Corneal Epithelial Wound Research Articles

Efficacy of Amniotic Membrane Transplantation for the Treatment of Corneal Ulcers.

To evaluate the outcome of amniotic membrane transplantation (AMTX) as a treatment for corneal ulcers. Patients treated with AMTX for refractory corneal ulcers between 2012 and 2017 were evaluated in a retrospective analysis. Primary outcome measure was complete reepithelialization. A total of 149 patients were included (mean age 68 ± 18 years). The mean duration between ulcer onset and AMTX was 42 ± 46 days. The longest time between ulcer diagnosis and AMTX was found in bacterial ulcers and the shortest time to AMTX in eyes with trauma/chemical burns (mean 65 ± 15 days and 14 ± 4 days, respectively). In 70% of the patients, a single AMTX procedure was sufficient to achieve epithelial closure (21% <1 month, 40% within 1 -3 months, and 9% within 3-6 months). Treatment failure was observed in 30% of all patients, and most of them underwent further interventions. Highest closure rates were found in bacterial ulcers, herpetic ulcers, and neurotrophic ulcers (80%, 85%, and 93%, respectively), whereas the lowest reepithelialization rates were found in ulcers after corneal surgery and ulcers associated with rheumatic disease (52% and 57%, respectively). AMTX is a valuable treatment option to achieve corneal epithelial wound healing in cases refractory to conventional treatment. Success rates differ depending on the etiology of ulcer.

Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model

The purpose of the current work was to utilize a three dimensional (3D) corneal epithelial tissue model to study dry eye disease and oxidative stress-related corneal epithelial injuries for the advancement of ocular therapeutics. Air-liquid interface cultures of normal human corneal epithelial cells were used to produce 3D corneal epithelial tissues appropriate for physiologically relevant exposure to environmental factors. Oxidative stress was generated by exposing the tissues to non-toxic doses of ultraviolet radiation (UV), hydrogen peroxide, vesicating agent nitrogen mustard, or desiccating conditions that stimulated morphological, cellular, and molecular changes relevant to dry eye disease. Corneal specific responses, including barrier function, tissue viability, reactive oxygen species (ROS) accumulation, lipid peroxidation, cytokine release, histology, and gene expression were evaluated. 3D corneal epithelial tissue model structurally and functionally reproduced key features of molecular responses of various types of oxidative stress-induced ocular damage. The most pronounced effects for different treatments were: UV irradiation - intracellular ROS accumulation; hydrogen peroxide exposure - barrier impairment and IL-8 release; nitrogen mustard exposure - lipid peroxidation and IL-8 release; desiccating conditions - tissue thinning, a decline in mucin expression, increased lipid peroxidation and IL-8 release. Utilizing a PCR gene array, we compared the effects of corneal epithelial damage on the expression of 84 oxidative stress-responsive genes and found specific molecular responses for each type of damage. The topical application of lubricant eye drops improved tissue morphology while decreasing lipid peroxidation and IL-8 release from tissues incubated at desiccating conditions. This model is anticipated to be a valuable tool to study molecular mechanisms of corneal epithelial damage and aid in the development of therapies against dry eye disease, oxidative stress- and vesicant-induced ocular injuries.

Neurokinin-1 Receptor Antagonism Ameliorates Dry Eye Disease by Inhibiting Antigen-Presenting Cell Maturation and T Helper 17 Cell Activation

Neuroinflammation plays an important role in the pathogenesis of ocular surface disease, including dry eye disease (DED), but little is known about the contribution of substance P (SP) to DED. In this study, we investigated the expression of SP at the ocular surface and evaluated its effect on maturation of antigen-presenting cells (APCs), the key cell component involved in the induction of type 17 helper T-cell (Th17) response in DED. The effect of topical blockade of SP signaling was further investigated using neurokinin-1 receptor (NK1R) inhibitors on APC maturation, Th17 cell activation, and disease severity in a mouse model of DED. The results demonstrate that SP is constitutively expressed at the ocular surface, and trigeminal ganglion neurons are the major source of SP in DED. SP derived from trigeminal ganglion enhanced the expression of major histocompatibility complex class II maturation marker by bone marrow-derived dendritic cells, an effect that is abrogated by blockade of SP signaling using NK1R antagonist spantide. Finally, using a well-established murine model of DED, topical treatment of DED mice with NK1R antagonists CP-99,994 and L-733,060 suppressed APC acquisition of major histocompatibility complex class II, reduced Th17 cell activity, and ameliorated DED severity. These findings are of translational value, as they suggest that antagonizing NK1R-mediated SP signaling may be an effective strategy in suppressing Th17-mediated ocular surface disease.

