Proliferation Of Corneal Epithelial Cells Research Articles

RGD and (−)-epigallocatechin-3-gallate modification enhanced biostability, bioactive and biofunctionality of acellular porcine cornea stroma crosslinked with oxidized dextran

The cornea is the main refractive element of the eye. Corneal transplantation is the foremost choice for addressing severe corneal diseases or trauma; however, corneal donors are difficult to meet the large clinical demand. Despite the clinical adoption of acellular porcine corneas stroma (APCS), their utility is limited by complications, such as post-implantation swelling, suture loosening, and rapid degradation. In this study, the APCS was crosslinked with oxidized dextran (ODEX) to enhance their biostability and preserve their original optical properties. Furthermore, to meet clinical indications in different pathological environments, it was double-modified with Arg-Gly-Asp (RGD) and (−)-epigallocatechin-3-gallate (EGCG) to prepare an artificial bio-cornea (OD-RE-APCS) with good bioactivity and biofunctionality. Preliminary experimental findings demonstrate that OD-RE-APCS effectively improves the mechanical, swelling, and degradation properties of APCS, enhances the adhesion, proliferation, and migration of corneal epithelial cells, and has anti-inflammatory, anti-oxidant and anti-angiogenic functions, which makes it a promising artificial bio-cornea.

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

BackgroundCorneal 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.MethodsTo 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.ResultsOur 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.ConclusionCCECs 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.

Elucidating the mechanism of corneal epithelial cell repair: unraveling the impact of growth factors.

The repair mechanism for corneal epithelial cell injuries encompasses migration, proliferation, and differentiation of corneal epithelial cells, and extracellular matrix remodeling of the stromal structural integrity. Furthermore, it involves the consequential impact of corneal limbal stem cells (LSCs). In recent years, as our comprehension of the mediating mechanisms underlying corneal epithelial injury repair has advanced, it has become increasingly apparent that growth factors play a pivotal role in this intricate process. These growth factors actively contribute to the restoration of corneal epithelial injuries by orchestrating responses and facilitating specific interactions at targeted sites. This article systematically summarizes the role of growth factors in corneal epithelial cell injury repair by searching relevant literature in recent years, and explores the limitations of current literature search, providing a certain scientific basis for subsequent basic research and clinical applications.

Topical Insulin for Ocular Surface Disease.

Background: Insulin and insulin-like growth factor (IGF)-1 receptors are present in ocular tissues such as corneal epithelium, keratocytes, and conjunctival cells. Insulin plays a crucial role in the growth, differentiation, and proliferation of corneal epithelial cells, as well as in wound healing processes in various tissues. Purpose: This review explores the potential role of topical insulin in the treatment of ocular surface diseases. Specifically, it examines its impact on corneal nerve regeneration, sub-basal plexus corneal nerves, and its application in conditions like corneal epithelial defects, dry eye disease, and diabetic keratopathy. Methods: The review analyzes studies conducted over the past decade that have investigated the use of topical insulin in ocular surface diseases. It focuses on indications, drug preparation methods, side effects, efficacy outcomes, and variations in insulin concentrations and dosages used. Results: While off-label use of topical insulin has shown promising results in refractory corneal epithelial defects, its efficacy in dry eye disease is yet to be demonstrated. Variations in concentrations, dilutions, and dosing guidelines have been reported. However, limited data on ocular penetration, ocular toxicity, and systemic side effects pose challenges to its widespread utility. Conclusion: This review synthesizes findings from ocular investigations on topical insulin to assess its potential applicability in treating ocular surface and corneal diseases. By highlighting indications, preparation methods, side effects, and efficacy outcomes, it aims to provide insights into the current status and future prospects of using topical insulin in ophthalmic practice.

Progranulin Facilitates Corneal Repair Through Dual Mechanisms of Inflammation Suppression and Regeneration Promotion.

