PAX6/CXCL14 regulatory axis promotes the repair of corneal injury by enhancing corneal epithelial cell proliferation.

BACKGROUND: Corneal injuries, often leading to severe vision loss or blindness, have traditionally been treated with the belief that limbal stem cells (LSCs) are essential for repair and homeostasis, while central corneal epithelial cells (CCECs) were thought incapable of such repair. However, our research reveals that CCECs can fully heal and maintain the homeostasis of injured corneas in rats, even without LSCs. We discovered that CXCL14, under PAX6's influence, significantly boosts the stemness, proliferation, and migration of CCECs, facilitating corneal wound healing and homeostasis. This finding introduces CXCL14 as a promising new drug target for corneal injury treatment. METHODS: To investigate the PAX6/CXCL14 regulatory axis's role in CCECs wound healing, we cultured human corneal epithelial cell lines with either increased or decreased expression of PAX6 and CXCL14 using adenovirus transfection in vitro. Techniques such as coimmunoprecipitation, chromatin immunoprecipitation, immunofluorescence staining, western blot, real-time PCR, cell colony formation, and cell cycle analysis were employed to validate the axis's function. In vivo, a rat corneal epithelial injury model was developed to further confirm the PAX6/CXCL14 axis's mechanism in repairing corneal damage and maintaining corneal homeostasis, as well as to assess the potential of CXCL14 protein as a therapeutic agent for corneal injuries. RESULTS: Our study reveals that CCECs naturally express high levels of CXCL14, which is significantly upregulated by PAX6 following corneal damage. We identified SDC1 as CXCL14's receptor, whose engagement activates the NF-κB pathway to stimulate corneal repair by enhancing the stemness, proliferative, and migratory capacities of CCECs. Moreover, our research underscores CXCL14's therapeutic promise for corneal injuries, showing that recombinant CXCL14 effectively accelerates corneal healing in rat models. CONCLUSION: CCECs play a critical and independent role in the repair of corneal injuries and the maintenance of corneal homeostasis, distinct from that of LSCs. The PAX6/CXCL14 regulatory axis is pivotal in this process. Additionally, our research demonstrates that the important function of CXCL14 in corneal repair endows it with the potential to be developed into a novel therapeutic agent for treating corneal injuries.

INOS ablation promotes corneal wound healing via activation of Akt signaling.

Corneal injury leads to impaired normal structure of the cornea. Improving the wound healing process in epithelial cells significantly contributes to ocular damage treatments. Here, we aimed to investigate the potential mechanisms of nitric oxide (NO) and its mediator, inducible nitric oxide synthase (iNOS), in the process of corneal wound healing. We established a corneal injury model of iNOS-/- mice, and treated human corneal epithelial cell lines (HCE-2) with the iNOS inhibitor L-INL, with or without NO replenishment by supplying sodium nitroferricyanide dihydrate (SNP). Our findings showed that inhibition of NO/iNOS accelerated corneal repair, enhanced uPAR (a receptor protein indicating the migration ability), and improved epithelial cell migration. Furthermore, NO/iNOS ablation activated Akt phosphorylation, reduced neutrophil marker protein MPO expression, and downregulated the transcription of inflammation cytokines CXCL-1, CXCL-2, IL-1ß, IL-6, and TNF-α. However, the protective effects of NO/iNOS inhibition are significantly reduced by NO replenishment when treated with SNP. Therefore, we confirmed that inhibiting NO/iNOS improved the corneal wound healing by facilitating epithelial cell migration and reducing inflammatory reactions, which might be related to the activation of the Akt signaling pathway.

Mesenchymal Stem Cell-Laden In Situ-Forming Hydrogel for Preventing Corneal Stromal Opacity.

