Regeneration Of Corneal Epithelium Research Articles

Targeting limbal epithelial stem cells: master conductors of corneal epithelial regeneration from the bench to multilevel theranostics.

The cornea is the outermost layer of the eye and plays an essential role in our visual system. Limbal epithelial stem cells (LESCs), which are localized to a highly regulated limbal niche, are the master conductors of corneal epithelial regeneration. Damage to LESCs and their niche may result in limbal stem cell deficiency (LSCD), a disease confused ophthalmologists so many years and can lead to corneal conjunctivalization, neovascularization, and even blindness. How to restore the LESCs function is the hot topic for ocular scientists and clinicians around the world. This review introduced LESCs and the niche microenvironment, outlined various techniques for isolating and culturing LESCs used in LSCD research, presented common diseases that cause LSCD, and provided a comprehensive overview of both the diagnosis and multiple treatments for LSCD from basic research to clinical therapies, especially the emerging cell therapies based on various stem cell sources. In addition, we also innovatively concluded the latest strategies in recent years, including exogenous drugs, tissue engineering, nanotechnology, exosome and gene therapy, as well as the ongoing clinical trials for treating LSCD in recent five years. Finally, we highlighted challenges from bench to bedside in LSCD and discussed cutting-edge areas in LSCD therapeutic research. We hope that this review could pave the way for future research and translation on treating LSCD, a crucial step in the field of ocular health.

Cell spray printing combined with Lycium barbarum glycopeptide promotes repair of corneal epithelial injury

The corneal epithelium, located as the outermost layer of the cornea, is inherently susceptible to injuries that may lead to corneal opacities and compromise visual acuity. Rapid restoration of corneal epithelial injury is crucial for maintaining the transparency and integrity of the cornea. Cell spray treatment emerges as an innovative and effective approach in the field of regenerative medicine. In our study, a cell spray printing platform was established, and the optimal printing parameters were determined to be a printing air pressure of 5 PSI (34.47 kPa) and a liquid flow rate of 30 ml/h. Under these conditions, the viability and phenotype of spray-printed corneal epithelial cells were preserved. Moreover, Lycium barbarum glycopeptide (LBGP), a glycoprotein purified from wolfberry, enhanced proliferation while simultaneously inhibiting apoptosis of the spray-printed corneal epithelial cells. We found that the combination of cell spray printing and LBGP facilitated the rapid construction of multilayered cell sheets on flat and curved collagen membranes in vitro. Furthermore, the combined cell spray printing and LBGP accelerated the recovery of the rat corneal epithelium in the mechanical injury model. Our findings offer a therapeutic avenue for addressing corneal epithelial injuries and regeneration.

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.

An adaptive drug-releasing contact lens for personalized treatment of ocular infections and injuries

This research introduces an innovative solution to address the challenges of bacterial keratitis and alkali burns. Current treatments for bacterial keratitis and alkali burns rely on the frequent use of antibiotics and anti-inflammatory eye drops. However, these approaches suffer from poor bioavailability and fluctuating concentrations, leading to limited efficacy and potential drug resistance. Our approach presents an adaptive drug-releasing contact lens responsive to reactive oxygen species (ROS) at ocular inflammation sites, synchronously releasing Levofloxacin and Diclofenac. During storage, minimal drug release occurred, but over 7 days of wear, the lens maintained a continuous, customizable drug release rate based on disease severity. This contact lens had strong antibacterial activity and biofilm prevention, effectively treating bacterial keratitis. When combined with autologous serum, this hydrophilic, flexible lens aids corneal epithelial regeneration, reducing irritation and promoting healing. In summary, this ROS-responsive drug-releasing contact lens combines antibacterial and anti-inflammatory effects, offering a promising solution for bacterial keratitis and alkali burns.

Deoxynivalenol Damages Corneal Epithelial Cells and Exacerbates Inflammatory Response in Fungal Keratitis.