Topical hyaluronan alone promotes corneal epithelial cell migration whereas combination with benzalkonium chloride impairs epithelial wound healing

Purpose: To evaluate the effects of topical hyaluronan (HA) on corneal epithelial wound healing when administered with or without benzalkonium chloride (BAC).Methods: A cultured human corneal epithelial cell line (HCE-T) was subjected to in vitro scratch assays and in situ epithelial migration was evaluated in organ-cultured rabbit corneas. The corneal epithelium of C57BL/6J mice was also evaluated to determine in vivo wound healing. An in vivo imaging system was also used to evaluate the effects of HA on eye drop retention on the ocular surface.Results: The findings revealed the promotion of HCE-T migration, in situ rabbit corneal epithelial migration, and in vivo wound healing in mouse corneal epithelium by HA. Pre-treatment with HA also protected against delayed epithelial wound healing in BAC in vitro. However, pre-treatment with 3 mg/mL HA did not show a protective effect against BAC in vivo, but instead delayed epithelial wound healing and increased detection of cleaved caspase-3. This suggested that HA promotes the retention of BAC on the ocular surface. The instilled HA was retained after 15 min, at a significantly higher rate than for phosphate-buffered saline.Conclusions: The combination of HA and BAC impaired wound healing in the corneal epithelium.

Oral Hyaluronic Acid Supplementation for the Treatment of Dry Eye Disease: A Pilot Study.

Purpose To evaluate the clinical efficacy of oral hyaluronic acid (HA) in patients with dry eye disease (DED). Study Design Prospective randomized controlled trial. Methods This trial enrolled 54 subjects and they were randomized into the study or control group. The inclusion criteria were as follows: (1) >18 years of age; (2) distance best-corrected visual acuity ≥ 20/40 Snellen equivalent in each eye; (3) IOP ≤ 21 mmHg in both eyes; (4) ocular surface disease index (OSDI) score of ≥18 and <65; (5) <10 seconds of tear break up time (TBUT); (6) >5 corneal spots of corneal fluorescein staining (CFS); and (7) ≤ 10 mm/5 min of the Schirmer test. All subjects were treated with a topical HA, and the study group was supplemented with oral HA. OSDI, TBUT, CFS, and the Schirmer test were evaluated for ocular surface parameters. Results 24 patients were assigned in the study group. Significant improvement of OSDI, TBUT, and CFS was observed at 1 month and 3 months after oral HA administration in the study group. At baseline and follow-up at 1 and 3 months, OSDI scores were 61.8 ± 16.2, 47.3 ± 11.6, and 42.3 ± 9.1, respectively (P < 0.001). TBUT was improved after treatment for 1 month and 3 months (4.2 ± 1.1; P=0.005 and 4.7 ± 1.1; P < 0.012). There were also statistically significant improvements in the CSF (1.8 ± 1.0, 0.8 ± 0.7; P < 0.001) at baseline compared with those at 1 month. Conclusions A combined supplement of both oral and topical HA more efficiently improves corneal epithelial wound healing and related symptoms than topical HA alone, in DED.

KLF4 prevents epithelial to mesenchymal transition in human corneal epithelial cells via endogenous TGF-β2 suppression.

IntroductionKrüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs via regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-β signalling pathway, which is involved in corneal epithelial wound healing.MethodsHCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-β secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-β2 treatment.ResultsKLF4-KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, keratin 12 and keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin–positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-β canonical signalling pathway, TGF-β2 secretion by HCECs increased up to 5 folds, and several TGF-β–associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-β signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 was overexpressed, cultivated HCECs showed upregulation of epithelial markers, keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium in vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-β2 treatment.ConclusionsThese results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-β2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-β signalling.