PURPOSE: The aims of this study were to construct a mesenchymal stem cell (MSC)-laden in situ-forming hydrogel and study its effects on preventing corneal stromal opacity. METHODS: The native gellan gum was modified by high temperature and pressure, and the rabbit bone marrow MSCs were encapsulated before adding Ca 2+ to initiate cross-linking. The effects of the hydrogel on 3D culture and gene expression of the rabbit bone marrow MSCs were observed in vitro. Then, the MSC-hydrogel was used to repair corneal stromal injury in New Zealand white rabbits within 28 days postoperation. RESULTS: The short-chain gellan gum solution has a very low viscosity (<0.1 Pa·s) that is ideal for encapsulating cells. Moreover, mRNA expressions of 3D-cultured MSCs coding for corneal stromal components (decorin, lumican, and keratocan) were upregulated (by 127.8, 165.5, and 25.4 times, respectively) ( P < 0.05) on day 21 in vitro and were verified by Western blotting results. For the in vivo study, the corneal densitometry of the experimental group was (20.73 ± 1.85) grayscale units which was lower than the other groups ( P < 0.05). The MSC-hydrogel downregulated mRNA expression coding for fibrosis markers (α-smooth muscle actin, vimentin, collagen type 5-α1, and collagen type 1-α1) in the rabbit corneal stroma. Furthermore, some of the 5-ethynyl-2'-deoxyuridine (EdU)-labeled MSCs integrated into the upper corneal stroma and expressed keratocyte-specific antigens on day 28 postoperation. CONCLUSIONS: The short-chain gellan gum allows MSCs to slowly release to the corneal stromal defect and prevent corneal stromal opacity. Some of the implanted MSCs can integrate into the corneal stroma and differentiate into keratocytes.

Endogenous TSG-6 modulates corneal inflammation following chemical injury.

PURPOSE: Tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) is upregulated in various pathophysiological contexts, where it has a diverse repertoire of immunoregulatory functions. Herein, we investigated the expression and function of TSG-6 during corneal homeostasis and after injury. METHODS: Human corneas, eyeballs from BALB/c (TSG-6+/+), TSG-6+/- and TSG-6-/- mice, human immortalized corneal epithelial cells and murine corneal epithelial progenitor cells were prepared for immunostaining and real time PCR analysis of endogenous expression of TSG-6. Mice were subjected to unilateral corneal debridement or alkali burn (AB) injuries and wound healing assessed over time using fluorescein stain, in vivo confocal microscopy and histology. RESULTS: TSG-6 is endogenously expressed in the human and mouse cornea and established corneal epithelial cell lines and is upregulated after injury. A loss of TSG-6 has no structural and functional effect in the cornea during homeostasis. No differences were noted in the rate of corneal epithelial wound closure between BALB/c, TSG-6+/- and TSG-6-/- mice. TSG-6-/- mice presented decreased inflammatory response within the first 24 h of injury and accelerated corneal wound healing following AB when compared to control mice. CONCLUSION: TSG-6 is endogenously expressed in the cornea and upregulated after injury where it propagates the inflammatory response following chemical injury.

BMP4 inhibits corneal neovascularization by interfering with tip cells in angiogenesis.

Corneal neovascularization (CNV) can lead to impaired corneal transparency, resulting in vision loss or blindness. The primary pathological mechanism underlying CNV is an imbalance between pro-angiogenic and anti-angiogenic factors, with inflammation playing a crucial role. Notably, a vascular endothelial growth factor（VEGF）-A gradient triggers the selection of single endothelial cells（ECs） into primary tip cells that guide sprouting, while a dynamic balance between tip and stalk cells maintains a specific ratio to promote CNV. Despite the central importance of tip-stalk cell selection and shuffling, the underlying mechanisms remain poorly understood. In this study, we examined the effects of bone morphogenetic protein 4 (BMP4) on VEGF-A-induced lumen formation in human umbilical vein endothelial cells (HUVECs) and CD34-stained tip cell formation. In vivo, BMP4 inhibited CNV caused by corneal sutures. This process was achieved by BMP4 decreasing the protein expression of VEGF-A and VEGFR2 in corneal tissue after corneal suture injury. By observing the ultrastructure of the cornea, BMP4 inhibited the sprouting of tip cells and brought forward the appearance of intussusception. Meanwhile, BMP4 attenuated the inflammatory response by inhibiting neutrophil extracellular traps （NETs）formation through the NADPH oxidase-2（NOX-2）pathway. Our results indicate that BMP4 inhibits the formation of tip cells by reducing the generation of NETs, disrupting the dynamic balance of tip and stalk cells and thereby inhibiting CNV, suggesting that BMP4 may be a potential therapeutic target for CNV.

Metabolomics for identifying pathways involved in vesicating agent lewisite-induced corneal injury.

