Mouse Corneal Epithelial Cells Research Articles

Mesenchymal Stem Cells-Derived Exosomal miR-223-3p Alleviates Ocular Surface Damage and Inflammation by Downregulating Fbxw7 in Dry Eye Models.

Our previous study indicated that exosomes derived from mouse adipose-derived mesenchymal stem cells (mADSC-Exos) alleviated the benzalkonium chloride (BAC)-induced mouse dry eye model. However, the specific active molecules in mADSC-Exos that contribute to anti-dry eye therapy remain unidentified. In this study, we aimed to investigate the efficacy and mechanisms of miR-223-3p derived from mADSC-Exos in dry eye models. Enzyme-linked immunosorbent assay (ELISA) experiments were conducted to determine miR-223-3p derived from mADSC-Exos that exerted anti-inflammatory effects on hyperosmolarity-induced mouse corneal epithelial cells (MCECs). The therapeutic efficacy of miR-223-3p was evaluated in mice with dry eye induced by either BAC or scopolamine (Scop). Mice were randomly assigned to 5 groups: sham, model, miR-223-3p overexpression, miR-223-3p knockdown, and 0.1% pranoprofen (positive group). Post-treatment, the severity of dry eye symptoms, and the pro-inflammatory cytokine levels were assessed. The effect of miR-223-3p on silencing the target gene was verified using ELISA and dual luciferase reporter assays. The mADSC-Exos that knocked out miR-223-3p did not reduce interleukin (IL)-6 content. Supplementing with miR-223-3p could restore the reduction of IL-6. The miR-223-3p effectively ameliorated ocular surface damage and decreased pro-inflammatory cytokines or chemokines in both BAC- and Scop-induced mouse dry eye models. Furthermore, miR-223-3p inhibited cell apoptosis. F-box and WD repeat domain-containing 7 (Fbxw7) was the potential direct target of miR-223-3p. The miR-223-3p suppressed the 3'-untranslated region of Fbxw7. The Fbxw7 knockdown suppressed hyperosmolarity-induced inflammation in MCECs. The mADSC-derived exosomal miR-223-3p mitigates ocular surface damage and inflammation, indicating its potential as a promising treatment option for dry eye.

MUC1‑ND interacts with TRPV1 to promote corneal epithelial cell proliferation in diabetic dry eye mice by partly activating the AKT signaling pathway.

Although both mucin1 (MUC1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) have been reported to be associated with dry eye (DE) disease, whether they interact and their regulatory roles in diabetic DE disease are unknown. Diabetic DE model mice were generated by streptozotocin induction and assessed by corneal fluorescein staining, tear ferning (TF) tests, phenol red thread tests, hematoxylin and eosin staining of corneal sections and periodic acid Schiff staining of conjunctival sections. Cell proliferation was measured by CCK8 assay. Western blotting was performed to measure protein expression. Primary mouse corneal epithelial cells (MCECs) were cultured after enzymatic digestion. Immunofluorescence staining of MCECs and frozen corneal sections was conducted to assess protein expression and colocalization. Coimmunoprecipitation was performed to detect protein‑protein interactions. It was found that, compared with control mice, diabetic DE mice exhibited increased corneal epithelial defects, reduced tear production, poorer TF pattern grades and impaired corneal and conjunctival tissues. In vivo and in vitro experiments showed that hyperglycemia impaired cell proliferation, accompanied by decreased levels of the MUC1 extracellular domain (MUC1‑ND) and TRPV1. Additionally, it was found that capsazepine (a TRPV1 antagonist) inhibited the proliferation of MCECs. Notably, MUC1‑ND was shown to interact with the TRPV1 protein in the control group but not in the diabetic DE group. It was also found that the AKT signaling pathway was attenuated in the diabetic DE mice and downstream of TRPV1. MUC1‑ND interacted with TRPV1, partly activating the AKT signaling pathway to promote MCEC proliferation. The present study found that the interaction of MUC1‑ND with TRPV1 promotes MCEC proliferation by partly activating the AKT signaling pathway, providing new insight into the pathogenesis of corneal epithelial dysfunction in diabetic DE disease.