Annexin A1 mimetic peptide Ac2-26 attenuates mechanical injury induced corneal scarring and inflammation

Annexin A1 (AnxA1) has been shown to exert potent anti-inflammatory and anti-fibrotic activities in a range of systemic inflammatory disorders. Corneal scarring is characterized by myofibroblast differentiation and disorganized extracellular matrix deposition. This study was aim to explore the potential therapeutic properties of Ac2-26, a mimetic peptide of AnnexinA1 (AnxA1), on TGF-β induced human corneal myofibroblast differentiation and mechanical injury-induced mouse corneal haze. The results found that Ac2-26 treatment dose dependently reduced α-SMA level and other fibrogenic gene expressions in HTK cells stimulated by exogenous TGF-β1. While this anti-fibrotic effect was abolished by an FPR2/ALX inhibitor WRW4. In mice, topical Ac2-26 application suppressed the development of corneal scarring, inhibited myofibroblast differentiation, while promoted the corneal epithelial wound healing. Moreover, Ac2-26 treatment inhibited Ly6G + neutrophil infiltration and reduced corneal inflammatory response. The results provided in vivo and in vitro supports the anti-fibrotic and anti-inflammatory effects of AnxA1 derived peptide Ac2-26, and suggest that AnxA1 mimetic agents might be a promising strategy for the treatment of corneal scarring.

Association of Cyclin Dependent Kinase 10 and Transcription Factor 2 during Human Corneal Epithelial Wound Healing in vitro model

Proper wound healing is dynamic in order to maintain the corneal integrity and transparency. Impaired or delayed corneal epithelial wound healing is one of the most frequently observed ocular defect and difficult to treat. Cyclin dependen kinase (cdk), a known cell cycle regulator, required for proper proliferating and migration of cell. We therefore investigated the role of cell cycle regulator cdk10, member of cdk family and its functional association with transcriptional factor (ETS2) at active phase of corneal epithelial cell migration. Our data showed that cdk10 was associated with ETS2, while its expression was upregulated at the active phase (18 hours) of cell migration and gradually decrease as the wound was completely closed. Topical treatment with anti-cdk10 and ETS2 antibodies delayed the wound closure time at higest concentration (10 µg/ml) compared to control. Further, our results also showed increased mRNA expression of cdk10 and ETS2 at active phase of migration at approximately 2 fold. Collectively, our data reveals that cdk10 and ETS2 efficiently involved during corneal wound healing. Further studies are warranted to better understand the mechanism and safety of topical cdk10 and ETS2 proteins in corneal epithelial wound-healing and its potential role for human disease treatment.

Knockdown of Tumour Necrosis Factor-α-Induced Protein 8-Like 2 Promotes Normal and Diabetic Corneal Epithelial Wound Healing and Attenuates Corneal Apoptosis via Upregulation of Nuclear Factor-κB p50

Background: Corneal epithelial defects are frequently observed in ocular surface disorders. Diabetic keratopathy impairs wound healing after corneal injury. The expression and roles of tumour necrosis factorα-induced protein 8-like 2 (TIPE2), a novel regulator of proliferation, apoptosis, inflammation, and immunity in the cornea during normal and diabetic corneal epithelial wound healing are unknown. Here, we evaluated the roles of TIPE2 in the cornea. Methods: The expression of TIPE2, nuclear factor (NF)-κB p50, and AKT was measured in intact and healing corneal epithelia in normal and streptozotocin-induced diabetic mice. Corneal epithelial wound healing and corneal apoptosis were determined in normal, TIPE2-deficient, and diabetic mice with or without NF-κB p50 inhibitor, AKT inhibitor, or TIPE2 siRNA treatment. Findings: TIPE2 levels were upregulated after corneal epithelial injury. TIPE2 deficiency accelerated corneal epithelial wound healing, decreased corneal apoptosis, and increased NF-κB p50 and phospho-AKT levels in intact and healing corneal epithelia. AKT inhibitor treatment suppressed NF-κB p50 expression, and NF-κB p50 or AKT inhibition impaired epithelial wound healing and increased corneal apoptosis. Diabetic mice showed more dramatic TIPE2 upregulation, NF-κB p50 downregulation, and phospho-AKT suppression. TIPE2 deficiency, and TIPE2 siRNA treatment improved corneal epithelial wound healing in diabetic mice. Interpretation: TIPE2 knockdown ameliorated corneal epithelial wound healing under normal and diabetic conditions by reducing apoptosis via upregulation of NF-κB p50 through AKT signalling. TIPE2 may be a therapeutic target in normal and diabetic corneal epithelial defects. Funding Statement: This research was supported by the National Natural Science Foundation of China (81670828, 81570820) and the Innovation Project of Shandong Academy of Medical Sciences. Lingling Yang are partially supported by the Taishan Scholar Youth Professional Program (tsqn20161059). Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: All animal experiments were carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Ethics Committee of Shandong Eye Institute.