Lewisite (LEW) is an arsenical vesicant that can be a potentially dangerous chemical warfare agent (CWA). Eyes are particularly susceptible to vesicant induced injuries and ocular LEW exposure can act swiftly, causing burning of eyes, edema, inflammation, cell death and even blindness. In our previous studies, we developed a LEW exposure-induced corneal injury model in rabbit and showed increased inflammation, neovascularization, cell death, and structural damage to rabbit corneas upon LEW exposure. In the present study, we further assessed the metabolomic changes to delineate the possible mechanisms underlying the LEW-induced corneal injuries. This information is vital and could help in the development of effective targeted therapies against ocular LEW injuries. Thus, the metabolomic changes associated with LEW exposures in rabbit corneas were assessed as a function of time, to delineate pathways from molecular perturbations at the genomic and proteomic levels. New Zealand white rabbit corneas (n = 3-6) were exposed to LEW vapor (0.2 mg/L; flow rate: 300 ml/min) for 2.5 min (short exposure; low dose) or 7.5 min (long-exposure; high dose) and then collected at 1, 3, 7, or 14 days post LEW exposure. Samples were prepared using the automated MicroLab STAR® system, and proteins precipitated to recover the chemically diverse metabolites. Metabolomic analysis was carried out by reverse phase UPLC-MS/MS and gas chromatography (GC)-MS. The data obtained were analyzed using Metabolon's software. The results showed that LEW exposures at high doses were more toxic, particularly at the day 7 post exposure time point. LEW exposure was shown to dysregulate metabolites associated with all the integral functions of the cornea and cause increased inflammation and immune response, as well as generate oxidative stress. Additionally, all important metabolic functions of the cells were also affected: lipid and nucleotide metabolism, and energetics. The high dose LEW exposures were more toxic, particularly at day 7 post LEW exposure (>10-fold increased levels of histamine, quinolinate, N-acetyl-ß-alanine, GMP, and UPM). LEW exposure dysregulated integral functions of the cornea, caused inflammation and heightened immune response, and generated oxidative stress. Lipid and nucleotide metabolism, and energetics were also affected. The novel information about altered metabolic profile of rabbit cornea following LEW exposure could assist in delineating complex molecular events; thus, aid in identifying therapeutic targets to effectively ameliorate ocular trauma.

Reducing Sialylation Enhances Electrotaxis of Corneal Epithelial Cells.

Corneal wound healing is a complex biological process that integrates a host of different signals to coordinate cell behavior. Upon wounding, there is the generation of an endogenous wound electric field that serves as a powerful cue to guide cell migration. Concurrently, the corneal epithelium reduces sialylated glycoforms, suggesting that sialylation plays an important role during electrotaxis. Here, we show that pretreating human telomerase-immortalized corneal epithelial (hTCEpi) cells with a sialyltransferase inhibitor, P-3FAX-Neu5Ac (3F-Neu5Ac), improves electrotaxis by enhancing directionality, but not speed. This was recapitulated using Kifunensine, which inhibits cleavage of mannoses and therefore precludes sialylation on N-glycans. We also identified that 3F-Neu5Ac enhanced the responsiveness of the hTCEpi cell population to the electric field and that pretreated hTCEpi cells showed increased directionality even at low voltages. Furthermore, when we increased sialylation using N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), hTCEpi cells showed a decrease in both speed and directionality. Importantly, pretreating enucleated eyes with 3F-Neu5Ac significantly improved re-epithelialization in an ex vivo model of a corneal injury. Finally, we show that in hTCEpi cells, sialylation is increased by growth factor deprivation and reduced by PDGF-BB. Taken together, our results suggest that during corneal wound healing, reduced sialylated glycoforms enhance electrotaxis and re-epithelialization, potentially opening new avenues to promote corneal wound healing.

ROCK Inhibitors as an Alternative Therapy for Corneal Grafting: A Systematic Review.