The cornea serves as a vital protective barrier for the eye; however, it is prone to injury and damage that can disrupt corneal epithelium and nerves, triggering inflammation. Therefore, understanding the biological effects and molecular mechanisms involved in corneal wound healing and identifying drugs targeting these pathways is crucial for researchers in this field. This study aimed to investigate the therapeutic potential of progranulin (PGRN) in treating corneal injuries. Our findings demonstrated that PGRN significantly enhanced corneal wound repair by accelerating corneal re-epithelialization and re-innervation. In vitro experiments with cultured epithelial cells and trigeminal ganglion cells further revealed that PGRN stimulated corneal epithelial cell proliferation and promoted axon growth in trigeminal ganglion cells. Through RNA-sequencing (RNA-seq) analysis and other experimental techniques, we discovered that PGRN exerted its healing effects modulating Wnt signaling pathway, which played a critical role in repairing epithelial cells and promoting axon regeneration in trigeminal neurons. Importantly, our study highlighted the anti-inflammatory properties of PGRN by inhibiting the NF-κB signaling pathway, leading to decreased infiltration of macrophages. In conclusion, our findings underscored the potential of PGRN in facilitating corneal wound healing by promoting corneal epithelial cell proliferation, trigeminal ganglion cell axon regeneration, and suppressing ocular inflammation. These results suggest that PGRN could potentially expedite the healing process and improve visual outcomes in patients with corneal injuries.

Unraveling the Intricate Network of lncRNAs in Corneal Epithelial Wound Healing: Insights Into the Regulatory Role of linc17500.

Epigenetic mechanisms orchestrate a harmonious process of corneal epithelial wound healing (CEWH). However, the precise role of long non-coding RNAs (lncRNAs) as key epigenetic regulators in mediating CEWH remains elusive. Here, we aimed to elucidate the functional contribution of lncRNAs in regulating CEWH. We used a microarray to characterize lncRNA expression profiling during mouse CEWH. Subsequently, the aberrant lncRNAs and their cis-associated genes were subjected to comprehensive Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blot analyses were performed to determine the expression profiles of key markers during CEWH. The in vivo effects of linc17500 on this process were investigated through targeted small interfering RNA (siRNA) injection. Post-siRNA treatment, corneal re-epithelialization was assessed, alongside the expression of cytokeratins 12 and 14 (Krt12 and Krt14) and Ki67. Effects of linc17500 on mouse corneal epithelial cell (TKE2) proliferation, cell cycle, and migration were assessed by multicellular tumor spheroids (MTS), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and scratch-wound assay, respectively. Microarray analysis revealed dysregulation of numerous lncRNA candidates during CEWH. Bioinformatic analysis provided valuable annotations regarding the cis-associated genes of these lncRNAs. In vivo experiments demonstrated that knockdown of linc17500 resulted in delayed CEWH. Furthermore, the knockdown of linc17500 and its cis-associated gene, CDC28 protein kinase regulatory subunit 2 (Cks2), was found to impede TKE2 cell proliferation and migration. Notably, downregulation of linc17500 in TKE2 cells led to suppression of the activation status of Akt and Rb. This study sheds light on the significant involvement of lncRNAs in mediating CEWH and highlights the regulatory role of linc17500 on TKE2 cell behavior. These findings provide valuable insights for future therapeutic research aimed at addressing corneal wound complications.

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.

Topical Insulin in Neurotrophic Keratopathy: A Review of Current Understanding of the Mechanism of Action and Therapeutic Approach.

Neurotrophic keratopathy is a corneal disease characterized by impaired corneal innervation. It can lead to corneal epithelial defects, ulcerations, and perforations. Topical insulin has been shown to be effective in treating this disorder. Insulin is a growth factor that can promote corneal epithelial cell proliferation and migration. In addition, it can also inhibit corneal epithelial cell apoptosis. Topical insulin has previously been found to enhance corneal wound healing. This article reviews the current understanding of the mechanism of action of topical insulin in the treatment of neurotrophic keratopathy.

Calreticulin accelerates corneal wound closure and mitigates fibrosis: Potential therapeutic applications.

The processes involved in regeneration of cutaneous compared to corneal tissues involve different intrinsic mechanisms. Importantly, cutaneous wounds involve healing by angiogenesis but vascularization of the cornea obscures vision. Previous studies showed that topically applied calreticulin (CALR) healed full-thickness excisional animal wounds by a tissue regenerative process markedly enhancing repair without evoking angiogenesis. In the current study, the application of CALR in a rabbit corneal injury model: (1) accelerated full wound closure by 3 days (2) accelerated delayed healing caused by corticosteroids, routinely used to prevent post-injury inflammation, by 6 days and (3) healed wounds without vascularization or fibrosis/hazing. Invitro, CALR stimulated proliferation of human corneal epithelial cells (CE) and corneal stromal cells (keratocytes) by 1.5-fold and 1.4-fold, respectively and induced migration of CE cells and keratocytes, by 72% and 85% compared to controls of 44% and 59%, respectively. As a marker of decreased fibrosis, CALR treated corneal wounds showed decreased immunostaining for α-smooth muscle actin (α-SMA) by keratocytes and following CALR treatment invitro, decreased the levels of TGF-β2 in human CE cells and α-SMA in keratocytes. CALR has the potential to be a novel therapeutic both, to accelerate corneal healing from various injuries and in conjunction with corticosteroids.

