Microglial Function Is Distinct in Different Anatomical Locations during Retinal Homeostasis and Degeneration.

Microglia from different nervous system regions are molecularly and anatomically distinct, but whether they also have different functions is unknown. We combined lineage tracing, single-cell transcriptomics, and electrophysiology of the mouse retina and showed that adult retinal microglia shared a common developmental lineage and were long-lived but resided in two distinct niches. Microglia in these niches differed in their interleukin-34 dependency and functional contribution to visual-information processing. During certain retinal-degeneration models, microglia from both pools relocated to the subretinal space, an inducible disease-associated niche that was poorly accessible to monocyte-derived cells. This microglial transition involved transcriptional reprogramming of microglia, characterized by reduced expression of homeostatic checkpoint genes and upregulation of injury-responsive genes. This transition was associated with protection of the retinal pigmented epithelium from damage caused by disease. Together, our data demonstrate that microglial function varies by retinal niche, thereby shedding light on the significance of microglia heterogeneity.

Identification of the regulatory circuit governing corneal epithelial fate determination and disease.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.

Redefining the human corneal immune compartment using dynamic intravital imaging.

The healthy human cornea is a uniquely transparent sensory tissue where immune responses are tightly controlled to preserve vision. The cornea contains immune cells that are widely presumed to be intraepithelial dendritic cells (DCs). Corneal immune cells have diverse cellular morphologies and morphological alterations are used as a marker of inflammation and injury. Based on our imaging of corneal T cells in mice, we hypothesized that many human corneal immune cells commonly defined as DCs are intraepithelial lymphocytes (IELs). To investigate this, we developed functional in vivo confocal microscopy (Fun-IVCM) to investigate cell dynamics in the human corneal epithelium and stroma. We show that many immune cells resident in the healthy human cornea are T cells. These corneal IELs are characterized by rapid, persistent motility and interact with corneal DCs and sensory nerves. Imaging deeper into the corneal stroma, we show that crawling macrophages and rare motile T cells patrol the tissue. Furthermore, we identify altered immune cell behaviors in response to short-term contact lens wear (acute inflammatory stimulus), as well as in individuals with allergy (chronic inflammatory stimulus) that was modulated by therapeutic intervention. These findings redefine current understanding of immune cell subsets in the human cornea and reveal how resident corneal immune cells respond and adapt to chronic and acute stimuli.

Dry eye disease in mice activates adaptive corneal epithelial regeneration distinct from constitutive renewal in homeostasis.

Many epithelial compartments undergo constitutive renewal in homeostasis but activate unique regenerative responses following injury. The clear corneal epithelium is crucial for vision and is renewed from limbal stem cells (LSCs). Using single-cell RNA sequencing, we profiled the mouse corneal epithelium in homeostasis, aging, diabetes, and dry eye disease (DED), where tear deficiency predisposes the cornea to recurrent injury. In homeostasis, we capture the transcriptional states that accomplish continuous tissue turnover. We leverage our dataset to identify candidate genes and gene networks that characterize key stages across homeostatic renewal, including markers for LSCs. In aging and diabetes, there were only mild changes with <15 dysregulated genes. The constitutive cell types that accomplish homeostatic renewal were conserved in DED but were associated with activation of cell states that comprise "adaptive regeneration." We provide global markers that distinguish cell types in homeostatic renewal vs. adaptive regeneration and markers that specifically define DED-elicited proliferating and differentiating cell types. We validate that expression of SPARC, a marker of adaptive regeneration, is also induced in corneal epithelial wound healing and accelerates wound closure in a corneal epithelial cell scratch assay. Finally, we propose a classification system for LSC markers based on their expression fidelity in homeostasis and disease. This transcriptional dissection uncovers the dramatically altered transcriptional landscape of the corneal epithelium in DED, providing a framework and atlas for future study of these ocular surface stem cells in health and disease.

Diverse calcium signaling profiles regulate migratory behavior in avascular wound healing and aberrant signal hierarchy occurs early in diabetes.