Currently, corneal blindness is affecting >10 million individuals worldwide, and there is a significant unmet medical need because only 1.5% of transplantation needs are met globally due to a lack of high-quality grafts. In light of this global health disaster, researchers are developing corneal substitutes that can resemble the human cornea in vivo and replace human donor tissue. Thus, this review examines ROCK (Rho-associated coiled-coil containing protein kinases) inhibitors as a potential corneal wound-healing (CWH) therapy by reviewing the existing clinical and nonclinical findings. The systematic review was done from PubMed, Scopus, Web of Science, and Google Scholar for CWH, corneal injury, corneal endothelial wound healing, ROCK inhibitors, Fasudil, Netarsudil, Ripasudil, Y-27632, clinical trial, clinical study, case series, case reports, preclinical study, in vivo, and in vitro studies. After removing duplicates, all downloaded articles were examined. The literature search included the data till January 2023. This review summarized the results of ROCK inhibitors in clinical and preclinical trials. In a clinical trial, various ROCK inhibitors improved CWH in individuals with open-angle glaucoma, cataract, iris cyst, ocular hypertension, and other ocular diseases. ROCK inhibitors also improved ocular wound healing by increasing cell adhesion, migration, and proliferation in vitro and in vivo. ROCK inhibitors have antifibrotic, antiangiogenic, anti-inflammatory, and antiapoptotic characteristics in CWH, according to the existing research. ROCK inhibitors were effective topical treatments for corneal infections. Ripasudil, Y-27632, H-1152, Y-39983, and AMA0526 are a few new ROCK inhibitors that may help CWH and replace human donor tissue.

Rational design and synthesis of lumican stapled peptides for promoting corneal wound healing.

PURPOSE: Lumican is a major extracellular matrix (ECM) component in the cornea that is upregulated after injury and promotes corneal wound healing. We have recently shown that peptides designed based on the 13 C-terminal amino acids of lumican (LumC13 and LumC13C-A) are able to recapitulate the effects of lumican on promoting corneal wound healing. Herein we used computational chemistry to develop peptide mimetics derived from LumC13C-A with increased stability and half-life that are biologically active and non-toxic, thereby promoting corneal wound healing with increased pharmacological potential. METHODS: Different peptides staples were rationalized using LumC13C-A sequence by computational chemistry, docked to TGFßRI and the interface binding energies compared. Lowest scoring peptides were synthesized, and the toxicity of peptides tested using CCK8-based cell viability assay. The efficacy of the stapled peptides at promoting corneal wound healing was tested using a proliferation assay, an in vitro scratch assay using human corneal epithelial cells and an in vivo murine corneal debridement wound healing model. RESULTS: Binding free energies were calculated using MMGBSA algorithm, and peptides LumC13C and LumC13S5 displayed superior binding to ALK5 compared to the non-stapled peptide LumC13C-A. The presence of the hydrocarbon staple in LumC13C enhances the stability of the α-helical conformation, thereby facilitating more optimal interactions with the ALK5 receptor. The stapled peptides do not present cytotoxic effects on human corneal epithelial cells at a 300 nM concentration. Similar to lumican and LumC13C-A, both C13C and LumC13S5 significantly promote corneal wound healing both in vitro and in vivo. CONCLUSIONS: Highly stable and non-toxic stapled peptides designed based on LumC13, significantly promote corneal wound healing. As a proof of principle, our data shows that more stable and pharmacologically relevant peptides can be designed based on endogenous peptide sequences for treating various corneal pathologies.

Long Noncoding RNA MIATNB Regulates Hyperosmotic Stress-induced Corneal Epithelial Cell Injury by Inhibiting Autophagy in Dry Eye Disease.