Fungal keratitis (FK) is a kind of infectious keratopathy with a high rate of blindness worldwide. Deoxynivalenol (DON) has been proven to have multiple toxic effects on humans and animals. The aim of this study was to explore a possible pathogenic role of DON in FK. We first made an animal model of FK in New Zealand white rabbits, and then attempted to detect DON in a culture medium in which Fusarium solani had been grown and also in the corneal tissue of the animal model of Fusarium solani keratitis. Next, a model of DON damage in human corneal epithelial cells (HCECs) was constructed to evaluate effects of DON on the activity, migration ability, cell cycle, and apoptosis in the HCECs. Then, putative the toxic damaging effects of DON on rabbit corneal epithelial cells and the impact of the repair cycle were studied. The expression levels of inflammatory factors in the corneas of the animal model and in the model of DON-damaged HCECs were measured. The Fusarium solani strain used in this study appeared to have the potential to produce DON, since DON was detected in the corneal tissue of rabbits which had been inoculated with this Fusarium solani strain. DON was found to alter the morphology of HCECs, to reduce the activity and to inhibit the proliferation and migration of HCECs. DON also induced the apoptosis and S-phase arrest of HCECs. In addition, DON was found to damage rabbit corneal epithelial cells, to prolong the corneal epithelial regeneration cycle, and to be associated with the upregulated expression of inflammatory factors in HCECs and rabbit corneas. DON appears to have a toxic damaging effect on HCECs in FK, and to induce the expression of inflammatory factors, leading to the exacerbation of keratitis and the formation of new blood vessels. Future studies will explore the possibility of developing a test to detect DON in ophthalmic settings to aid the rapid diagnosis of FK, and to develop DON neutralizers and adsorbents which have the potential to improve keratocyte status, inhibit apoptosis, and alleviate inflammation, therein providing new thinking for therapy of clinical FK.

Effect of Topical Recombinant Human Nerve Growth Factor on Corneal Epithelial Regeneration in Refractory Epithelial Keratopathy

ABSTRACT Purpose To report the effect of topical application of recombinant human nerve growth factor (rhNGF) eye drops on corneal epithelial regeneration in patients with refractory epitheliopathy. Methods A retrospective chart review was conducted on patients treated with topical rhNGF for refractory epithelial keratopathy due to stage I neurotrophic keratitis (NK). Data regarding demographics and ocular/systemic past medical history was extracted from patient charts. Visual acuity and corneal staining scores were recorded at baseline and subsequent follow-up visits at 8 weeks and 3 months. Measurements from the worse eye were used to compare before and after treatment values. Results We identified 14 patients (median age 68 years, 21% male) who received rhNGF treatment for refractory epithelial keratopathy. After an 8-week treatment with topical rhNGF, the median corneal staining score in the worse eye improved from 4 to 1 (p = 0.001). All patients showed at least one-grade improvement in corneal staining at 8 weeks, with sustained effect in seven patients at 3 months. A better response was observed in eyes with post-radiation epithelial keratopathy, LASIK, and Sjogren’s disease. Those with chronic use of other topical treatments and uncontrolled diabetes mellitus demonstrated incomplete responses. Eight patients reported mild-to-moderate ocular discomfort from drop application that fully resolved after completion of treatment. Conclusions Topical rhNGF was effective and safe for refractory epithelial keratopathy in our small cohort, but sustained effects were seen only in certain etiologies for up to 3 months. Further studies are needed for optimal dosing and duration based on underlying causes.

Dopamine Receptor 1 Treatment Promotes Epithelial Repair of Corneal Injury by Inhibiting NOD-Like Receptor Protein 3-Associated Inflammation.