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound edge are highly ordered, with groups of cells engaging in cyclical patterns of initiation and propagation. The cells in these groups exhibit a diverse range of signaling behavior, and dominant "conductor cells" drive activity in groups of lower-signaling neighbors. Ex vivo model systems reveal that conductor cells are present in wing cell layers of the corneal epithelium and that signaling propagates both within and between wing and basal layers. There are significant aberrations in conductor phenotype and interlayer propagation in type II diabetic murine models, indicating that signal hierarchy breakdown is an early indicator of disease. In vitro models reveal that signaling profile diversity and conductor cell phenotype is eliminated with P2X7 inhibition and is altered in Pannexin-1 or P2Y2 but not Connexin-43 inhibition. Conductor cells express significantly less P2X7 than their lower-signaling neighbors and exhibit significantly less migratory behavior after injury. Together, our results show that the postinjury calcium signaling cascade exhibits significantly more ordered and hierarchical behavior than previously thought, that proteins previously shown to be essential for regulating motility are also essential for determining signaling phenotype, and that loss of signal hierarchy integrity is an early indicator of disease state. NEW & NOTEWORTHY Calcium signaling in corneal epithelial cells after injury is highly ordered, with groups of cells engaged in cyclical patterns of event initiation and propagation driven by high-signaling cells. Signaling behavior is determined by P2X7, Pannexin-1, and P2Y2 and influences migratory behavior. Signal hierarchy is observed in healthy ex vivo models after injury and becomes aberrant in diabetes. This represents a paradigm shift, as signaling was thought to be random and determined by factors in the environment.

rhFGF-21 accelerates corneal epithelial wound healing through the attenuation of oxidative stress and inflammatory mediators in diabetic mice.

Diabetic keratopathy, commonly associated with a hyperactive inflammatory response, is one of the most common eye complications of diabetes. The peptide hormone fibroblast growth factor-21 (FGF-21) has been demonstrated to have anti-inflammatory and antioxidant properties. However, whether administration of recombinant human (rh) FGF-21 can potentially regulate diabetic keratopathy is still unknown. Therefore, in this work, we investigated the role of rhFGF-21 in the modulation of corneal epithelial wound healing, the inflammation response, and oxidative stress using type 1 diabetic mice and high glucose-treated human corneal epithelial cells. Our experimental results indicated that the application of rhFGF-21 contributed to the enhancement of epithelial wound healing. This treatment also led to advancements in tear production and reduction in corneal edema. Moreover, there was a notable reduction in the levels of proinflammatory cytokines such as TNF-α, IL-6, IL-1ß, MCP-1, IFN-γ, MMP-2, and MMP-9 in both diabetic mouse corneal epithelium and human corneal epithelial cells treated with high glucose. Furthermore, we found rhFGF-21 treatment inhibited reactive oxygen species production and increased levels of anti-inflammatory molecules IL-10 and SOD-1, which suggests that FGF-21 has a protective role in diabetic corneal epithelial healing by increasing the antioxidant capacity and reducing the release of inflammatory mediators and matrix metalloproteinases. Therefore, we propose that administration of FGF-21 may represent a potential treatment for diabetic keratopathy.

TRPV4 activation in human corneal epithelial cells promotes membrane mucin production.

Given that the corneal epithelium is situated on the outermost part of the eye, its functions can be influenced by external temperatures and chemical substances. This study aimed to elucidate the expression profile of chemosensory receptors in corneal epithelial cells and analyze their role in eye function regulation. A comprehensive analysis of 425 chemosensory receptors in human corneal epithelial cells-transformed (HCE-T) revealed the functional expression of TRPV4. The activation of TRPV4 in HCE-T cells significantly increased the expression of membrane-associated mucins MUC1, MUC4, and MUC16, which are crucial for stabilizing tear films, with efficacy comparable to the active components of dry eye medications. The present study suggests that TRPV4, which is activated by body temperature, regulates mucin expression and proposes it as a novel target for dry eye treatment.

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.

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.

Safety and Efficacy of Epithelium-Off Corneal Collagen Cross-Linking for the Treatment of Corneal Ectasia: A Report by the American Academy of Ophthalmology.