Oxidation enhances the toxicity of polyethylene microplastics to mouse eye: Perspective from in vitro and in vivo

Microplastics (MPs) are ubiquitously dispersed in the environment, and undergoing the process of oxidation that alters their physical and chemical properties. Eyes, which directly interface with the external milieu, inevitably encounter MPs. Nonetheless, the ophthalmic toxicity of MPs towards organisms remains unclear. In this study, primary mouse corneal epithelial cells (MCECs), C57BL/6 mice, and CX3CrlGFP/+ mice were utilized to evaluate the toxicity and differences between oxidized low-density polyethylene MPs (modified-MPs) and low-density polyethylene MPs (virgin-MPs) on eyes. The results manifested that virgin-MPs and modified-MPs could be endocytosed by primary MCECs, resulting in a range of cellular damage. Furthermore, they could diminish tear secretion, increase intraocular pressure, and could be internalized into cornea and retina in mice, instigating a series of detrimental reactions. Importantly, modified-MPs exhibited heightened toxicity towards mouse eyes, seemingly due to oxidation enhances the interaction between virgin-MPs/modified-MPs and tissues/cells, and leading to the release of toxic substances increased. In conclusion, our discoveries demonstrate that oxidation exacerbates the harm of virgin-MPs to eyes, and are of great significance for evaluating the risk of MPs to ocular health.

Reticulated Retinoic Acid Synthesis is Implicated in the Pathogenesis of Dry Eye in Aqp5 Deficiency Mice.

Abnormalities in aquaporins are implicated in the pathological progression of dry eye syndrome. Retinoic acid (RA) regulates cellular proliferation, differentiation, and apoptosis in the cornea, thereby being associated with dry eye disease (DED). The objective of this study is to explore the underlying mechanisms responsible for RA metabolic abnormalities in corneas lacking aquaporin 5 (AQP5). Dry eye (DE) models were induced via subcutaneous scopolamine hydrobromide. Aqp5 knockout (Aqp5-/-) mice and DE mice were utilized to assess corneal epithelial alterations. Tear secretion, goblet cell counts, and corneal punctate defects were evaluated. The impact of Aqp5 on RA-related enzymes and receptors was investigated using pharmacological RA or SR (A JunB inhibitor), a transcription factor JunB inhibitor, treatment in mouse corneal epithelial cells (CECs), or human corneal epithelial cells (HCECs). The HCECs and NaCl-treated HCECs underwent quantitative real-time PCR (qRT-PCR), immunofluorescent, Western blot, and TUNEL assays. The regulation of transcription factor JunB on Aldh1a1 was explored via ChIP-PCR. Aqp5 and Aldh1a1 were reduced in both CECs of DE mice and NaCl-induced HCECs. Aqp5-/- mice exhibited DE phenotype and reduced Aldh1a1. RA treatment reduced apoptosis, promoted proliferation, and improved the DE phenotype in Aqp5-/- mice. JunB enrichment in the Aldh1a1 promoter was identified by ChIP-PCR. SR significantly increased Aldh1a1 expression, Ki67, and ΔNp63-positive cells, and decreased TUNEL-positive cells in CECs and HCECs. Our findings demonstrated the downregulation of Aqp5 expression and aberrant RA metabolism in DE conditions. Knockout of Aqp5 resulted in reduced production of RA through activation of JunB, subsequently leading to the manifestation of DE symptoms.

The Role of SLIT3-ROBO4 Signaling in Endoplasmic Reticulum Stress-Induced Delayed Corneal Epithelial and Nerve Regeneration.

In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration. Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells. Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment. ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.

BMSC Alleviates Dry Eye by Inhibiting the ROS-NLRP3-IL-1β Signaling Axis by Reducing Inflammation Levels