Nonivamide inhibits proliferation of human corneal epithelial cells by inducing cell cycle arrest and oxidative stress

Nonivamide, an agonist of transient receptor potential vanilloid type 1 (TRPV1), is widely used as a riot control agent, police incapacitant spray and pesticide. Although generally considered non-fatal, eye discomfort and even ocular injuries caused by such products are common. Little research has been conducted on the effects of nonivamide on corneal epithelial cells. Cell viability, impedance, flow cytometry, western blotting, and real-time fluorescence analyses were performed to investigate the effects of nonivamide on human corneal epithelial cells (HCE-T cells). We found that nonivamide impaired proliferation at subtoxic doses (100 μM and 200 μM) in HCE-T cells. Next, we described the mechanisms of action of nonivamide. Nonivamide caused cell cycle arrest by increasing p21 and decreasing cyclin D1. TRPV1 was activated by nonivamide, leading to an influx of Ca2+. Enhanced Ca2+ influx partially contributed to oxidative stress. Mitochondrial membrane potential (MMP) also decreased. All combined stress resulted in the inhibition of cell proliferation in HCE-T cells. In summary, nonivamide inhibited the proliferation of HCE-T cells at sub-toxic doses by inducing cell cycle arrest and oxidative stress. Our data demonstrate the corneal toxicity of nonivamide and explain the mechanisms underlying nonivamide-induced corneal injury.

Establishing a Mouse Model of Chlorpromazine-Induced Corneal Trigeminal Denervation.

This study aimed to establish a mouse model of chlorpromazine-induced corneal trigeminal denervation (CCTD). Retrobulbar chlorpromazine injections were administered to 6- to 8-week-old C57BL/6j mice to induce corneal denervation. Additionally, apoptosis was assessed in isolated primary trigeminal ganglion cells after culturing in a conditioned medium containing chlorpromazine. Finally, the success rate of model generation, mortality and complication rates, and model-preparation learning curves were compared between the CCTD model and the electrocoagulation and axotomy models. Chlorpromazine retrobulbar injections resulted in trigeminal denervation, leading to a reduced blink reflex, corneal nerve density, and corneal epithelium thickness. Furthermore, 90% (9/10) of the mice developed epithelial defects, accompanied by increased apoptosis and inhibited proliferation of corneal epithelial cells. In vitro, trigeminal ganglion cell apoptosis increased after culturing in a conditioned medium containing chlorpromazine. Moreover, the CCTD model exhibited a higher success rate, longer survival rate, and lower complication rate compared to the electrocoagulation and axotomy models. Crucially, the learning curve demonstrated that the method used to generate the CCTD model was easy to learn. The CCTD model is a user-friendly mouse model for studying corneal trigeminal denervation that offers a less invasive alternative to existing models. The CCTD model serves as a valuable tool for investigating the functional mechanisms of corneal trigeminal nerves and their interactions with corneal cells.

Dopamine‐Based High‐Transparent Hydrogel as Bioadhesive for Sutureless Ocular Tissue Repair

AbstractOcular injuries and their complications represent the most common causes of visual impairment. For ocular surgery, there is an unmet need for highly transparent bioadhesives with superior adhesion, biocompatibility, and regenerative properties. Herein, a novel high‐transparent bioadhesive hydrogel composed of gelatin methacryloyl (GelMA) and dopamine methacrylamide (DMA) is developed by in situ oxidative free‐radical polymerization. This bioadhesive hydrogel overcomes the fundamental weakness of mussel‐inspired adhesive copolymers in clinical practice by combining multiple favorable properties, including high light transmission, mechanical strength, adhesive strength, and biocompatibility. DMA significantly enhances corneal epithelial cells adhesion, proliferation, and migration on GelMA, and prevents the accumulation of reactive oxygen species (ROS) in corneal epithelial cells. In rabbit models of corneal and conjunctiva transplantation, the bioadhesive is able to decrease the inflammatory response and fibrosis formation induced by suture surgical trauma. In addition, the rabbit corneal stromal defect model reveals that the Gel/DMA bioadhesive could effectively seal corneal defects, accelerates corneal re‐epithelialization, and promotes wound healing. Thus, given the advantages of high bioactivity and simple preparation, the Gel/DMA bioadhesive represents a promising strategy for suture‐free ocular repair.

