study was to specifically determine the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR-/-) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)2D3 or 24R,25(OH)2D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca++ (Ca++i) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)2D3 and 24R,25(OH)2D3. Cx30 and Cx43 protein levels were also significantly increased in VDR-/- MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)2D3 and 1,25(OH)2D3. Ca++i was not affected by 1,25(OH)2D3 or 24R,25(OH)2D3 in HCEC or MPCEC. We conclude that both 1,25(OH)2D3 and 24R,25(OH)2D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Ca++i.

Crab cuticle membrane application for treatment of corneal lamellar laceration in rats: a preliminary study

Corneal lamellar laceration defined as a partial thickness injury to the cornea that may affect vision in various degree. Prompt management is needed to maintain integrity of the cornea and to avoid possible complications resulting from the injury. Material such as amniotic membrane had been widely use to promote reepithelization after corneal injury. Result from previous studies revealed that chitin, the most abundant substance in the cuticle layer of crab shell, is contribute to reepithelization after injury. Crab cuticle membrane preparation were made similar to amniotic membrane that is widely use in the management of corneal injury, therefore chitin might be sought as alternative material in the management of corneal injury which promote reepithelization. This study aimed to assess corneal epithelial proliferation profile and the number of keratocyte in rats after corneal lamellar laceration injury with and without application of crab cuticle membrane. Rats with corneal lamellar laceration injury were divided into groups treated with and without crab cuticle membrane application. Eyes were enucleated on the first and third days after treatment. Histologic preparations were made to assess corneal epithelial proliferation process and number of keratocyte in all groups. Cellular migration of more than 50 % were found in 66.7% of samples on 1 day after treatment and 61.1% of samples on the third days after treatment with crab cuticle membrane. The average number of keratocyte were 33.89 ± 5.27 cells/LPF on 1 day after treatment and 46.22 ± 9.55 cells/LPF on the third days after treatment with crab cuticle membrane. Epithelial proliferation after corneal lamellar laceration in rats treated with crab cuticle membrane application is faster and the number of keratocyte is more abundant compared to treatment without the application of crab cuticle membrane.

Effects of HGF and KGF gene silencing on vascular endothelial growth factor and its receptors in rat ultraviolet radiation‑induced corneal neovascularization.

Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), two paracrine growth factors, modulate corneal epithelial cell metabolism, apoptosis and survival. Vascular endothelial growth factor (VEGF) serves as a proangiogenic factor in corneal neovascularization (CNV), which is a major cause of vision impairment and corneal blindness. The aim of the present study was to evaluate the ability of HGF and KGF to influence VEGF and its receptor, kinase insert domain receptor (Flk‑1) in corneal injury and CNV in rats induced by ultraviolet radiation (UVR). An UVR‑induced corneal injury rat model was successfully established to characterize the expression patterns of KGF, HGF, VEGF and Flk‑1 in corneal tissues. Corneal epithelial cells were extracted and treated with small interfering RNAs (siRNAs) targeting KGF, HGF or both (si‑KGF, si‑HGF or si‑HGF/KGF). The effects of HGF and KGF were examined through detection of the expression of KGF, HGF, VEGF and Flk‑1, and the evaluation of cell proliferation, cell cycle and cell apoptosis. The expression levels of KGF, HGF, VEGF and Flk‑1 in corneal tissues were increased in the rat model. In the cell experiments, the transfection of si‑HGF/KGF resulted in reductions in VEGF, Flk‑1, KGF and HGF. In addition, decreased cell proliferation and elevated cell apoptosis were found in the corneal epithelial cells from the rat model following KGF and HGF gene silencing. Taken together, these findings suggest that HGF and KGF gene silencing inhibits UVR‑induced corneal epithelial proliferation and CNV and may function as novel targets for corneal wound healing.