PURPOSE: To review the evidence on the safety and effectiveness of epithelium-off corneal collagen cross-linking (CXL) for the treatment of progressive corneal ectasia. METHODS: A literature search of the PubMed database was most recently conducted in March 2024 with no date restrictions and limited to studies published in English. The search identified 359 citations that were reviewed in abstract form, and 43 of these were reviewed in full text. High-quality randomized clinical trials comparing epithelium-off CXL with conservative treatment in patients who have keratoconus (KCN) and post-refractive surgery ectasia were included. The panel deemed 6 articles to be of sufficient relevance for inclusion, and these were assessed for quality by the panel methodologist; 5 were rated level I, and 1 was rated level II. There were no level III studies. RESULTS: This analysis includes 6 prospective, randomized controlled trials that evaluated the use of epithelium-off CXL to treat progressive KCN (5 studies) and post-laser refractive surgery ectasia (1 study), with a mean postoperative follow-up of 2.4 years (range, 1-5 years). All studies showed a decreased progression rate in treated patients compared with controls. Improvement in the maximum keratometry (Kmax) value, corrected distance visual acuity (CDVA), and uncorrected distance visual acuity (UDVA) was observed in the treatment groups compared with control groups. A decrease in corneal thickness was observed in both groups but was greater in the CXL group. Complications were rare. CONCLUSIONS: Epithelium-off CXL is effective in reducing the progression of KCN and post-laser refractive surgery ectasia in most treated patients with an acceptable safety profile. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Production and Limbal Lineage Commitment of Aniridia Patient-Derived Induced Pluripotent Stem Cells.

Congenital aniridia is caused by heterozygous mutations on the PAX6 gene leading to reduced amount of PAX6 protein (haploinsufficiency), abnormal eye development, and aniridia-associated keratopathy (AAK). This progressive corneal opacification resembles late-onset limbal stem cell (LSC) deficiency, leading to disrupted corneal epithelial renewal. The factors leading to AAK are not known and defects in native LSC differentiation and/or features leading to ocular surface dysfunction like inflammation and loss of innervation could contribute to development of AAK. Here, we produced induced pluripotent stem cells (hiPSC) from 3 AAK patients and examined whether PAX6 haploinsufficiency affects LSC lineage commitment. During LSC differentiation, characterization of the AAK lines showed lowered PAX6 expression as compared to wild type (WT) controls and expression peak of PAX6 during early phase of differentiation was detected only in the WT hiPSC lines. Whether it reflects developmental regulation remains to be studied further. Nevertheless, the AAK-hiPSCs successfully differentiated toward LSC lineage, in line with the presence of LSCs in young patients before cell loss later in life. In addition, patient-specific LSCs showed similar wound healing capacity as WT cells. However, extensive batch-related variation in the LSC marker expression and wound healing efficacy was detected without clear correlation to AAK. As development and maintenance of corneal epithelium involves an interplay between LSCs and their environment, the AAK-hiPSCs generated here can be further used to study the crosstalk between LSCs and limbal niche including, eg, corneal immune cells, stroma cells, and neurons.

Krupple-like factor 4 (KLF4) methylation signature in host cell in active viral keratitis with epithelial manifestation.

HSV1 presents as epithelial or stromal keratitis or keratouveitis and can lead to sight-threatening complications. KLF4, a critical transcription factor, and regulator of cell growth and differentiation, is essential in corneal epithelium stratification and homeostasis. Here, we want to understand the epigenetic modification specifically the methylation status of KLF4 in epithelium samples of HSV1 keratitis patients. After obtaining consent, epithelial scrapes were collected from 7 patients with clinically diagnosed HSV1 keratitis and 7 control samples (patients undergoing photorefractive keratectomy). Genomic DNA was isolated from the collected samples using the Qiagen DNeasy Kit. Subsequently, bisulfite modification was performed. The bisulphite-modified DNA was then subjected to PCR amplification using specific primers designed to target the KLF4, ACTB gene region, allowing for the amplification of methylated and unmethylated DNA sequences. The amplified DNA products were separated and visualized on a 3% agarose gel. KLF4 hypermethylation was found in 6 out of 7 (85.71%) eyes with viral keratitis, while 1 eye showed hypomethylation compared to PRK samples. Out of these 6, there were 2 each of epithelial dendritic keratitis, epithelial geographical keratitis, and neurotrophic keratitis. The patient with hypomethylated KLF4 had a recurrent case of HSV1 keratitis with multiple dendrites and associated vesicular lesions of the lip along with a history of fever. KLF4 hypermethylation in most viral keratitis cases indicated the under functioning of KLF4 and could indicate a potential association between KLF4 hypermethylation and the development or progression of HSV1 keratitis.

Wnt/ß-catenin signaling in corneal epithelium development, homeostasis, and pathobiology.