Corneal Healing: Tetrahedral Framework Nucleic Acids Promote Corneal Epithelial Wound Healing in Vitro and in Vivo (Small 31/2019)

Corneal Healing: Tetrahedral Framework Nucleic Acids Promote Corneal Epithelial Wound Healing in Vitro and in Vivo (Small 31/2019)

DNase I Improves Corneal Epithelial and Nerve Regeneration in Diabetic Mice

Background: Neutrophils aggregation, usually in the form of neutrophils extracellular traps (NETs), contributes to the delayed wound healing in diabetic condition. DNase I plays a vital role in NETs clearance, while the function and mechanism of DNase I on diabetes-related corneal wound healing is still remain unclear. Methods: Intraperitoneal streptozocin injection was used to induce type 1 diabetic mouse models, and 1mg/ml DNase I or PAD4 inhibitor (Cl-amidine) topical administration were used as treatments for tissue regeneration after corneal epithelial removal. Corneal epithelial defects, inflammatory response, regeneration related signaling pathways, oxidative stress, corneal nerve innervation and sensation were examined and quantized during the corneal epithelial and nerve regeneration both in diabetic and normal mice. Findings: Topical DNase I or Cl-amidine administration improved the corneal wound healing rate in diabetic mice. Meanwhile, DNase I application significantly improved inflammation resolution, reactivated epithelial regeneration-related signaling pathways, and attenuated the accumulation of reactive oxygen species (ROS). Moreover, DNase I application also prominently promoted corneal nerve regeneration, and restored the impaired corneal sensitivity in diabetic mice. Interpretation: DNase I promoted corneal epithelial wound healing and the restoration of mechanical sensation in diabetic mice, thereby topical application of DNase I may serve as a novel therapy for the treatment of delayed corneal wound healing in diabetic condition. Funding Statement: This work was partially funded by National Natural Foundation of China (81770904, 81800805, 81570820), Shandong Medical and Health Science Technology Development Project (2018WS233), Shandong Academy of Medical Sciences Technology Project (2018-21), Qingjun Zhou is partially supported by the Taishan Scholar Program (20150215, 20161059) and the Innovation Project of Shandong Academy of Medical Sciences. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal experiments were conducted with the approval of the Ethics Committee of Shandong Eye Institute according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

Preliminary study of the antibacterial effect on the conjunctival sac and the influence on the corneal epithelium of domestic 5% povidone-iodine before cataract extraction