Aberrant expression of a stabilized \u03b2-catenin mutant in keratocytes inhibits mouse corneal epithelial stratification

We previously reported that genetic deletion of β-catenin in mouse corneal keratocytes resulted in precocious corneal epithelial stratification. In this study, to strengthen the notion that corneal keratocyte-derived Wnt/β-catenin signaling regulates corneal epithelial stratification during mouse development, we examined the consequence of conditional overexpression of a stabilized β-catenin mutant (Ctnnb1ΔE3) in corneal keratocytes via a doxycycline (Dox)-inducible compound transgenic mouse strain. Histological analysis showed that conditional overexpression of Ctnnb1ΔE3 in keratocytes inhibited corneal epithelial stratification during postnatal development. Unlike the corneal epithelium of the littermate controls, which consisted of 5-6 cell layers at postnatal day 21 (P21), the mutant corneal epithelium contained 1-2 or 2-3 cell layers after Dox induction from embryonic day 0 (E0) to P21 and from E9 to P21, respectively. X-gal staining revealed that Wnt/β-catenin signaling activity was significantly elevated in the corneal keratocytes of the Dox-induced mutant mice, compared to the littermate controls. Furthermore, RT-qPCR and immunostaining data indicated that the expression of Bmp4 and ΔNp63 was downregulated in the mutant corneas, which was associated with reduced corneal epithelial proliferation in mutant epithelium, as revealed by immunofluorescent staining. However, the expression of Krt12, Krt14 and Pax6 in the mutant corneas was not altered after overexpression of Ctnnb1ΔE3 mutant protein in corneal keratocytes. Overall, mutant β-catenin accumulation in the corneal keratocytes inhibited corneal epithelial stratification probably through downregulation of Bmp4 and ΔNp63 in the corneal epithelium.

Microplasma Jet Arrays as a Therapeutic Choice for Fungal Keratitis

The clinical impact of microplasma jets on rabbit eyes infected by Candida albicans has been investigated. Arrays of such jets produce low-temperature plasma micro-columns suitable for ophthalmic therapeutics and fungal infections, in particular, and the technology is capable of being scaled to surface areas of at least 10 cm2. Keratitis was induced in the right central corneas of rabbits, whereas the left eyes served as a normal group. The rabbits were divided into the plasma non-treated group (control) and plasma treatment group. Histologic analyses of both groups showed marked reductions in the thickness, angiogenesis, and opacity of all rabbit corneas following plasma treatment. Indeed, for treatment times beyond 14 days, infected eyes exhibited no significant differences from the normal group. Healing of rabbit eyes infected by Candida albicans apparently proceeds by disrupting corneal epithelial proliferation, and by reducing fibrotic changes in the stroma. This study demonstrates that low-temperature plasma jets are remarkably effective in healing Candida albicans-infected corneas, thereby providing a promising medical treatment option for keratitis.

The corneal nerves in glaucoma and ocular surface diseases

SummaryIn addition to their important sensory function, corneal nerves provide protective and trophic functions and also regulate corneal epithelial integrity, proliferation and wound healing. The well‐defined in vivo confocal microscopy (IVCM) appearance of the subbasal corneal nerves has facilitated their quantitative in vivo analysis in ocular surface diseases. Although patients with dry eyes and patients treated for glaucoma experience similar ocular surface signs and symptoms, different correlations between corneal sensation and nerve morphology were observed in these two groups of patients. The relationship between corneal nerve function and structure remains complex, and nerve alteration and/or dysfunction could be different among different ocular surface diseases. Improvements in IVCM image acquisition and analysis will be an important step to further evaluate the role of corneal nerves in physiological and pathological ocular surface conditions.

Combinatorial protective effects of α-melanocyte-stimulating hormone and carboxymethylcellulose on ocular surface in dry eye rat models