PURPOSE: Dry eye disease (DED) has a complex etiology and the roles of long noncoding RNAs (lncRNAs) in its pathophysiology are not completely understood. Autophagy is a self-eating process important for cell survival and homeostasis. The present study explored the role of myocardial infarction-associated transcript neighbor (MIATNB) long non-coding RNA in hyperosmolarity-induced autophagy and apoptosis in human corneal epithelial cell (HCEC)-based model of dry eye disease. METHODS: In vitro assays were performed with a human SV40 immortalized corneal epithelial cell line. Different concentrations of NaCl were used to create hyperosmolarity. HCECs were cultured in presence of 70-120 mM NaCl for 24 h to create an in vitro model of dry eye. RT-qPCR was performed to assess the expression of dry eye related LC3B, ATG16L, BECN1, ATG1, ATG7, ATG13, ATG5, ATG10, and ATG101 mRNAs and western blot analysis of LC3B and P62 and RFP -GFP-tagged LC3. Flow cytometry and western blot analysis of caspase 3, BCL2 and BAX were performed to detect apoptosis. Chloroquine (CQ) was used to inhibit autophagy pharmacologically. RESULTS: Autophagy flux was activated in HCECs subjected to hyperosmotic stress. Hyperosmolarity activated apoptosis and inhibited HCEC migration and autophagy. Hyperosmolarity upregulated MIATNB expression, while MIATNB knockdown inhibited autophagosome degradation and promoted HCEC apoptosis. Under hyperosmolar conditions, MIATNB knockdown also inhibited the degradation of autophagolysosomes and stimulated HCEC apoptosis. CONCLUSION: MIATNB plays a vital role in dry eye pathogenesis and serves as a bridge between autophagy and apoptosis. Targeting MIATNB for DED treatment should be further evaluated.

Rapamycin inhibits corneal inflammatory response and neovascularization in a mouse model of corneal alkali burn.

Alkali burn-induced corneal injury often causes inflammation and neovascularization and leads to compromised vision. We previously reported that rapamycin ameliorated corneal injury after alkali burns by methylation modification. In this study, we aimed to investigate the rapamycin-medicated mechanism against corneal inflammation and neovascularization. Our data showed that alkali burn could induce a range of different inflammatory response, including a stark upregulation of pro-inflammatory factor expression and an increase in the infiltration of myeloperoxidase- and F4/80-positive cells from the corneal limbus to the central stroma. Rapamycin effectively downregulated the mRNA expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), toll-like receptor 4 (TLR4), nucleotide binding oligomerization domain-like receptors (NLR) family pyrin domain-containing 3 (NLRP3), and Caspase-1, and suppressed the infiltration of neutrophils and macrophages. Inflammation-related angiogenesis mediated by matrix metalloproteinase-2 (MMP-2) and rapamycin restrained this process by inhibiting the TNF-α upregulation in burned corneas of mice. Rapamycin also restrained corneal alkali burn-induced inflammation by regulating HIF-1α/VEGF-mediated angiogenesis and the serum cytokines TNF-α, IL-6, Interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The findings of this study indicated rapamycin may reduce inflammation-associated infiltration of inflammatory cells, shape the expression of cytokines, and balance the regulation of MMP-2 and HIF-1α-mediated inflammation and angiogenesis by suppressing mTOR activation in corneal wound healing induced by an alkali injury. It offered novel insights relevant for a potent drug for treating corneal alkali burn.

Enhanced adipose-derived stem cells with IGF-1-modified mRNA promote wound healing following corneal injury.

The cornea serves as an important barrier structure to the eyeball and is vulnerable to injuries, which may lead to scarring and blindness if not treated promptly. To explore an effective treatment that could achieve multi-dimensional repair of the injured cornea, the study herein innovatively combined modified mRNA (modRNA) technologies with adipose-derived mesenchymal stem cells (ADSCs) therapy, and applied IGF-1 modRNA (modIGF1)-engineered ADSCs (ADSCmodIGF1) to alkali-burned corneas in mice. The therapeutic results showed that ADSCmodIGF1 treatment could achieve the most extensive recovery of corneal morphology and function when compared not only with simple ADSCs but also IGF-1 protein eyedrops, which was reflected by the healing of corneal epithelium and limbus, the inhibition of corneal stromal fibrosis, angiogenesis and lymphangiogenesis, and also the repair of corneal nerves. In vitro experiments further proved that ADSCmodIGF1 could more significantly promote the activity of trigeminal ganglion cells and maintain the stemness of limbal stem cells than simple ADSCs, which were also essential for reconstructing corneal homeostasis. Through a combinatorial treatment regimen of cell-based therapy with mRNA technology, this study highlighted comprehensive repair in the damaged cornea and showed the outstanding application prospect in the treatment of corneal injury.

C1q/TNF-Related Proteins 1, 6 and 8 Are Involved in Corneal Epithelial Wound Closure by Targeting Relaxin Receptor RXFP1 In Vitro.