To elucidate the influence of dopamine receptor 1 (DRD1) on the proliferation of mouse corneal epithelial cells (MCECs) under inflammatory conditions. In vitro, immortalized MCECs (iMCECs) were treated with IL-1β, with and without pcDNA3.1_DRD1. Primary MCECs (pMCECs) were exposed to IL-1β, with and without DRD1 agonist (A68930). Cell proliferation was quantified using the Cell Counting Kit-8 (CCK-8) assay and immunofluorescence staining for Ki-67 and p63. Expression levels of NOD-like receptor protein 3 (NLRP3), IL-1β, and IL-6 were assessed. To establish a corneal injury model in mice, a 2-mm superficial keratectomy was performed. Either 0.1% A68930 or PBS was topically administered three times daily to the injured eyes for up to 5days post-injury. Immunofluorescence analysis was employed to evaluate the expression of Ki-67, p63, and CD45 in mouse corneas. Western blotting and real-time quantitative PCR were utilized for quantitative analysis of DRD1, NLRP3, IL-1β, and IL-6 in mouse corneas. Corneal epithelial regeneration was monitored through fluorescein sodium staining for a duration of up to 5days following the injury. Overexpression of DRD1 and A68930 promoted MCEC proliferation and suppressed the expression of NLRP3, IL-1β, and IL-6 in vitro. Topical application of the 0.1% A68930 following mechanical corneal injury in mice led to increased Ki-67 and p63 expression compared to PBS treatment. Furthermore, topical administration of the 0.1% A68930 reduced the expression of CD45, NLRP3, IL-1β, and IL-6. Analysis with fluorescein sodium indicated accelerated corneal epithelial regeneration in the 0.1% A68930 treatment group. DRD1 treatment counteracts NLRP3-associated inflammation and facilitates epithelial repair of corneal injury.

Evaluation of the Possible Influence of Povidone Iodine (PVI) Solution and Polyhexanide (PHMB) on Wound Healing in Corneal Epithelial Regeneration.

This study investigates the possible toxic effects of the preoperative antiseptic substances povidone iodine (PVI) and polyhexanide (PHMB; Serasept® 2) on wound healing in ophthalmology. To assess this impact, human telomerase-immortalized corneal epithelial (hTCEpi) cells and human telomerase-immortalized conjunctival epithelial (hCjE) cells were exposed to 1% and 5% PVI or 0.04% PHMB for different periods to evaluate the cytotoxicity of these two antiseptics. Furthermore, the toxicity of these antiseptics was investigated in a human tissue-specific corneal epithelial construct and porcine eye culture model. The results reveal the high cytotoxicity of PVI and PHMB in the hTCEpi and hCjE in monolayer cell culture models, independent of the incubation time and concentration of these substances. However, after hTCEpi cell differentiation into a tissue-specific corneal epithelial construct, contact with these antiseptics for the relevant preoperative time did not alter cPARP1 or Ki67 expression. Furthermore, the wound-healing process in the porcine cornea was not significantly influenced after incubation with these antiseptics. In summary, corneal and conjunctival epithelial cell lines are very sensitive to PVI and PHMB, whereas no significant alterations were found in intact tissue-specific corneal epithelial constructs or porcine corneas. Therefore, we could not identify PVI and PHMB as reasons for postoperative eye irritation.

3D printing sequentially strengthening high-strength natural polymer hydrogel bilayer scaffold for cornea regeneration.

3D printing of high-strength natural polymer biodegradable hydrogel scaffolds simultaneously resembling the biomechanics of corneal tissue and facilitating tissue regeneration remains a huge challenge due to the inherent brittleness of natural polymer hydrogels and the demanding requirements of printing. Herein, concentrated aqueous solutions of gelatin and carbohydrazide-modified alginate (Gel/Alg-CDH) are blended to form a natural polymer hydrogel ink, where the hydrazides in Alg-CDH are found to form strong hydrogen bonds with the gelatin. The hydrogen-bonding-strengthened Gel/Alg-CDH hydrogel demonstrates an appropriate thickened viscosity and shear thinning for extrusion printing. The strong hydrogen bonds contribute to remarkably increased mechanical properties of Gel/Alg-CDH hydrogel with a maximum elongation of over 400%. In addition, sequentially Ca2+-physical crosslinking and then moderately chemical crosslinking significantly enhance the mechanical properties of Gel/Alg-CDH hydrogels that ultimately exhibit an intriguing J-shaped stress-strain curve (tensile strength of 1.068 MPa and the toughness of 677.6 kJ/m2). The dually crosslinked Gel-Alg-CDH-Ca2+-EDC hydrogels demonstrate a high transparency, physiological swelling stability and rapid enzymatic degradability, as well as suturability. The growth factor and drug-loaded biomimetic bilayer hydrogel scaffold are customized via a multi-nozzle printing system. This bioactive bilayer hydrogel scaffold considerably promotes regeneration of corneal epithelium and stroma and inhibits cornea scarring in rabbit cornea keratoplasty.