Background Reasearches showed that α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation and ameliorates the ocular surface abnormalities in a scopolamine-induced dry eye rat model, and the managing effect of sodium carboxymethylcellulose (CMC) on dry eyes also has been determined.However, whether α-MSH can enhance the therapeutic effects of CMC remains to be investigated. Objective This study was to investigate the protective effects of α-MSH combined with CMC on ocular surface in a scopolamine-induced dry eye rat model. Methods Sixty clean female Wistar rats were randomly divided into normal control group, model control group, NaCl group, CMC group, α-MSH group and α-MSH+ CMC group, and 10 rats for each group.The dry eye models were established by subcutaneous injection of scopolamine hydrobromide at 9: 00, 12: 00, 15: 00 and 18: 00 per day for 28 days.0.9% NaCl solution, 1×10-3 mg/ml α-MSH solution, 0.5% CMC eye drop, and 1×10-3 mg/ml α-MSH+ 0.5% CMC solution were topically administered twice a day (8: 00, 17: 00) since the initial day of modeling according to grouping.Shirmer Ⅰ test (SⅠt), breakup time of tear film (BUT) and corneal fluorescence staining were performed before and 7, 14, 21, 28 days after the application of drugs.At 28 days following the administration of drugs, the eyeballs of the rats were collected.Hemotoxylin and eosin staining was employed to examine the morphology of corneas, and periodic acid schiff (PAS) staining was used to count the conjunctival goblet cells.This study protocol was approved by Experimental Animal Ethic Committee of Tianjin Medical University(SYXK 2009-0001), and the use and care of the rats complied with ARVO Statement. Results The SⅠt and BUT values were significantly reduced, and the corneal fluorescence staining scores were significantly increased over time following modeling in the model control group (all at P 0.05). At 7, 14 and 21 days after intervention, the SⅠt values were (4.800±0.789), (4.100±0.516) and (4.300±0.856)mm in the α-MSH+ CMC group, which were considerably higher than (2.875±0.719), (2.375±0.619) and (2.532±0.957)mm in the NaCl group (all at P<0.01). At 7 days after intervention, the BUT values were (4.938±1.843) seconds and (5.000±1.491) seconds in the α-MSH group and α-MSH+ CMC group, which were significantly higher than (3.250±1.000) seconds in the NaCl group (both at P<0.01). The corneal fluorescence staining scores in the CMC group, α-MSH group and α-MSH+ CMC group were significantly lower than that in the NaCl group, with the lowest score in the α-MSH+ CMC group (all at P<0.05). The thickening of corneal epithelial layer, corneal edema and arrangement disorder of corneal stroma were found in the model control group and NaCl group; while slight corneal edema and epithelial cell proliferation were exhibited in the α-MSH+ CMC group by hemotoxylin and eosin staining.PAS staining showed that the number of goblet cells was much more in the CMC group, α-MSH group and α-MSH+ CMC group than that in the model control group and NaCl group (all at P<0.01). Conclusions The sole application of α-MSH or CMC alleviates ocular surface damage and morphological abnormality to certain extent, and the combination of α-MSH and CMC generates more effective protection in comparison with sole administration of α-MSH or CMC.The early application of the drugs plays an improvement role in tear secretion and tear film stability in dry eyes. Key words: Dry eyes/drug therapeutic; Ocular surface; Protection; Sodium carboxymethylcellulose; α-Melanocyte-stimulating hormone; Disease models; Rats, Wistar

Effects of interleukin-6 in promoting corneal epithelial stem/progenitor cell regeneration and accelerating corneal epithelial wound healing in diabetic mouse