Objective: To investigate the efficacy and adverse reactions of 5% povidone-iodine in removing bacteria from the conjunctival sac with different durations. Methods: Randomized controlled study. A total of 420 patients who underwent cataract surgery in Daping Hospital, Army Medical University from December 2017 to June 2018 were selected. Non-surgical eyes (420 eyes) were selected as the study subjects and divided into 4 groups randomly: 30-second group, 1.0-min group, 2.0-min group and 3.5-min group. On the day of surgery, domestic 5% povidone-iodine was used to flush the conjunctival sac for 30 seconds, 1.0 min, 2.0 min and 3.5 min, respectively. The conjunctival sac specimens were collected for bacterial culture and bacterial identification before and after flushing the conjunctival sac with povidone-iodine. The positive rates of bacterial culture and bacterial growth were compared. The patients' ocular surface was observed and the incidence of corneal epithelial injury was recorded at 1 hour and 1 day after surgery. The positive rates of bacterial culture and corneal epithelial injury between groups were compared by Pearson chi-square test. Results: After excluding 20 patients with suspected specimens contamination, 400 patients (400 non-surgical eyes) were enrolled, including 191 males and 209 females, with an average age of 66.8 years. Before flushing the conjunctival sac, the positive rates of bacterial culture in the 30-second group, 1.0-min group, 2.0-min group and 3.5-min group were 44.8% (43/96), 43.3% (39/90), 43.1% (47/109) and 43.8% (46/105), respectively, with no statistically significant difference (χ(2)=0.066, P=0.996). After flushing, the positive rates of conjunctival sac bacterial culture in the 4 groups were 29.2% (28/96), 31.1% (28/90), 13.8% (15/109) and 13.3% (14/105), respectively. The differences between the 30-second group and 2.0-min group (χ(2)=7.308, P=0.007), between the 1.0-min group and 2.0-min group (χ(2)=8.760, P=0.003), between the 30-second group and 3.5-min group (χ(2)=7.606, P=0.006), and between the 1.0-min group and 3.5-min group (χ(2)=9.063, P=0.003) were statistically significant. At 1 hour after surgery, mild corneal epithelial injury occurred in each group, with a rate of 16.7% (16/96), 18.9% (17/90), 20.2% (22/109) and 34.3% (36/105), respectively. The differences between the 30-second group and 3.5-min group (χ(2)=8.118, P=0.004), between the 1.0-min group and 3.5-min group (χ(2)=5.804, P=0.016), and between the 2.0-min group and 3.5-min group (χ(2)=5.383, P=0.020) were statistically significant. At 1 day after surgery, there was no occurrence of new injury, and the incidence of mild corneal injury in each group was 3.1% (3/96), 5.6% (5/90), 9.2% (10/109) and 15.2% (16/105), respectively. There was statistically significant difference between the 30-second group and 3.5-min group (χ(2)=8.597, P=0.003), and between the 1.0-min group and 3.5-min group (χ(2)=4.728, P=0.030). The corneal epithelial injury healed completely at 1 week after surgery. Conclusions: The preoperative bacterial load of the conjunctival sac is more effectively reduced with 5% povidone-iodine in the 2.0-min and 3.5-min than in the 30-second and 1.0-min, and the 2-min is superior to the 3.5-min in the occurrence of corneal epithelial injury at 1 hour after surgery. Irrigation of the conjunctival sac with 5% povidone-iodine for 2 min is effective and safe, which can be an alternative measure. (Chin J Ophthalmol, 2019, 55: 509-514).

Clinical effect of combined bandage contact lenses and artificial tears on corneal epithelial injury due to correction of blepharoptosis

Objective To explore the effect of the combination of bandage contact lenses and artificial tears on corneal epithelial injury, due to correction of blepharoptosis in patients. Methods Thirty-five patients (56 eyes) were included in this randomized controlled trial during May 2013 to May 2018, and randomly divided into observation group (combination of bandage contact lenses and artificial tears) and control group (combination of erythromycin eye ointment and artificial tears), and followed up for 3 months. The corneal wound healing, corneal fluorescent staining (FL), Schirmer test (Slt), break-up time (BUT) and the severity of eye pain were documented and compared by statistical method. Results The scores of FL in the observation group were reduced (t=-3.211, P 0.05; the scores of eye pain, t=-1.800, P>0.05; the corneal wound healing, χ2=1.018, P>0.05; the BUT score, t=1.661, P>0.05). Conclusions The combination of bandage contact lenses and artificial tears is recommended for the early use after correction of blepharoptosis, to alleviate eye symptoms and promote corneal wound healing. Key words: Blepharoptosis; Corneal injuries; Ptosis; Corneal epithelial injury; Contact lens

Tetrahedral Framework Nucleic Acids Promote Corneal Epithelial Wound Healing in Vitro and in Vivo.