Purpose Bone marrow mesenchymal stem cells (BMSC) have multiple biological functions and are widely involved in regulating inflammatory diseases, tissue repair and regeneration. However, the mechanism of their action in dry eye disease (DED) is currently unclear. The purpose of this study was to investigate the effect of BMSCs in the treatment of dry eye mice and to explore its specific therapeutic mechanism. Methods Mouse corneal epithelial cells (MCECs) were treated with 500 mOsM sodium chloride hypertonic solution to induce a DED cell model. The dry eye animal model was constructed by adding 5 μL 0.2% benzalkonium chloride solution to mouse eyes. Western blotting was used to detect the expression of related proteins, and flow cytometry, enzyme-linked immunosorbent assay (ELISA), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, hematoxylin-eosin (HE) staining, and periodic acid schiff (PAS) staining were used to detect cell and eye tissue damage. Results The experimental results showed that BMSCs can reduce the levels of reactive oxygen species (ROS) and inflammatory factors in MCECs, promote cell proliferation, inhibit cell apoptosis, improve the integrity of the corneal epithelial layer in vivo, promote an increase in the number of goblet cells, and alleviate DED. Further exploration of the molecular mechanism of BMSCs treatment revealed that BMSCs alleviate the progression of DED by inhibiting the ROS-NLRP3-IL-1β signaling pathway. Conclusion BMSCs inhibit ROS-NLRP3-IL-1β signaling axis, reducing inflammation levels and alleviating dry eye symptoms. These findings provide new ideas and a basis for the treatment of DED and provide an experimental basis for further research on the application value of BMSCs in alleviating DED.

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.

Transient plasma membrane disruption induced calcium waves in mouse and human corneal epithelial cells.

The purpose of this study was to examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (TPMD Ca++ Wvs) in human and mouse corneal epithelium (HCEC and MCEC). A multi-photon microscope was used to create laser-induced TPMDs in single cultured cells and in intact ex vivo and in vivo MCECs and ex vivo human cornea rim HCECs. Eye rubbing-induced TPMDs were studied by gentle rubbing with a cotton tipped applicator over a closed eyelid in ex vivo and in vivo MCECs. Ca++ sources for TPMD-induced Ca++ waves were explored using Ca++ channel inhibitors and Ca++-free media. TPMDs and TPMD Ca++ Wvs were observed in all cornea epithelial models examined, often times showing oscillating Ca++ levels. The sarcoplasmic reticulum Ca++ ATPase inhibitors thapsigargin and CPA reduced TPMD Ca++ Wvs. TRP V1 antagonists reduced TPMD Ca++ Wvs in MCECs but not HCECs. Ca++-free medium, 18α-GA (gap junction inhibitor), apyrase (hydrolyzes ATP), and AMTB (TRPM8 inhibitor) did not affect TPMD Ca++ Wvs. These results provide a direct demonstration of corneal epithelial cell TPMDs and TPMDs in in vivo cells from a live animal. TPMDs were observed following gentle eye rubbing, a routine corneal epithelial cell mechanical stress, indicating TPMDs and TPMD Ca++ Wvs are common features in corneal epithelial cells that likely play a role in corneal homeostasis and possibly pathophysiological conditions. Intracellular Ca++ stores are the primary Ca++ source for corneal epithelial cell TPMD Ca++ Wvs, with TRPV1 Ca++ channels providing Ca++ in MCECs but not HCECs. Corneal epithelial cell TPMD Ca++ Wv propagation is not influenced by gap junctions or ATP.

Corneal neuroepithelial compartmentalized microfluidic chip model for evaluation of toxicity-induced dry eye

Part of the lacrimal functional unit, the cornea protects the ocular surface from numerous environmental aggressions and xenobiotics. Toxicological evaluation of compounds remains a challenge due to complex interactions between corneal nerve endings and epithelial cells. To this day, models do not integrate the physiological specificity of corneal nerve endings and are insufficient for the detection of low toxic effects essential to anticipate Toxicity-Induced Dry Eye (TIDE).Using high-content imaging tool, we here characterize toxicity-induced cellular alterations using primary cultures of mouse trigeminal sensory neurons and corneal epithelial cells in a compartmentalized microfluidic chip.We validate this model through the analysis of benzalkonium chloride (BAC) toxicity, a well-known preservative in eyedrops, after a single (6h) or repeated (twice a day for 15 min over 5 days) topical 5.10−4% BAC applications on the corneal epithelial cells and nerve terminals.In combination with high-content image analysis, this advanced microfluidic protocol reveal specific and tiny changes in the epithelial cells and axonal network as well as in trigeminal cells, not directly exposed to BAC, with ATF3/6 stress markers and phospho-p44/42 cell activation marker. Altogether, this corneal neuroepithelial chip enables the evaluation of toxic effects of ocular xenobiotics, distinguishing the impact on corneal sensory innervation and epithelial cells. The combination of compartmentalized co-culture/high-content imaging/multiparameter analysis opens the way for the systematic analysis of toxicants but also neuroprotective compounds.