A Versatile Hydrogel with Antibacterial and Sequential Drug-Releasing Capability for the Programmable Healing of Infectious Keratitis.

Hydrogels have attracted tremendous attention as favorable corneal substitutes for treating severe infectious keratitis (IK). However, current hydrogel-based corneal substitutes were majorly designed to promote the single stage of corneal regeneration, which falls short in meeting the clinical management needs of severe IK including the multiple phases of corneal wound healing. Herein, we introduce a versatile hybrid hydrogel (SQPV) composed of silk fibroin and chitosan, which exhibits spatiotemporal properties for drug release. The SQPV is fabricated by incorporating verteporfin-loaded poly(lactic-co-glycolic)-polyethylene glycol-o-nitrobenzene micelles into a hydrogel network, which is formed from methacrylate silk fibroin and glycidyl methacrylate functionalized quaternized chitosan containing polydeoxyribonucleotide. This double network approach results in a material with exceptional anti-inflammatory, antibacterial, and proliferative stimulation and tissue remodeling regulation capabilities. Furthermore, SQPV showcases mechanical strength and transparency akin to those of native cornea. Extensive in vitro and in vivo studies validate SQPV's ability to effectively eliminate residual bacteria, mitigate inflammation, foster regeneration of corneal epithelium and stroma, prevent corneal scarring, and ultimately expedite wound healing. In summary, the SF/CS-based hybrid hydrogel may represent a promising substitute for comprehensive corneal repair and regeneration in severe IK.

Modulating Growth Factor Receptor Signaling to Promote Corneal Epithelial Homeostasis.

The corneal epithelium is the first anatomical barrier between the environment and the cornea; it is critical for proper light refraction onto the retina and prevents pathogens (e.g., bacteria, viruses) from entering the immune-privileged eye. Trauma to the highly innervated corneal epithelium is extremely painful and if not resolved quickly or properly, can lead to infection and ultimately blindness. The healthy eye produces its own growth factors and is continuously bathed in tear fluid that contains these proteins and other nutrients to maintain the rapid turnover and homeostasis of the ocular surface. In this article, we review the roles of growth factors in corneal epithelial homeostasis and regeneration and some of the limitations to their use therapeutically.

Limbal Epithelial Stem Cells in the Diabetic Cornea.

Continuous replenishment of the corneal epithelium is pivotal for maintaining optical transparency and achieving optimal visual perception. This dynamic process is driven by limbal epithelial stem cells (LESCs) located at the junction between the cornea and conjunctiva, which is otherwise known as the limbus. In patients afflicted with diabetes, hyperglycemia-induced impairments in corneal epithelial regeneration results in persistent epithelial and other defects on the ocular surface, termed diabetic keratopathy (DK), which progressively diminish vision and quality of life. Reports of delayed corneal wound healing and the reduced expression of putative stem cell markers in diabetic relative to healthy eyes suggest that the pathogenesis of DK may be associated with the abnormal activity of LESCs. However, the precise role of these cells in diabetic corneal disease is poorly understood and yet to be comprehensively explored. Herein, we review existing literature highlighting aberrant LESC activity in diabetes, focusing on factors that influence their form and function, and emerging therapies to correct these defects. The consequences of malfunctioning or depleted LESC stocks in DK and limbal stem cell deficiency (LSCD) are also discussed. These insights could be exploited to identify novel targets for improving the management of ocular surface complications that manifest in patients with diabetes.

Topical insulin, a novel corneal epithelial regeneration agent in dry eye disease.