Poor post-traumatic wound healing can affect the normal function of damaged tissues and organs. For example, poor healing of corneal epithelial injuries may lead to permanent visual impairment. It is of great importance to find a therapeutic way to promote wound closure. Tetrahedral framework nucleic acids (tFNAs) are new promising nanomaterials, which can affect the biological behavior of cells. In the experiment, corneal wound healing is used as an example to explore the effect of tFNAs on wound healing. Results show that the proliferation and migration of human corneal epithelial cells are enhanced by exposure to tFNAs in vitro, possibly relevant to the activation of P38 and ERK1/2 signaling pathway. An animal model of corneal alkali burn is established to further identify the facilitation effect of tFNAs on corneal wound healing in vivo. Clinical evaluations and histological analyses show that tFNAs can improve the corneal transparency and accelerate the re-epithelialization of wounds. Both in vitro and in vivo experiments show that tFNAs can play a positive role in corneal epithelial wound healing.

Re-epithelialization and remodeling of decellularized corneal matrix in a rabbit corneal epithelial wound model

This study investigated the in vivo correlation between re-epithelialization and remodeling of a decellularized corneal matrix prepared by a high-hydrostatic pressure (HHP) method in rabbits. Decellularized corneal matrices were transplanted in a 6-mm-diameter recipient corneal interlamellar pocket with a 2 mm epithelial defect. The time course of graft status in rabbits was examined daily for 6 months by biomicroscopy and scored for clarity and re-epithelialization, after which the rabbits were sacrificed for histological analysis. Fluorescein staining revealed that the corneal epithelial cells had migrated onto the decellularized corneal matrix. Histological analysis revealed that the implanted decellularized corneal matrix was completely integrated with the recipient rabbit cornea and the stratified corneal epithelia consisting of multiple layers were regenerated, similar to that in the normal cornea. The recipient keratocytes infiltrated into the decellularized corneal matrix at 6 months after the operation and the decellularized corneal matrix was gradually remodeled into the recipient tissue. Transmission electron microscopy revealed that the ultrastructure of the decellularized corneal matrix was rearranged, similar to the normal cornea. These findings suggest that the decellularized corneal matrix serves as a template for remodeling. The decellularized corneal matrix obtained through HHP is a useful graft for corneal tissue regeneration.

Evidence for a GPR18 Role in Chemotaxis, Proliferation, and the Course of Wound Closure in the Cornea.

We previously showed that cannabinoid-related GPR18 receptors are present in the murine corneal epithelium, but their function remains unknown. The related CB1 receptors regulate corneal healing, possibly via chemotaxis. We therefore examined a potential role for GPR18 in corneal epithelial chemotaxis and wound healing. We examined GPR18 messenger RNA (mRNA) and protein expression in the cornea. We additionally examined GPR18 action in cultured bovine corneal epithelial cells (bCECs) using Boyden and tracking assays, as well as proliferation and signaling. Finally, we examined wound closure in murine corneal explants. GPR18 mRNA was upregulated with injury in the mouse cornea. GPR18 protein was present in basal epithelial cells of the mouse and cow and redistributed to the wound site upon injury. GPR18 ligand N-arachidonoylglycine induced bCEC chemotaxis. The endocannabinoid arachidonoylethanolamine also induced chemotaxis via fatty acid amide hydrolase-mediated metabolism to N-arachidonoylglycine. GPR18 receptor activation additionally induced bCEC proliferation. In an explant model, the GPR18 antagonist O-1918 slowed corneal epithelial cell migration and the rate of corneal wound closure. Corneal GPR18 activation induced both chemotaxis and proliferation in corneal epithelial cells in vitro and impacted wound healing. GPR18 may contribute to the maintenance of corneal integrity.

Hyaluronic Acid Combined with Serum Rich in Growth Factors in Corneal Epithelial Defects.