A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures

Purpose This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures. Methods Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method. Methods evaluated included 0.25% trypsin-EDTA, low temperature, mitomycin-C, and dispase. Degree of fibroblast contamination was examined using light microscopy evaluation of cell phenotype, immunofluorescence and western blotting using cell type-specific markers. Anti-pancytokeratin (PanCK) was used as the epithelial immunofluorescence label, and anti-α smooth muscle actin (αSMA) as the fibroblast immunofluorescence label. Epithelial western blot antibodies included PanCK, keratin 12, and E-cadherin, while αSMA, collagen 1A1 and collagen 3A1 were used to identify fibroblasts. Results Fibroblast contamination of human and mouse primary cornea epithelial cell cultures was best controlled using the 0.25% trypsin-EDTA method. The other methods examined were not effective at eliminating cornea fibroblast contamination. Conclusions Trypsin-EDTA digestion is a simple and effective method for controlling fibroblast contamination of cultured primary human and mouse corneal epithelial cells.

Interleukin-36 Receptor Signaling Attenuates Epithelial Wound Healing in C57BL/6 Mouse Corneas.

The IL-36 cytokines are known to play various roles in mediating the immune and inflammatory response to tissue injury in a context-dependent manner. This study investigated the role of IL-36R signaling in mediating epithelial wound healing in normal (NL) and diabetic (DM) C57BL/6 mouse corneas. The rate of epithelial wound closure was significantly accelerated in IL-36 receptor-deficient (IL-36R-/-) compared to wild-type (WT) mice. Wounding increased IL-36α and -36γ but repressed IL-36R antagonist (IL-36Ra) expression in B6 mouse corneal epithelial cells. The wound-induced proinflammatory cytokines CXCL1 and CXCL2 were dampened, while the antimicrobial peptides (AMPs) S100A8 and A9 were augmented in IL-36R-/- mouse corneas. Intriguingly, the expression of AMP LCN2 was augmented at the mRNA level. LCN2 deficiency resulted in an acceleration of epithelial wound healing. IL-36R deficiency also greatly increased the healing rate of the corneal epithelial wound in DM mice. IL-36R deficiency also suppressed IL-1β, IL-1Ra, and ICAM expression in unwounded-DM mice and wounded NL corneas. Opposing IL-1β and ICAM, the expression of IL-Ra in DM corneas of IL-36R-/- mice was augmented. The presence of recombinant IL-1Ra and IL-36Ra accelerated epithelial wound closure in T1DM corneas of B6 mice. Our study revealed an unprecedented role of IL-36R signaling in controlling corneal epithelial wound healing in normal (NL) and diabetic (DM) mice. Our data suggest that IL-36Ra, similar to IL-1Ra, might be a therapeutic reagent for improving wound healing and reducing wound-associated ulceration, particularly in the cornea and potentially in the skin of DM patients.

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Mouse Corneal Epithelial Cells.

Corneal epithelial homeostasis is maintained by coordinated gene expression across distinct cell populations, but the gene regulatory programs underlying this cellular diversity remain to be characterized. Here we applied single-cell multi-omics analysis to delineate the gene regulatory profile of mouse corneal epithelial cells under normal homeostasis. Single cells isolated from the cornea epithelium (with marginal conjunctiva) of adult mice were subjected to scRNA-seq and scATAC-seq using the 10×Genomics platform. Cell types were clustered by the graph-based visualization method uniform manifold approximation and projection and unbiased computational informatics analysis. The scRNA-seq and scATAC-seq datasets were integrated following the integration pipeline described in ArchR and Seurat. We characterized diverse corneal epithelial cell types based on gene expression signatures and chromatin accessibility. We found that cell type-specific accessibility regions were mainly located at distal regions, suggesting essential roles of distal regulatory elements in determining corneal epithelial cell diversity. Trajectory analyses revealed a continuum of cell state transition and higher coordination between transcription factor (TF) motif accessibility and gene expression during corneal epithelial cell differentiation. By integrating transcriptomic and chromatin accessibility analysis, we identified cell type-specific and shared gene regulation programs. We also uncovered critical TFs driving corneal epithelial cell differentiation, such as nuclear factor I (NFI) family members, Rarg, Elf3. We found that nuclear factor-κB (NF-κB) family members were positive TFs in limbal cells and some superficial cells, but they were involved in regulating distinct biological processes. Our study presents a comprehensive gene regulatory landscape of mouse cornea epithelial cells, and provides valuable foundations for future investigation of corneal epithelial homeostasis in the context of cornea pathologies and regenerative medicine.