Effects of Tricalcium Silicate Cement on Corneal Cell Proliferation and Its Relationship with Vascular Endothelial Growth Factor Level and Receptor Typing

This research analyzed the effects of tricalcium silicate (C3S) cement and hypoxia on proliferation of human corneal epithelial cells (HCEpiCs) and the levels of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). Nanoscale C3S was prepared using a combustion method and characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), and laser particle size (LPS) analyzer. HCEpiCs were cultured, and influences of C3S with changed concentrations on proliferation of (HCEpiCs were analyzed. The cells were treated with hypoxia or with low-concentration (0.5 mg/mL, LC-C3S), medium-concentration (5 mg/mL, MC-C3S), or high-concentration (50 mg/mL, HC-C3S) of C3S. Meanwhile, normal HCEpiCs were undertaken as controls (Ctrl group). Cell proliferation, apoptosis, and the expression of target genes were detected using CCK-8, Annexin V-FITC/PI, fluorescent quantitative polymerase chain reaction (fqPCR), and Western blotting (WB). The results suggested that nanoscale C3S had multiple morphologies and an average particle size (APS) of (231.5±8.3) nm. With increasing nanoscale C3S concentration, proliferation of HCEpiCs increased (P < 0.05), and the highest proliferation was visualized at 5 mg/mL. Based on the conditions in the Ctrl group, the hypoxia group exhibited a decreased proliferation rate (PR), an increased apoptosis rate (AR), downshifted VEGF, VEGFR-2, and VEGFR-3, and elevated VEGFR-1 (P < 0.05). Based on the hypoxia group, the LCC3S, MC-C3S, and HC-C3S groups presented increased cell PRs, decreased APs, upshifted VEGF, VEGFR-2, and VEGFR-3, and downregulated VEGFR-1 (P < 0.05). The MC-C3S group showed an increased cell PR, a decreased AP, upregulated VEGF, VEGFR-2, and VEGFR-3, and downregulated VEGFR-1 to the LC-C3S group (P < 0.05). Additionally, the HC-C3S group had a decreased cell PR, an increased AP, upregulated VEGF, VEGFR-2, and VEGFR-3, and a downshifted VEGFR-1 to the MC-C3S group (P < 0.05). Therefore, C3S promoted proliferation of HCEpiCs, upregulated VEGF, VEGFR-2, and VEGFR-3, and downregulated VEGFR-1.

Tetraspanin CD9-derived peptides inhibit Pseudomonas aeruginosa corneal infection and aid in wound healing of corneal epithelial cells

Pseudomonas aeruginosa is a leading cause of corneal infection both within India and globally, often causing a loss of vision. Increasing antimicrobial resistance among the bacteria is making its treatment more difficult. Preventing initial bacterial adherence to the host membrane has been explored here to reduce infection of the cornea. Synthetic peptides derived from human tetraspanin CD9 have been shown to reduce infection in corneal cells both in vitro, ex vivo and in vivo. We found constitutive expression of CD9 in immortalized human corneal epithelial cells by flow cytometry and immunocytochemistry. The synthetic peptides derived from CD9 significantly reduced bacterial adherence to cultured corneal epithelial cells and ex vivo human cadaveric corneas as determined by colony forming units. The peptides also significantly reduced bacterial burden in a murine model of Pseudomonas keratitis and lowered the cellular infiltration in the corneal stroma. Additionally, the peptides aided corneal wound healing in uninfected C57BL/6 mice compared to control mice. These potential therapeutics had no effect on cell viability or proliferation of corneal epithelial cells and have the potential to be developed as an alternative therapeutic intervention.

Sodium hyaluronate promotes proliferation, autophagy, and migration of corneal epithelial cells by downregulating miR-18a in the course of corneal epithelial injury