Inadequate wound healing of ocular surface injuries can lead to permanent visual impairment. The relaxin ligand-receptor system has been demonstrated to promote corneal wound healing through increased cell migration and modulation of extracellular matrix formation. Recently, C1q/tumor necrosis factor-related protein (CTRP) 8 was identified as a novel interaction partner of relaxin receptor RXFP1. Additional data also suggest a role for CTRP1 and CTRP6 in RXFP1-mediated cAMP signaling. However, the role of CTRP1, CTRP6 and CTRP8 at the ocular surface remains unclear. In this study, we investigated the effects of CTRP1, CTRP6, and CTRP8 on epithelial ocular surface wound closure and their dependence on the RXFP1 receptor pathway. CTRP1, CTRP6, and CTRP8 expression was analyzed by RT-PCR and immunohistochemistry in human tissues and cell lines derived from the ocular surface and lacrimal apparatus. In vitro ocular surface wound modeling was performed using scratch assays. We analyzed the effects of recombinant CTRP1, CTRP6, and CTRP8 on cell proliferation and migration in human corneal and conjunctival epithelial cell lines. Dependence on RXFP1 signaling was established by inhibiting ligand binding to RXFP1 using a specific anti-RXFP1 antibody. We detected the expression of CTRP1, CTRP6, and CTRP8 in human tissue samples of the cornea, conjunctiva, meibomian gland, efferent tear ducts, and lacrimal gland, as well as in human corneal, conjunctival, and meibomian gland epithelial cell lines. Scratch assays revealed a dose-dependent increase in the closure rate of surface defects in human corneal epithelial cells after treatment with CTRP1, CTRP6, and CTRP8, but not in conjunctival epithelial cells. Inhibition of RXFP1 fully attenuated the effect of CTRP8 on the closure rate of surface defects in human corneal epithelial cells, whereas the CTRP1 and CTRP6 effects were not completely suppressed. Conclusions: Our findings demonstrate a novel role for CTRP1, CTRP6, and CTRP8 in corneal epithelial wound closure and suggest an involvement of the relaxin receptor RXFP1 signaling pathway. This could be a first step toward new approaches for pharmacological and therapeutic intervention.

Extracellular vesicles derived from mouse adipose-derived mesenchymal stem cells promote diabetic corneal epithelial wound healing through NGF/TrkA pathway activation involving dendritic cells.

Diabetic keratopathy (DK) is a common ocular complication of diabetes in which the dendritic cells (DCs)-mediated inflammatory response plays an important role. Nerve growth factor (NGF)/Tropomyosin receptor kinase A (TrkA)-mediated inhibition of the nuclear factor kappa B (NF-κB) pathway can reduce inflammatory cytokine production. Extracellular vesicles (EVs) derived from mouse adipose-derived mesenchymal stem cells (mADSC-EVs) have been explored extensively as treatments for degenerative eye disease. However, mADSC-EVs is poorly studied in the DK models. In this study, we investigated the anti-inflammatory effects of mADSC-EVs and explored the underlying mechanisms in vitro and in vivo DK models. Our results showed that mADSC-EVs have significant therapeutic effects including increasing tear volume and the ratio of lacrimal gland/body weight, promoting corneal nerve regeneration, and sensation recovery in streptozotocin (STZ)-induced DK mice. In addition, mADSC-EVs significantly reduced the inflammatory response involving DCs, consistently up-regulated protein expression of the NGF/TrkA pathway, and importantly, reduced lipopolysaccharide (LPS)-mediated IL-6 and TNF-α expression and directly dependent on TrkA in the induced culture of bone marrow-derived DCs (BMDCs). Taken together, our findings revealed that mADSC-EVs promoted diabetic corneal epithelial wound healing through NGF/TrkA pathway activation involving DCs. Given the significant therapeutic efficacy of mADSC-EVs and its clinical application, mADSC-EVs appears to be a promising new therapy for DK.

Zeb1 facilitates corneal epithelial wound healing by maintaining corneal epithelial cell viability and mobility.