To evaluate the efficacy of insulin eye drops for dry eye disease in reducing corneal staining and improving symptoms. In this retrospective case series, patients with dry eye disease treated with off-label use of insulin eye drops were collected. The main inclusion criterion was diagnosis of dry eye disease with epithelial damage and acceptance of the off-label use of topical insulin. Age, sex, type of dry eye disease, time since diagnosis, previous ocular surgeries, concomitant treatment, best corrected visual acuity, symptoms, conjunctival hyperemia and corneal staining were recorded. Data from the 1 and 3-month visit were included. 16 patients (32 eyes) were treated with insulin (14 females and 2 males; mean age 61.3 ± 16.8 years). 12 patients (71%) were also on autologous serum and 10 patients (63%) on cyclosporine. Symptoms were 3.4 ± 1.3 (range 2-5) when scaled from 0 to 5. Mean hyperemia was 1.0 ± 0.9 (range 0-3) and corneal staining was 2.5 ± 1.3 (range 0-5). After 3 months, 5 patients (31%) referred to be much better, 6 (38%) better, 3 (19%) slightly better and 2 patients (13%) were subjectively similar, mean symptoms being 2.3 ± 1.0 (range 1-4; p = 0.001). Hyperemia was 0.3 ± 0.4 (range 0-1) and corneal staining was 1.1 ± 1.0 (range 0-3; both p < 0.001). Topical insulin was well tolerated with no adverse events. The excellent results presented in these case series illustrate topical insulin as a promising treatment in dry eye disease with refractory epithelial damage.

Development of Anterior Segment Focused Biologic Therapies to Regenerate Corneal Tissue for the Treatment of Disease: Drug Development Experience.

On February 24-27, 2021, the Association for Ocular Pharmacology and Therapeutics (AOPT) held its 15th biennial scientific meeting online. The meeting was organized by Dr. Sanjoy Bhattacharya of the University of Miami in conjunction with the board of trustees of the AOPT. The 3-day conference was attended by academic scientists, clinicians, and industry and regulatory professionals. The theme of the meeting was Restoring Vision through Regeneration and it was sponsored, in part, by the National Institutes of Health, Bright Focus, Regeneron, and Santen (USA). During the 3 days of the meeting, presentations from several sessions explored different aspects of regenerative medicine in ophthalmology, including optic nerve regeneration, drugs and devices in glaucoma, retinal neuroprotection and plasticity, visual perception, and degeneration of trabecular meshwork. This article summarizes the proceedings of the session on corneal regenerative medicine research and discusses emerging concepts in drug development for corneal epithelial and endothelial regeneration. Since the meeting in 2021, several of these concepts have advanced to clinical-stage therapies, but so far as of 2023, none has been approved by regional regulatory authorities in the United States. One form of corneal endothelial cell therapy has been approved in Japan and only for bullous keratopathy. Ongoing work is proceeding in the United States and other countries. Clinical Registration No: National Clinical Trials 04894110, 04812667; Japan Registry for Clinical Trials a031210199.

The role of neutrophils in corneal nerve regeneration

BackgroundTo investigate the role of neutrophils in corneal nerve regeneration.MethodsA mouse model simulating corneal nerve injury was established and samples from corneal scraping with and without neutrophil closure were collected. These samples were used for corneal nerve staining, ribonucleic acid sequencing, and bioinformatics. Differential expression analysis was used to perform enrichment analysis to identify any significant differences between these two groups. The differential genes were then intersected with neutrophil-associated genes and a protein-protein interaction network was constructed using the intersected genes. The immune infiltration between the two groups was examined along with the immune cell variation between the high and low gene expression groups.ResultsNeutrophil removal delays corneal epithelial and nerve regeneration. A total of 546 differential genes and 980 neutrophil-associated genes, with 27 genes common to both sets were obtained. Molecular Complex Detection analysis yielded five key genes, namely integrin subunit beta 2 (ITGB2), matrix metallopeptidase 9 (MMP9), epidermal growth factor (EGF), serpin family E member 1 (SERPINE1), and plasminogen activator urokinase receptor (PLAUR). Among these genes, ITGB2, SERPINE1, and PLAUR exhibited increased expression in the neutrophil-confined group, while MMP9 and EGF showed decreased expression, with MMP9 and EGF displaying a more significant difference. Immune infiltration was also observed between the two groups, revealing significant differences in the infiltration of M0 macrophages, activated mast cells, and neutrophils. Moreover, the neutrophil levels were lower in the groups with low MMP9 and EGF expressions and higher in the high-expression group.ConclusionNeutrophil confinement might significantly affect the MMP9 and EGF expression levels. Strategies to inhibit MMP9 could potentially yield therapeutic benefits.

Mechanism and application of β-adrenoceptor blockers in soft tissue wound healing.