The aim of this study is to assess if an adhesive biopolymer, sodium hyaluronate (NaHA), has synergistic effects with s-PRGF (a serum derived from plasma rich in growth factors and a blood derivative that has already shown efficacy in corneal epithelial wound healing), to reduce time of healing or posology. In vitro proliferation and migration studies, both in human corneal epithelial (HCE) cells and in rabbit primary corneal epithelial (RPCE) cultures, were carried out. In addition, we performed studies of corneal wound healing in vivo in rabbits treated with s-PRGF, NaHA, or the combination of both. We performed immunohistochemistry techniques (CK3, CK15, Ki67, ß4 integrin, ZO-1, α-SMA) in rabbit corneas 7 and 30 days after a surgically induced epithelial defect. In vitro results show that the combination of NaHA and s-PRGF offers the worst proliferation rates in both HCE and RPCE cells. Addition of NaHA to s-PRGF diminishes the re-epithelializing capability of s-PRGF. In vivo, all treatments, given twice a day, showed equivalent efficacy in corneal epithelial healing. We conclude that the combined use of s-PRGF and HaNA as an adhesive biopolymer does not improve the efficacy of s-PRGF alone in the wound healing of corneal epithelial defects.

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.

Anti-inflammatory potential of human corneal stroma-derived stem cells determined by a novel in vitro corneal epithelial injury model.

BACKGROUNDAn in vitro injury model mimicking a corneal surface injury was optimised using human corneal epithelial cells (hCEC).AIMTo investigate whether corneal-stroma derived stem cells (CSSC) seeded on an amniotic membrane (AM) construct manifests an anti-inflammatory, healing response.METHODSTreatment of hCEC with ethanol and pro-inflammatory cytokines were compared in terms of viability loss, cytotoxicity, and pro-inflammatory cytokine release, in order to generate the in vitro injury. This resulted in an optimal injury of 20% (v/v) ethanol for 30 s with 1 ng/mL interleukin-1 (IL-1) beta. Co-culture experiments were performed with CSSC alone and with CSSC-AM constructs. The effect of injury and co-culture on viability, cytotoxicity, IL-6 and IL-8 production, and IL1B, TNF, IL6, and CXCL8 mRNA expression were assessed.RESULTSCo-culture with CSSC inhibited loss of hCEC viability caused by injury. Enzyme linked immunosorbent assay and polymerase chain reaction showed a significant reduction in the production of IL-6 and IL-8 pro-inflammatory cytokines, and reduction in pro-inflammatory cytokine mRNA expression during co-culture with CSSC alone and with the AM construct. These results confirmed the therapeutic potential of the CSSC and the possible use of AM as a cell carrier for application to the ocular surface.CONCLUSIONCSSC were shown to have a potentially therapeutic anti-inflammatory effect when treating injured hCEC, demonstrating an important role in corneal regeneration and wound healing, leading to an improved knowledge of their potential use for research and therapeutic purposes.

DNA Methylation Regulates Corneal Epithelial Wound Healing by Targeting miR-200a and CDKN2B

DNA methylation is a key epigenetic modification involved in various biological processes and diseases. Corneal epithelial wound healing (CEWH) is essential for restoring corneal integrity and transparency after injury. However, the role of DNA methylation in CEWH remains elusive. Here, we investigate the function and underlying mechanism of DNA methylation in regulating CEWH. Dot blots and global methylation assays determined DNA methylation levels during CEWH. Quantitative RT-PCR and Western blot analysis examined the expression of DNA methyltransferases (DNMTs), cyclin-dependent kinase inhibitor 2B (CDKN2B), and miR-200a during CEWH, respectively. MTS assays and flow cytometry were used to analyze human corneal epithelial cell (HCEC) proliferation and cell cycle, respectively. The in vitro scratch wound assay assessed HCEC migration and an in vivo murine corneal epithelial debridement model evaluated wound healing. Using bisulfite sequencing PCR, we determined the DNA methylation status of the candidate genes. Transfection of miR-200a mimic or inhibitor assessed the function of miR-200a in HCECs. Rescue experiments were performed to clarify the correlation between DNMT1 and miR-200a/CDKN2B during CEWH. DNMT1 and DNMT3B expression was significantly upregulated during CEWH, resulting in a significant global DNA hypermethylation. DNMT1 downregulation dramatically delayed CEWH in vivo, and suppressed HCEC proliferation and migration. MiR-200a inhibited HCEC migration. Furthermore, miR-200a and CDKN2B were identified as molecular targets of DNA methylation and as having a causal connection with DNMT1. DNMT1-mediated DNA hypermethylation can enhance the process of CEWH by directly targeting miR-200a and CDKN2B. This insight pinpoints novel potential drug targets for promoting CEWH.