The corneal epithelium is located on the most anterior surface of the eyeball and protects against external stimuli. The development of the corneal epithelium and the maintenance of corneal homeostasis are essential for the maintenance of visual acuity. It has been discovered recently via the in-depth investigation of ocular surface illnesses that the Wnt/ß-catenin signaling pathway is necessary for the growth and stratification of corneal epithelial cells as well as the control of endothelial cell stability. In addition, the Wnt/ß-catenin signaling pathway is directly linked to the development of common corneal illnesses such as keratoconus, fungal keratitis, and corneal neovascularization. This review mainly summarizes the role of the Wnt/ß-catenin signaling pathway in the development, homeostasis, and pathobiology of cornea, hoping to provide new insights into the study of corneal epithelium and the treatment of related diseases.

Dynasore modulates store-operated calcium entry and mitochondrial calcium release in corneal epithelial cells.

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Isolation and characterization of rabbit limbal niche cells.

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRß) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRß and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRß in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRß+).

S100A9-TLR4 axis aggravates dry eye through the blockage of autophagy.

This research focused on how upregulation of S100A9 contributed to the pathogenesis of the dry eye disease (DED) and whether S100A9 served as a promising therapeutic target in DED. Public single-cell RNA sequencing (scRNA-seq) data of a lacrimal gland excision (LGE) murine DED model was analyzed. LGE model was established and expression of protein was measured through immunofluorescence and Western blot. DED-related signs were evaluated through tear secretion and fluorescent staining. TUNEL was performed to detect the level of cell death. Briefly, S100A9 was recognized as a highly variable gene in the DED group. LGE model was successfully established, and S100A9 showed a time-dependent increase in the corneal epithelia. Autophagic blockage was predicted by the scRNA-seq data in DED, and further verified by decrease of LC3B-II/LC3B-I and increase of SQSTM1 and p-mTOR/mTOR, while S100A9 inhibitor paquinimod (PAQ) reversed the changes. PAQ also downregulated TLR4, and inhibition of TLR4 also alleviated autophagic blockage in DED. Finally, signs of DED, chronic corneal inflammation and cell death got a remission after either inhibition of S100A9 or TLR4. In general, we deduced a S100A9-TLR4-Autophagic blockage pathway in the pathogenesis of DED.

Comparative effectiveness of nitro dihydrocapsaicin, new synthetic derivative capsaicinoid, and capsaicin in alleviating oxidative stress and inflammation on lipopolysaccharide-stimulated corneal epithelial cells.

Loss of tear homeostasis, characterized by hyperosmolarity of the ocular surface, induces cell damage through inflammation and oxidation. Transient receptor potential vanilloid 1 (TRPV1), a sensor for osmotic changes, plays a crucial role as a calcium ion channel in the pathogenesis of hypertonic-related eye diseases. Capsaicin (CAP), a potent phytochemical, alleviates inflammation during oxidative stress events by activating TRPV1. However, the pharmacological use of CAP for eye treatment is limited by its pungency. Nitro dihydrocapsaicin (NDHC) was synthesized with aromatic ring modification of CAP structure to overcome the pungent effect. We compared the molecular features of NDHC and CAP, along with their biological activities in human corneal epithelial (HCE) cells, focusing on antioxidant and anti-inflammatory activities. The results demonstrated that NDHC maintained cell viability, cell shape, and exhibited lower cytotoxicity compared to CAP-treated cells. Moreover, NDHC prevented oxidative stress and inflammation in HCE cells following lipopolysaccharide (LPS) administration. These findings underscore the beneficial effect of NDHC in alleviating ocular surface inflammation, suggesting that NDHC may serve as an alternative anti-inflammatory agent targeting TRPV1 for improving hyperosmotic stress-induced ocular surface damage.

Necrostatin-1 protects corneal epithelial cells by inhibiting the RIPK1/RIPK3/MLKL cascade in a benzalkonium chloride-induced model of necroptosis.