Soft tissue damage stimulates sympathetic nerves to release large amounts of catecholamine hormones which bind to β-adrenergic receptors (β-ARs) on the cell membrane surface. It activates the downstream effector molecules and impairs soft tissue wound healing. β-blockers specifically inhibit β-ARs activation in acute/chronic skin lesions and ulcerative hemangiomas. They also accelerate soft tissue wound healing by shortening the duration of inflammation, speeding keratinocyte migration and reepithelialization, promoting wound contraction and angiogenesis, and inhibiting bacterial virulence effects. In addition, β-blockers shorten wound healing periods in patients with severe thermal damage by reducing the hypermetabolic response. While β-blockers promote/inhibit corneal epithelial cell regeneration and restores limbal stem/progenitor cells function, it could well accelerate/delay corneal wound healing. Given these meaningful effects, a growing number of studies are focused on examining the efficacy and safety of β-blockers in soft tissue wound repair, including acute and chronic wounds, severe thermal damage, ulcerated infantile hemangioma, corneal wounds, and other soft tissue disorders. However, an intensive investigation on their acting mechanisms is imperatively needed. The purpose of this article is to summerize the roles of β-blockers in soft tissue wound healing and explore their clinical applications.

MiRNA 24-3p-rich exosomes functionalized DEGMA-modified hyaluronic acid hydrogels for corneal epithelial healing.

The damage of corneal epithelium may lead to the formation of irreversible corneal opacities and even blindness. The migration rate of corneal epithelial cells directly affects corneal repair. Here, we explored ocu-microRNA 24-3p (miRNA 24-3p) that can promote rabbit corneal epithelial cells migration and cornea repair. Exosomes, an excellent transport carrier, were exacted from adipose derived mesenchymal stem cells for loading with miRNA 24-3p to prepare miRNA 24-3p-rich exosomes (Exos-miRNA 24-3p). It can accelerate corneal epithelial migration in vitro and in vivo. For application in cornea alkali burns, we further modified hyaluronic acid with di(ethylene glycol) monomethyl ether methacrylate (DEGMA) to obtain a thermosensitive hydrogel, also reported a thermosensitive DEGMA-modified hyaluronic acid hydrogel (THH) for the controlled release of Exos-miRNA 24-3p. It formed a highly uniform and clear thin layer on the ocular surface to resist clearance from blinking and extended the drug-ocular-epithelium contact time. The use of THH-3/Exos-miRNA 24-3p for 28 days after alkali burn injury accelerated corneal epithelial defect healing and epithelial maturation. It also reduced corneal stromal fibrosis and macrophage activation. MiRNA 24-3p-rich exosomes functionalized DEGMA-modified hyaluronic acid hydrogel as a multilevel delivery strategy has a potential use for cell-free therapy of corneal epithelial regeneration.

Targeting Ca2+-dependent pathways to promote corneal epithelial wound healing induced by CISD2 deficiency

Chronic epithelial defects of the cornea, which are usually associated with severe dry eye disease, diabetes mellitus, chemical injuries or neurotrophic keratitis, as well as aging, are an unmet clinical need. CDGSH Iron Sulfur Domain 2 (CISD2) is the causative gene for Wolfram syndrome 2 (WFS2; MIM 604928). CISD2 protein is significantly decreased in the corneal epithelium of patients with various corneal epithelial diseases. Here we summarize the most updated publications and discuss the central role of CISD2 in corneal repair, as well as providing new results describing how targeting Ca2+-dependent pathways can improve corneal epithelial regeneration. This review mainly focuses on the following topics. Firstly, an overview of the cornea and of corneal epithelial wound healing. The key players involved in this process, such as Ca2+, various growth factors/cytokines, extracellular matrix remodeling, focal adhesions and proteinases, are briefly discussed. Secondly, it is well known that CISD2 plays an essential role in corneal epithelial regeneration via the maintenance of intracellular Ca2+ homeostasis. CISD2 deficiency dysregulates cytosolic Ca2+, impairs cell proliferation and migration, decreases mitochondrial function and increases oxidative stress. As a consequence, these abnormalities bring about poor epithelial wound healing and this, in turn, will lead to persistent corneal regeneration and limbal progenitor cell exhaustion. Thirdly, CISD2 deficiency induces three distinct Ca2+-dependent pathways, namely the calcineurin, CaMKII and PKCα signaling pathways. Intriguingly, inhibition of each of the Ca2+-dependent pathways seems to reverse cytosolic Ca2+ dysregulation and restore cell migration during corneal wound healing. Notably, cyclosporin, an inhibitor of calcineurin, appears to have a dual effect on both inflammatory and corneal epithelial cells. Finally, corneal transcriptomic analyses have revealed that there are six major functional groupings of differential expression genes when CISD2 deficiency is present: (1) inflammation and cell death; (2) cell proliferation, migration and differentiation; (3) cell adhesion, junction and interaction; (4) Ca2+ homeostasis; (5) wound healing and extracellular matrix; and (6) oxidative stress and aging. This review highlights the importance of CISD2 in corneal epithelial regeneration and identifies the potential of repurposing venerable FDA-approved drugs that target Ca2+-dependent pathways for new uses, namely treating chronic epithelial defects of the cornea.