Corneal epithelium can resist the invasion of external pathogenic factors to protect the eye from external pathogens. Sodium hyaluronate (SH) has been confirmed to promote corneal epithelial wound healing. However, the mechanism by which SH protects against corneal epithelial injury (CEI) is not fully understood. CEI model mice were made by scratching the mouse corneal epithelium, and in vitro model of CEI were constructed via curettage of corneal epithelium or ultraviolet radiation. The pathologic structure and level of connective tissue growth factor (CTGF) expression were confirmed by Hematoxylin and Eosin staining and immunohistochemistry. CTGF expression was detected by an IHC assay. The levels of CTGF, TGF-β, COLA1A, FN, LC3B, Beclin1, and P62 expression were monitored by RT-qPCR, ELISA, Western blotting or immunofluorescence staining. Cell proliferation was detected by the CCK-8 assay and EdU staining. Our results showed that SH could markedly upregulate CTGF expression and downregulate miR-18a expression in the CEI model mice. Additionally, SH could attenuate corneal epithelial tissue injury, and enhance the cell proliferation and autophagy pathways in the CEI model mice. Meanwhile, overexpression of miR-18a reversed the effect of SHs on cell proliferation and autophagy in CEI model mice. Moreover, our data showed that SH could induce the proliferation, autophagy, and migration of CEI model cells by downregulating miR-18a. Down-regulation of miR-18a plays a significant role in the ability of SH to promote corneal epithelial wound healing. Our results provide a theoretical basis for targeting miR-18a to promote corneal wound healing.

Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells.

Understanding the regulatory mechanisms underlying corneal epithelial cell (CEC) proliferation in vitro may provide the means to boost CEC production in cell therapy for ocular disorders. The transcription factor ΔNp63 plays a crucial role in the proliferation of CECs, but the underlying mechanisms is yet to be elucidated. TP63 and ΔNp63 are encoded by the TP63 gene via alternative promoters. We previously reported that both ΔNp63 and activating transcription factor (ATF3) are substantially expressed in cultured CECs, but the regulatory relationship between ΔNp63 and ATF3 is unknown. In the present study, we found that ΔNp63 increased ATF3 expression and ATF3 promoter activity in cultured CECs. The deletion of the p63 binding core site reduced ATF3 promoter activity. CECs overexpressing ATF3 exhibited significantly greater proliferation than control CECs. ATF3 knockdown suppressed the ΔNp63-induced increase in cell proliferation. Overexpression of ATF3 in CECs significantly elevated protein and mRNA levels of cyclin D. The protein levels of keratin 3/14, integrin β1, and involucrin did not differ between ATF3-overexpressing CECs, ATF3-downregulated CECs, and control cells. In conclusion, our results suggest that ΔNp63 increases CEC proliferation via the ΔNp63/ATF3/CDK pathway.

Administration of serotonin and norepinephrine reuptake inhibitors tends to have less ocular surface damage in a chronic stress-induced rat model of depression than selective serotonin reuptake inhibitors

Depressed patients who medicate with selective serotonin reuptake inhibitors (SSRIs) often report ocular dryness. Epidemiological studies have found that serotonin and norepinephrine reuptake inhibitors (SNRIs) are not risk factors for dry eye in depressed patients. However, the effect of SNRIs on the ocular surface is unknown. A depression rat model was induced by chronic unpredictable mild stress (CUMS), and SNRIs or SSRIs were administered to the rats for 3 or 6 weeks. The levels of norepinephrine (NE) and serotonin in tear fluid were tested by ELISA. The corneal fluorescence and lissamine green staining were used to evaluate ocular surface damage. NE and/or serotonin were administered to human corneal epithelial cells in vitro. RNA sequencing (RNA-seq) analysis was performed to investigate the mRNA expression profiles. Tear NE levels were higher in the SNRIs group, and ocular surface inflammation and apoptosis were significantly reduced compared to the SSRIs group. RNA-Seq indicated that NE significantly activate MAPK signaling pathway. NE can inhibit serotonin-induced activation of the NF-κB signaling pathway through α-1 adrenergic receptors and promotes the proliferation of corneal epithelial cells through activation of the MAPK signaling pathway. SNRIs administration have less ocular surface damage than SSRIs. NE protects human corneal epithelial cells from damage, and reduce inflammation on the ocular surface via activating the MAPK signaling pathway. SNRIs might be used as an appropriate treatment for depression-related DED.

Subconjunctival administration of mesenchymal stem cells alleviates ocular inflammation in a murine model of corneal alkali burn.