Background Interleukin-6 (IL-6) is a pleiotropic cytokine involving in inflammation and wound healing.Previous report found that IL-6 increases phosphorylated STAT3 (p-STAT3) level and promotes corneal epithelial wound healing by stimulating migration.However, the essential role of IL-6 in corneal epithelial wound healing and the expression changes in diabetic mellitus remains unknown. Objective This study was to explore the roles of IL-6 in corneal epithelial proliferation and wound healing in both normal and diabetic mice. Methods Fifty-two normal C57BL/6 mice were randomized into normal control group (32 mice) and diabetic group (20 mice). Type 1 diabetic mellitus was induced by intraperitoneal injections of 50 mg/kg streptozotocin once per day for consecutive 5 days in the mice of the diabetic group.Whole corneal epithelium was scraped in all mice, and the corneal epithelial defect area was examined by fluorescein staining in 24, 48 and 72 hours after corneal epithelium removal.Recombinant mouse IL-6 or anti-IL-6 blocking antibody of 5 μl were subconjunctivally injected according to the grouping and contrasted with PBS injection group or isotype control antibody group, respectively.TKE2 cells, a mouse corneal epithelial stem/progenitor cell line, were trypsinized and incubated in the KSFM with different concentrations of IL-6 or without IL-6, and colony formation efficency (CFE) was examined by crystal violet staining.The expressions of ΔNP63 and Ki67, specific makers of stem cells, were detected by immunofluorescine technology.The expressions of ΔNP63, Ki67 and p-STAT3 proteins were assayed in the cells by Western blot, respectively.The expression of IL-6 mRNA and protein in the regenerated corneal epithelium was detected by real time quantitative PCR and ELISA.The use and care of the mice complied with the Statement of Association for Research in Vision and Ophthalmology. Results The percentage of residual corneal epithelium defect area with initial detect area was gradually shrinked over time after PBS and IL-6 injection in both normal control mice and diabetic mice, and the percentage of residual corneal epithelium defect area was significantly reduced in the IL-6 injected group compared with the PBS injected group (normal control group: Fgroup=19.982, P<0.01; Ftime =589.350, P<0.01; Diabetic group: Fgroup =25.411, P<0.01; Ftime =334.807, P<0.01). The CFE was (13.23±1.12)%, (15.87±1.30)%, (21.69±1.62)%, (25.33±1.28)% and (18.67±1.54)% in the blank control group and 10, 20, 50, 100 ng/ml IL-6-treated groups, respectively, showing a gradual increase of CFE dependent upon IL-6 concetrations (F=35.547, P<0.01). The expressions of ΔNP63, Ki67, p-STAT3 proteins in the cells were gradually increased over time after 50 ng/ml IL-6 treated for 5, 10, 15, 30 and 60 minutes, and the relative expression level of the cytokines was significnatly higher in the IL-6 cultured groups than that without IL-6 culture group (all at P<0.05). The relative expression of IL-6 mRNA in the regenerated corneal epithelilum was 0.45±0.21 and 1.00±0.16 in the diabetic group and normal control group, respectively, and compared with the normal control group, the expression of IL-6 mRNA reduced by 56% (t=3.42, P=0.03). The content of IL-6 protein in regenerated corneal epithelium of the diabetic group was (257±12) ng/μl, which was significantly lower than (323±17) ng/μl of the normal control group (t=5.60, P<0.01). Conclusions IL-6 promotes the proliferation and regeneration of corneal limbal stem cells to repair defected corneal epithelium by activating STAT3 signaling pathway in both normal and diabetic mice, while the blocking of endogenous IL-6 impairs the corneal epithelial cell activation and wound healing. Key words: Interleukin-6; Corneal injuries; Cell proliferation/drug effects; STAT3 transcription factor/metabolism; Wound healing/drug effects; Diabetes mellitus; Corneal epithelial stem/progenitor cells; Mice, inbred C57BL

Silk-Derived Protein Enhances Corneal Epithelial Migration, Adhesion, and Proliferation.

PurposeThe corneal surface is vulnerable to a myriad of traumatic insults including mechanical, chemical, and thermal injuries. The resulting trauma may render the naturally occurring regenerative properties of the cornea incapable of restoring a healthy epithelial surface, and may result in the loss of corneal transparency and vision. Healing of the corneal epithelium requires a complex cascade of biological processes that work to restore the tissue after injury. New therapeutic agents that act on the multiple steps of the corneal wound-healing process would offer a potential for improving patient outcomes. Here, a novel silk fibroin–derived protein (SDP) was studied for potential impacts on wound healing through studying an in vitro model.MethodsSolubilized SDP, produced from the Bombyx mori silkworm cocoon, was added to human corneal limbal-epithelial (hCLE) cultures to evaluate the material's effects on epithelial cell migration, proliferation, and adhesion through the use of various scratch wound assays and flow chamber studies.ResultsResults indicated that the addition of SDP to culture increased hCLE migration rate by over 50%, and produced an approximate 60% increase in cell proliferation. This resulted in a nearly 30% enhancement of in vitro scratch wound closure time. In addition, cultures treated with SDP experienced increased cell-matrix focal adhesion formation by over 95% when compared to controls.ConclusionsThe addition of SDP to culture media significantly enhanced hCLE cell sheet migration, proliferation, and attachment when compared to untreated controls, and indicates SDP's potential utility as an ophthalmic therapeutic agent.

Effects of tryptophan, kynurenine and kynurenic acid exerted on human reconstructed corneal epithelium in vitro.