The cornea is the outmost ocular tissue and plays an important role in protecting the eye from environmental insults. Corneal epithelial wounding provokes pain and fear and contributes to the most ocular trauma emergency assessments worldwide. ZEB1 is an essential transcription factor in development; but its roles in adult tissues are not clear. We identify Zeb1 is an intrinsic factor that facilitates corneal epithelial wound healing. In this study, we demonstrate that monoallelic deletion of Zeb1 significantly expedites corneal cell death and inhibits corneal epithelial EMT-related cell migration upon an epithelial debridement. We provide evidence that Zeb1-regulation of corneal epithelial wound healing is through the repression of genes required for Tnfa-induced epithelial cell death and the induction of genes beneficial for epithelial cell migration. We suggest utilizing TNF-α antagonists would reduce TNF/TNFR1-induced cell death in the corneal epithelium and inflammation in the corneal stroma to help corneal wound healing.

Upadacitinib inhibits corneal inflammation and neovascularization by suppressing M1 macrophage infiltration in the corneal alkali burn model.

Alkali burn-induced corneal inflammation and subsequent corneal neovascularization (CNV) are major causes of corneal opacity and vision loss. M1 macrophages play a central role in inflammation and CNV. Therefore, modulation of M1 macrophage polarization is a promising strategy for corneal alkali burns. Here, we illustrate the effect and underlying mechanisms of upadacitinib on corneal inflammation and CNV induced by alkali burns in mice. The corneas of BALB/c mice were administered with 1 M NaOH for 30 s and randomly assigned to the vehicle group and the upadacitinib-treated group. Corneal opacity and corneal epithelial defects were assessed clinically. Quantitative real-time PCR (qRT-PCR), immunohistochemistry, and western blot analysis were performed to detect M1 macrophage polarization and CD31+ corneal blood vessels. The results showed that upadacitinib notably decreased corneal opacity, and promoted corneal wound healing. On day 7 and 14 after alkali burns, upadacitinib significantly suppressed CNV. Corneal alkali injury caused M1 macrophage recruitment in the cornea. In contrast to the vehicle, upadacitinib suppressed M1 macrophage infiltration and decreased the mRNA expression levels of inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1ß, and vascular endothelial growth factor A (VEGF-A) in alkali-injured corneas. Moreover, upadacitinib dose-dependently inhibited M1 macrophage polarization by suppressing interferon (IFN)-γ-/lipopolysaccharide-stimulated STAT1 activation in vitro. Our findings reveal that upadacitinib can efficiently alleviate alkali-induced corneal inflammation and neovascularization by inhibiting M1 macrophage infiltration. These data demonstrate that upadacitinib is an effective drug for the treatment of corneal alkali burns.

Effect of TRPM8 Functional Loss on Corneal Epithelial Wound Healing in Mice.

Purpose: To reveal the role of cold-sensing transient receptor potential melastatin 8 (TRPM8) channels in corneal epithelial wound healing. Methods: Cold sensitivity, tear production, corneal thickness, and corneal opacity assessments were used to evaluate the effect of Trpm8 knockout on the ocular surface. A corneal epithelial wounding model was generated by scraping the corneal epithelium once or multiple times using C57BL/6J (wild-type [WT]) and Trpm8-/- mice. The processes of corneal epithelial repair and corneal epitheliopathy were observed and recorded. Corneas were collected for sequencing, immunofluorescence staining, hematoxylin and eosin staining, and quantitative PCR. Results: The perception of coldness, basal tear secretion, and corneal thickness were decreased in young Trpm8-/- mice compared with those in WT mice, except for the corneal sensitivity. Corneal opacity and increased corneal thickness were observed in aged Trpm8-/- mice. TRPM8 deficiency promoted corneal epithelial wound closure, consistent with the observed increase in Ki67-positive epithelial cells, and the pharmacological activation of TRPM8 in WT mice delayed corneal re-epithelization. After subjecting mice to multiple injuries, squamous metaplasia emerged in Trpm8-/- corneas, as verified by cytokeratin-1 and small proline-rich protein 1B-positive staining. The IFN-ß and IFN-γ signaling pathways were significantly activated in Trpm8-/- mice, which was confirmed based on the up-regulated expression of the key mediators, signal transducer and activator of transcription-1 and phosphor-signal transducer and activator of transcription-1, as well as the induction of IFN-stimulated genes, compared with levels in WT mice. Conclusions: In corneal wound healing, the loss of TRPM8 function could promote epithelial repair, but predispose the cornea to epithelial lesions.

Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model.

FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.