PURPOSE: Benzalkonium chloride (BAC) is commonly used as a preservative in ophthalmic medications, despite its potential to induce chemical injury. Extensive research has demonstrated that BAC can lead to adverse effects, including injuries to the ocular surface. Our study aimed to elucidate the underlying mechanism of necroptosis induced by BAC. METHODS: Human corneal epithelial (HCE) cells and mouse corneas were subjected to chemical injury, and the necrostatin-1 (Nec1) group was compared to the dimethylsulfoxide (DMSO) group. The extent of damage to HCE cells was assessed using CCK-8 and flow cytometry. Hematoxylin and eosin staining, as well as fluorescein sodium staining, were used to detect and characterize corneal injury. The activation of inflammatory cytokines and necroptosis-related proteins and genes was evaluated using Western blotting, immunofluorescence staining, and quantitative RT‒PCR. RESULTS: In our study, the induction of necroptosis by a hypertonic solution was not observed. However, necroptosis was observed in HCE cells exposed to NaOH and BAC, which activated the receptor-interacting protein kinase 1 (RIPK1) - receptor-interacting protein kinase 3 (RIPK3) - mixed lineage kinase domain-like protein (MLKL) signaling pathway. In mouse corneal tissues, BAC could induce necroptosis and inflammation. The administration of Nec1 mitigated the inflammatory response and ocular surface damage caused by BAC-induced necroptosis in our experimental models. Furthermore, our in vivo experiments revealed that the severity of necroptosis was greater in the 3-day group than in the 7-day group. CONCLUSIONS: Necroptosis plays a role in the pathological development of ocular surface injury caused by exposure to BAC. Furthermore, our study demonstrated that the administration of Nec1 could mitigate the pathological effects of necroptosis induced by BAC in clinical settings.

Mesenchymal vs. epithelial extracellular vesicles in corneal epithelial repair, apoptosis, and immunomodulation: An in vitro study.

Corneal injuries often lead to epithelial damage, apoptosis, and inflammation which impact visual function. Effective epithelial healing is critical for optimal vision and functioning of the cornea. Mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs) present promising avenues for cell-free therapy, however, evaluation of their specific roles in corneal epithelial injury requires further investigations with due consideration to the endogenous human corneal epithelial cell-derived EVs (HCEC-EVs). This study aims to isolate and characterize the EVs from a commonly available human corneal epithelial cell line (HCE-2 [50. B1], ATCC) and evaluate their corneal epithelial repair, anti-apoptotic, and immunomodulatory potential in comparison with human bone marrow mesenchymal stem cell-derived EVs (BM-MSC-EVs) in vitro. Both the BM-MSC- and HCEC-EVs exhibited similar morphology with a diameter <150 nm. However, the yield of EVs from HCECs was higher than that of BM-MSCs. Nanoparticle tracking analysis revealed an average EV size of ∼120 nm, while western blotting confirmed the presence of CD63, CD81, and TSG101, whereas Calnexin could not be detected in the BM-MSC- and HCEC-EVs. The corneal epithelial repair was monitored through in vitro wound healing assay, whereas apoptosis was studied through flow cytometry-based Propidium iodide staining in H2O2-treated cells. IL-1ß-stimulated HCECs were treated with BM-MSC- and HCEC-EVs for 24 h and expression of pro- (IL-6 and TNF-α) and anti-inflammatory (IL-10 and TGF-ß) cytokines was evaluated through ELISA. Our results, limited to in vitro investigations, suggest that compared with HCEC-EVs, BM-MSC-EVs showed: i) accelerated corneal epithelial healing, ii) enhanced anti-apoptotic potential, and iii) improved anti-inflammatory properties, in cultured HCECs.

Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

Severe corneal injury can lead to blindness even after prompt treatment. 14-3-3zeta, a member of an adaptor protein family, contributes to tissue repair by enhancing cellular viability and inhibiting fibrosis and inflammation in renal disease or arthritis. However, its role in corneal regeneration is less studied. In this study, filter disc of 2-mm diameter soaked in sodium hydroxide with a concentration of 0.5 N was placed at the center of the cornea for 30 s to establish a mouse model of corneal alkali injury. We found that 14-3-3zeta, which is mainly expressed in the epithelial layer, was upregulated following injury. Overexpression of 14-3-3zeta in ocular tissues via adeno-associated virus-mediated subconjunctival delivery promoted corneal wound healing, showing improved corneal structure and transparency. In vitro studies on human corneal epithelial cells showed that 14-3-3zeta was critical for cell proliferation and migration. mRNA-sequencing in conjunction with KEGG analysis and validation experiments revealed that 14-3-3zeta regulated the mRNA levels of ITGB1, PIK3R1, FGF5, PRKAA1 and the phosphorylation level of Akt, suggesting the involvement of the PI3K-Akt pathway in 14-3-3zeta-mediated tissue repair. 14-3-3zeta is a potential novel therapeutic candidate for treating severe corneal injury.