Tryptophan metabolites formed along kynurenine pathway may affect cell proliferation and tissue function. This pathway presents potential sites for drug discovery. Tryptophan and its metabolites kynurenine and kynurenic acid may be involved in the physiology and pathology of the ocular surface. The purpose of this work is to investigate the effect exerted by tryptophan, kynurenine and kynurenic acid upon corneal epithelium. A SkinEthic™ HCE human reconstructed corneal epithelium model was used. WST-1 test was used to examine cell proliferation and viability, and the Griess reaction for nitric oxide determination. The levels of IL-6 and IL-10 were measured by means of ELISA assay. All analyses were carried out after the cells were exposed to tryptophan, kynurenine and kynurenic acid at concentrations of 5, 50 or 100μM for 0-24h and 24-48h. Tryptophan (100μM), kynurenine (100μM) and kynurenic acid (5-100μM) slightly increased the viability and proliferation of corneal epithelium. All of the tested compounds decreased cellular NO release. Kynurenine (50-100μM) and tryptophan (50-100μM) decreased while tryptophan (5μM) and kynurenic acid (100μM) increased the release of IL-6. All of the tested substances increased the level of IL-10 and decreased the IL-6 to IL-10 ratio. Tryptophan, kynurenine and kynurenic acid affect physiological processes in corneal epithelium and therefore may play a significant role in the physiology and pathology of the ocular surface. Our results indicate that the use of these compounds may be considered in the treatment of ocular surface diseases.

Chitosan Promoted the Corneal Epithelial Wound Healing via Activation of ERK Pathway

ABSTRACTPurpose: To investigate the mechanism of chitosan promoting corneal wound healing though evaluating its effect on extracellular signal-regulated kinases (ERK) and p38 pathway activity in a rabbit animal model.Methods: Cell proliferation and migration assay were performed 24 hours after chitosan treatment. The activity of ERK and p38 pathways was detected by using immunofluorescence and Western blotting in the presence of chitosan and an ERK inhibitor. In vivo study of epithelial debridement wounds was performed on 8 mm rabbit corneas in the presence of chitosan and an ERK pathway inhibitor.Results: Immunostaining with Ki67 and migrating assay showed that chitosan could upregulate the cell proliferation and promote the cell migration. Chitosan activated the ERK pathway in 5 min to 30 min after treatment but did not affect the p38 pathway. ERK inhibitor PD98059 can inhibit the chitosan-stimulated ERK phosphorylation. Chitosan increased the corneal epithelial wound closure in organ culture and ERK inhibition with PD98059 blocked the effect of chitosan on wound healing.Conclusions: Chitosan promoted corneal epithelial proliferation and migration during the wound healing in rabbits’ eye. Chitosan-stimulated epithelial wound healing is partially mediated through the activation of the ERK pathway but not the p38 pathway.

Cofactors of LIM Domains Associate with Estrogen Receptor α to Regulate the Expression of Noncoding RNA H19 and Corneal Epithelial Progenitor Cell Function

Cofactors of LIM domain proteins, CLIM1 and CLIM2, are widely expressed transcriptional cofactors that are recruited to gene regulatory regions by DNA-binding proteins, including LIM domain transcription factors. In the cornea, epithelium-specific expression of a dominant negative (DN) CLIM under the keratin 14 (K14) promoter causes blistering, wounding, inflammation, epithelial hyperplasia, and neovascularization followed by epithelial thinning and subsequent epidermal-like differentiation of the corneal epithelium. The defects in corneal epithelial differentiation and cell fate determination suggest that CLIM may regulate corneal progenitor cells and the transition to differentiation. Consistent with this notion, the K14-DN-Clim corneal epithelium first exhibits increased proliferation followed by fewer progenitor cells with decreased proliferative potential. In vivo ChIP-sequencing experiments with corneal epithelium show that CLIM binds to and regulates numerous genes involved in cell adhesion and proliferation, including limbally enriched genes. Intriguingly, CLIM associates primarily with non-LIM homeodomain motifs in corneal epithelial cells, including that of estrogen receptor α. Among CLIM targets is the noncoding RNA H19 whose deregulation is associated with Silver-Russell and Beckwith-Wiedemann syndromes. We demonstrate here that H19 negatively regulates corneal epithelial proliferation. In addition to cell cycle regulators, H19 affects the expression of multiple cell adhesion genes. CLIM interacts with estrogen receptor α at the H19 locus, potentially explaining the higher expression of H19 in female than male corneas. Together, our results demonstrate an important role for CLIM in regulating the proliferative potential of corneal epithelial progenitors and identify CLIM downstream target H19 as a regulator of corneal epithelial proliferation and adhesion.