Corneal alkali burns cause extensive damage not only on the cornea but also into the intraocular tissues. As an anti-inflammatory therapy, subconjunctival administration of mesenchymal stem cells (MSCs) for corneal protection after corneal alkali burn has been explored. Little evidence demonstrates the potential of subconjunctival MSCs delivery in protecting the post-burn intraocular tissues. This study aimed to evaluate the therapeutic efficacy of subconjunctival injection of human placental (hP)-MSCs in protecting against ocular destruction after the burn. hP-MSCs were subconjunctivally administered to C57/BL mice after corneal alkali burn. Western blot of iNOS and CD206 was performed to determine the M1, M2 macrophage infiltration in the cornea. Infiltration of inflammatory cells in the anterior uvea and retina were analyzed by flow cytometry. The TUNEL assay or Western blot of Bax and Bcl2 was used to evaluate the anti-apoptotic effects of MSCs. MSCs could effectively facilitate cornea repair by suppressing inflammatory cytokines IL-1β, MCP-1, and MMP9, and polarizing CD206 positive M2 macrophages. Anterior uveal and retinal inflammatory cytokines expression and inflammatory cells infiltration were inhibited in the MSC-treated group. Reduced TUNEL positive staining and Bax/Bcl2 ratio indicated the anti-apoptosis of MSCs. MSC-conditioned medium promoted human corneal epithelial cell proliferation and regulated LPS-stimulated inflammation in RAW 264.7 macrophages, confirming the trophic and immunoregulatory effects of MSCs. Our findings demonstrate that subconjunctival administration of MSCs exerted anti-inflammatory and anti-apoptotic effects in the cornea, anterior uvea, and retina after corneal alkali burn. This strategy may provide a new direction for preventing post-event complications after corneal alkali burn.

Fibroblast Growth Factor 5 Promotes the Proliferation of Corneal Epithelium Through Phosphoinositide 3-Kinase/Protein Kinase B Signaling Pathway Activation

The recovery of corneal epithelial wounds is a complex process involving several physiological and pathological mechanisms that require further investigation. Our study has unveiled that the primary expression of FGF5 and FGFR1 occurs in the basal epithelium of the limbus. In the early 48 hours of the corneal epithelial wound healing process, FGF5 expression gradually rose and subsequently returned to baseline levels. The mice corneal epithelial cells (TKE2) proliferation was promoted by rhFGF5. Furthermore, the stemness-related genes K14, K15, P63, and the proliferation marker Ki67 levels expression increased after treatment with rhFGF5. In vivo, rhFGF5 promoted corneal epithelial wound healing. The RNA sequencing analysis of rhFGF5 treated TKE2 cells revealed 1512 differentially expressed genes. Different genes related to the cell cycle, proliferation, and regulatory signaling pathways were enriched. Moreover, the activation of the phosphoinositide 3-kinase/protein kinase B (PI3K-AKT) signaling pathway due to FGF5 led to the amplification of corneal progenitor cells. When FGF5 expression was curbed using siRNA, both proliferation and PI3K-AKT signaling pathway activation decreased. Our investigation has demonstrated that FGF5 stimulates the expansion of corneal limbal stem cells by activating the PI3K-AKT signaling pathway. Therefore, FGF5 has the potential to be an efficacious therapeutic intervention for treating corneal epithelial injuries.

The RNA m5C Methylase NSUN2 Modulates Corneal Epithelial Wound Healing.

The emerging epitranscriptomics offers insights into the physiopathological roles of various RNA modifications. The RNA methylase NOP2/Sun domain family member 2 (NSUN2) catalyzes 5-methylcytosine (m5C) modification of mRNAs. However, the role of NSUN2 in corneal epithelial wound healing (CEWH) remains unknown. Here we describe the functional mechanisms of NSUN2 in mediating CEWH. RT-qPCR, Western blot, dot blot, and ELISA were used to determine the NSUN2 expression and overall RNA m5C level during CEWH. NSUN2 silencing or overexpression was performed to explore its involvement in CEWH both in vivo and in vitro. Multi-omics was integrated to reveal the downstream target of NSUN2. MeRIP-qPCR, RIP-qPCR, and luciferase assay, as well as in vivo and in vitro functional assays, clarified the molecular mechanism of NSUN2 in CEWH. The NSUN2 expression and RNA m5C level increased significantly during CEWH. NSUN2 knockdown significantly delayed CEWH in vivo and inhibited human corneal epithelial cells (HCEC) proliferation and migration in vitro, whereas NSUN2 overexpression prominently enhanced HCEC proliferation and migration. Mechanistically, we found that NSUN2 increased ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) translation through the binding of RNA m5C reader Aly/REF export factor. Accordingly, UHRF1 knockdown significantly delayed CEWH in vivo and inhibited HCEC proliferation and migration in vitro. Furthermore, UHRF1 overexpression effectively rescued the inhibitory effect of NSUN2 silencing on HCEC proliferation and migration. NSUN2-mediated m5C modification of UHRF1 mRNA modulates CEWH. This finding highlights the critical importance of this novel epitranscriptomic mechanism in control of CEWH.