Phosphorylcholine and KR12-Containing Corneal Implants in HSV-1-Infected Rabbit Corneas.

Severe HSV-1 infection can cause blindness due to tissue damage from severe inflammation. Due to the high risk of graft failure in HSV-1-infected individuals, cornea transplantation to restore vision is often contraindicated. We tested the capacity for cell-free biosynthetic implants made from recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) to suppress inflammation and promote tissue regeneration in the damaged corneas. To block viral reactivation, we incorporated silica dioxide nanoparticles releasing KR12, the small bioactive core fragment of LL37, an innate cationic host defense peptide produced by corneal cells. KR12 is more reactive and smaller than LL37, so more KR12 molecules can be incorporated into nanoparticles for delivery. Unlike LL37, which was cytotoxic, KR12 was cell-friendly and showed little cytotoxicity at doses that blocked HSV-1 activity in vitro, instead enabling rapid wound closure in cultures of human epithelial cells. Composite implants released KR12 for up to 3 weeks in vitro. The implant was also tested in vivo on HSV-1-infected rabbit corneas where it was grafted by anterior lamellar keratoplasty. Adding KR12 to RHCIII-MPC did not reduce HSV-1 viral loads or the inflammation resulting in neovascularization. Nevertheless, the composite implants reduced viral spread sufficiently to allow stable corneal epithelium, stroma, and nerve regeneration over a 6-month observation period.

Interference of sympathetic overactivation restores limbal stem/progenitor cells function and accelerates corneal epithelial wound healing in diabetic mice

Diabetic keratopathy (DK), the diabetic complication in the cornea, is characterized by the delayed epithelial regeneration and sensory nerve degeneration. The involvement of limbal stem/progenitor cells (LSPCs) dysfunction has been reported, however the pathogenic mechanisms remain unclear. Here, we confirmed the dysfunction of LSPCs in diabetic mouse and human corneas. The sympathetic nerve in the cornea was adjacent to LSPCs, and the sympathetic overactivation was found in diabetic mice. Surgical and pharmacological ablation of sympathetic nerves rescued the LSPCs function and promoted corneal epithelial regeneration in diabetic mice. In contrast, both topical norepinephrine (NE) application and chemogenetic sympathetic overactivation directly impaired the stemness and proliferation characteristics of LSPCs, as well as the normal epithelial regeneration. Moreover, we identified that β2-adrenoceptor (Adrb2) was the predominant adrenergic receptor expressed in LSPCs by corneal limbal single-cell sequencing and real time PCR (RT-PCR) analysis of sorted LSPCs. The Adrb2 knockout mice exhibited the enhancement of epithelial regeneration and LSPCs function, compared with the wild-type mice. Similarly, topical application of the Adrb2 specific antagonist ICI 118, 551 effectively accelerated diabetic corneal epithelial regeneration with the restored LSPCs function. Mechanistically, sonic hedgehog (Shh) activity mediated the downstream effects of NE-Adrb2 signaling pathway in regulating LSPCs and epithelial regeneration. Taken together, our data revealed the involvement of sympathetic overactivation in the impairment of diabetic LSPCs function and corneal epithelial regeneration through the NE-Adrb2-Shh signaling pathway. The interference of sympathetic overactivation may provide novel treatment strategies for diabetic keratopathy.