ALDH3A1 Plays a Functional Role in Maintenance of Corneal Epithelial Homeostasis

Aldehyde dehydrogenase 1A1 (ALDH1A1) and ALDH3A1 are corneal crystallins. They protect inner ocular tissues from ultraviolet radiation (UVR)-induced oxidative damage through catalytic and non-catalytic mechanisms. Additionally, ALDH3A1 has been postulated to play a regulatory role in the corneal epithelium based on several studies that report an inverse association between ALDH3A1 expression and corneal cell proliferation. The underlying molecular mechanisms and the physiological significance of such association remain poorly understood. In the current study, we established Tet-On human corneal epithelial cell (hTCEpi) lines, which express tetracycline-inducible wild-type (wt) or catalytically-inactive (mu) ALDH3A1. Utilizing this cellular model system, we confirmed that human ALDH3A1 decreases corneal cell proliferation; importantly, this effect appears to be partially mediated by its enzymatic activity. Mechanistically, wt-ALDH3A1, but not mu-ALDH3A1, promotes sequestering of tumor suppressor p53 in the nucleus. In the mouse cornea, however, augmented cell proliferation is noted only in Aldh1a1-/-/3a1-/- double knockout (DKO) mice, indicating in vivo the anti-proliferation effect of ALDH3A1 can be rescued by the presence of ALDH1A1. Interestingly, the hyper-proliferative epithelium of the DKO corneas display nearly complete loss of p53 expression, implying that p53 may be involved in ALDH3A1/1A1-mediated effect. In hTCEpi cells grown in high calcium concentration, mRNA levels of a panel of corneal differentiation markers were altered by ALDH3A1 expression and modulated by its enzyme activity. In conclusion, we show for the first time that: (i) ALDH3A1 decreases corneal epithelial proliferation through both non-enzymatic and enzymatic properties; (ii) ALDH1A1 contributes to the regulation of corneal cellular proliferation in vivo; and (iii) ALDH3A1 modulates corneal epithelial differentiation. Collectively, our studies indicate a functional role of ALDH3A1 in the maintenance of corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation through both enzymatic and non-enzymatic mechanisms.

Comparison of cytotoxicity and wound healing effect of carboxymethylcellulose and hyaluronic acid on human corneal epithelial cells.

To investigate the cytotoxic effect on human corneal epithelial cells (HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose (CMC) and hyaluronic acid (HA). HCECs were exposed to 0.5% CMC (Refresh plus(®), Allergan, Irvine, California, USA) and 0.1% and 0.3% HA (Kynex(®), Alcon, Seoul, Korea, and Hyalein mini(®), Santen, Osaka, Japan) for the period of 30min, and 4, 12, and 24h. Methyl thiazolyl tetrazolium (MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase (LDH) leakage assay to assess the cytotoxicity. Apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24h after confluent HCECs were scratch wounded. The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells were demonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC. CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.

Anti-neovascular effect of chondrocyte-derived extracellular matrix on corneal alkaline burns in rabbits.

We investigated the effect of a chondrocyte-derived extracellular matrix (CDECM) on experimental corneal alkaline burns in rabbits. Corneal neovascularization (NV) was induced by applying an 8-mm filter paper soaked in 1N NaOH to the right central corneas of rabbits for 1minute. Ten days later, the rabbits were randomly divided into three groups: the alkaline burn group, the CDECM transplantation group, and the human amniotic membrane (HAM) transplantation group. The left eyes were used as controls. CDECM and HAM were transplanted onto the corneal surface to completely cover the resected area and were subsequently sutured. On the 10th day after transplantation, the structural changes of the cornea were analyzed histologically. We examined the effects of CDECM on clinical NV features and on the expression of corneal NV markers. The alkaline burn produced significant NV and increased the corneal thickness. On day 10 after transplantation, the thickness, NV and opacity of the cornea were markedly decreased in the CDECM group (p < 0.001). However, the HAM transplantation group did not exhibit improvements in these clinical parameters, and there were no significant differences relative to the burn group. In addition, the use of CDECM improved the healing of the cornea following the alkaline burn by disrupting the corneal epithelial proliferation and reducing the fibrotic changes of the stroma. The hallmarks of NV were significantly induced in the subepithelium by the alkaline burn, and these levels were also suppressed by CDECM. The CDECM suppressed corneal NV by inhibiting nuclear factor-kappa B (NF-κB) activation by blocking the PKC and Akt signaling pathways. CDECM transplantation was markedly effective in healing alkali-burned corneas by modulating the translocation of NF-κB to the nucleus, thereby representing a promising material for the noninvasive treatment of ocular surface disease.