Development of In Vitro Dry Eye Models to Study Proliferative and Anti-Inflammatory Effects of Allogeneic Serum Eye Drops.

This study aimed to develop valid in vitro models for preclinical evaluation of proliferative and anti-inflammatory effects of human allogeneic serum eye drops for dry eye disease (DED) treatment. A DED wound healing model was developed by analyzing the influence of coating and serum concentrations on human corneal epithelial (HCE-T) wound closure. Further, intralaboratory variance, freeze-thaw cycle effects, donor variability and stability assays were conducted. Interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) were used to induce the gene expression of matrix metalloproteinase 9 (MMP9), cyclooxygenase 2 (COX2), transforming growth factor-ß (TGFß) and IL-1ß. MMP9 induction was optimized using a design-of-experiments (DoE) approach and applied to examine serum under static and dynamic conditions. MMP9 protein expression was analyzed by ELISA. The DED wound healing model detected proliferative effects of serum down to 1% with a small intralaboratory variance. Serum stability was shown over six months, donor variance could be detected, and freeze-thaw cycle effects did not affect wound closure. Serum decreased MMP9 expression on the gene and protein levels. The induction method was successfully optimized using DoE modeling and transferred to a dynamic setting mimicking tear film fluidics. The DED wound healing and inflammatory DED model present useful in vitro models for the preclinical evaluation of allogeneic serum eye drops without the use of animal experiments.

Characterization of C-X-C chemokine receptor type 5 in the cornea and role in the inflammatory response after corneal injury.

C-X-C chemokine receptor type 5 (CXCR5) regulates inflammatory responses in ocular and non-ocular tissues. However, its expression and role in the cornea are still unknown. Here, we report the expression of CXCR5 in human cornea in vitro and mouse corneas in vivo, and its functional role in corneal inflammation using C57BL/6J wild-type (CXCR5+/+) and CXCR5-deficient (CXCR5-/-) mice, topical alkali injury, clinical eye imaging, histology, immunofluorescence, PCR, qRT-PCR, and western blotting. Human corneal epithelial cells, stromal fibroblasts, and endothelial cells demonstrated CXCR5 mRNA and protein expression in PCR, and Western blot analyses, respectively. To study the functional role of CXCR5 in vivo, mice were divided into four groups: Group-1 (CXCR5+/+ alkali injured cornea; n = 30), Group-2 (CXCR5-/- alkali injured cornea; n = 30), Group-3 (CXCR5+/+ naïve cornea; n = 30), and Group-4 (CXCR5-/- naïve cornea; n = 30). Only one eye was wounded with alkali. Clinical corneal evaluation and imaging were performed before and after injury. Mice were euthanized 4 h, 3 days, or 7 days after injury, eyes were excised and used for histology, immunofluorescence, and qRT-PCR. In clinical eye examinations, CXCR5-/- mouse corneas showed ocular health akin to the naïve corneas. Alkali injured CXCR5+/+ mouse corneas showed significantly increased mRNA (p < 0.001) and protein (p < 0.01 or p < 0.0001) levels of the CXCR5 compared to the naïve corneas. Likewise, alkali injured CXCR5-/- mouse corneas showed remarkably amplified inflammation in clinical eye exams in live animals. The histological and molecular analyses of these corneas post euthanasia exhibited markedly augmented inflammatory cells in H&E staining and significant CD11b + cells in immunofluorescence (p < 0.01 or < 0.05); and tumor necrosis factor-alpha (TNFα; p < 0.05), cyclooxygenase 2 (COX-2; p < 0.0001), interleukin (IL)-1ß (p < 0.0001), and IL-6 (p < 0.0001 or < 0.01) mRNA expression compared to the CXCR5+/+ mouse corneas. Interestingly, CXCR5-/- alkali injured corneas also showed altered mRNA expression of fibrotic alpha smooth muscle actin (α-SMA; p > 0.05) and angiogenic vascular endothelial growth factor (VEGF; p < 0.01) compared to the CXCR5+/+ alkali injured corneas. In summary, the CXCR5 gene is expressed in all three major layers of the cornea and appears to influence corneal inflammatory and repair events post-injury in vivo. More studies are warranted to tease the mechanistic role of CXCR5 in corneal inflammation and wound healing.