Spry1 and Spry2 are necessary for eyelid closure

Sproutys (Sprys) are downstream targets and negative feedback regulators of the FGF–Ras–ERK signaling pathway. Our previous studies have shown that Spry1 and Spry2, through negative modulation of FGF–ERK signaling, allow lens vesicle separation from the overlying ectoderm and regulate corneal epithelial proliferation. Here we show that Spry1 and Spry2 are necessary for eyelid closure. Murine palpebral conjunctival epithelial cells that differentiate as inner eyelids and adjacent mesenchymal cells express Spry1 and Spry2 prior to eyelid closure. Conditional deletion of both Spry1 and Spry2, but not either one alone, in the ocular surface epithelial cells result in the “EOB” (eyes open at birth) phenotype suggesting redundant roles for these proteins during eyelid closure. Spry mutant eyelids show increased proliferation of conjunctival epithelial cells with concomitant induction of FGF targets, Erm, Pea3 and Dusp6 and elevated ERK phosphorylation. Peridermal cells at the leading edge of Spry-mutant eyelids showed reduced c-Jun, but not ERK, phosphorylation, reduced F-actin polymerization and reduced motility in vitro. Spry mutant eyelids also showed disruptions in epithelial mesenchymal interactions reflected in the enhanced mesenchymal Spry1 and Spry4 expression, disaggregation of BMP4-positive mesenchymal cells and loss of Shh in the eyelid epithelium. Spry mutant eyelids also showed increased Wnt signaling and reduced expression of Foxc1 and Foxc2, two transcription factors previously shown to be necessary for eyelid closure. Collectively, our results show that conjunctival epithelial Spry1 and Spry2 redundantly promote eyelid closure by (a) stimulating ERK-independent, c-Jun-mediated peridermal migration, (b) suppressing conjunctival epithelial proliferation through FGF–ERK signaling, (c) mediating conjunctival epithelial-mesenchymal interactions and (d) maintaining expression of Foxc1 and Foxc2.

The cellular mechanisms of dry eye: From pathogenesis to treatment

Dry eye is a complex disease characterized by changes in the ocular surface epithelia related to reduced quality and/or quantity of tears, inflammatory reaction, and impairment of ocular surface sensitivity. It has recently been proposed that increased tear osmolarity represents a main trigger to the altered cellular mechanisms leading to epithelial damage in dry eye. However, dry eye pathogenesis is multifactorial, with cytotoxic inflammatory mediators, altered lacrimal gland secretion and nerve function, squamous metaplasia of the conjunctival epithelium and decrease of goblet cells density, all playing a role in a detrimental loop that perpetuates and worsens damage to the corneal and conjunctival epithelia. Current topical treatments for dry eye patients include the use of lubricants and anti-inflammatory drugs. However, lubricants only improve symptoms temporarily, and chronic use of topical steroids is associated to severe ocular side effects such as cataract and glaucoma. The deeper understanding of the cellular mechanisms that are altered in dry eye is opening novel perspectives for patients and physicians, who are seeking treatments capable not only of improving symptoms but also of restoring the homeostasis of the ocular surface. In this review, we will focus on novel anti-inflammatory agents and on nerve growth factor, a neurotrophin that is altered in dry eye and has been suggested as a main player in the neuroimmune cross-talk of the ocular surface as well as in the stimulation of corneal sensitivity, epithelial proliferation and differentiation, and stimulation of mucin production by goblet cells. J. Cell. Physiol. 228: 2253-2256, 2013. © 2013 Wiley Periodicals, Inc.