Mouse Corneal Epithelial and Stromal Cell Isolation and Culture.

Corneal epithelium and stroma are the major cellular structures for ocular protection and vision accuracy; they play important roles in corneal wound healing and inflammation under pathological conditions. Unlike human, murine corneal and stromal fibroblast cells are difficult to isolate for cell culture. In our laboratory, we successfully used an ex vivo culture procedure and an enzymatic procedure to isolate, purify, and culture mouse corneal epithelial and stromal fibroblast cells. Key features • Primary cell culture models of a disease are critical for cellular and molecular mechanism studies. • Corneal tissues with the limbus contain stem cells to generate both epithelial and stromal cells. • An ex vivo corneal culture provides a constant generation of primary corneal cells for multiple passages. • The isolated cells are validated by the corneal epithelial cell markers Krt12 and Cdh1 and the stromal fibroblast marker Vim.

Molecular mechanisms regulating wound repair: Evidence for paracrine signaling from corneal epithelial cells to fibroblasts and immune cells following transient epithelial cell treatment with Mitomycin C

In this paper, we use RNAseq to identify senescence and phagocytosis as key factors to understanding how mitomyin C (MMC) stimulates regenerative wound repair. We use conditioned media (CM) from untreated (CMC) and MMC treated (CMM) human and mouse corneal epithelial cells to show that corneal epithelial cells indirectly exposed to MMC secrete elevated levels of immunomodulatory proteins including IL-1α and TGFβ1 compared to cells exposed to CMC. These factors increase epithelial and macrophage phagocytosis and promote ECM turnover. IL-1α supplementation can increase phagocytosis in control epithelial cells and attenuate TGFβ1 induced αSMA expression by corneal fibroblasts. Yet, we show that epithelial cell CM contains factors besides IL-1α that regulate phagocytosis and αSMA expression by fibroblasts. Exposure to CMM also impacts the activation of bone marrow derived dendritic cells and their ability to present antigen. These in vitro studies show how a brief exposure to MMC induces corneal epithelial cells to release proteins and other factors that function in a paracrine way to enhance debris removal and enlist resident epithelial and immune cells as well as stromal fibroblasts to support regenerative and not fibrotic wound healing.

Integrated Analysis of lncRNA-miRNA-mRNA ceRNA Network in Mixed Dry Eye Disease.

In order to investigate the relationship between inflammation and lncRNA in mixed dry eye disease (DED), this study establishes competitive endogenous RNA (ceRNA) network in mixed DED. Microarray analysis of cornea from mixed DED mice is performed to screen for differences in lncRNA and target genes, and miRNA bioinformatics were predicted based on the ceRNA hypothesis. The ceRNA network, which consists of 96 relationship pairs, is constructed using the top 10 upregulated lncRNAs and all upregulated mRNAs and two pairs of lncRNA-miRNA-mRNA pairs (NONMMUT047964.2-miR-671-5p-Egr-1andNONMMUT054540.2-miR-1934-5p-Grm2) are selected for RT-qRCR verification in mouse corneal epithelial cells under high osmotic pressure and the samples for microarray. Meanwhile, mouse corneal epithelial cell lines (MCECs), transfected siRNA of NONMMUT047964.2 under high osmotic pressure, shows a decrease in apoptosis rate and a decrease in expression of IL-1β and IL-6. The experimental results show that the NONMMUT047964.2-miR-671-5p-Egr-1 axis may regulate the inflammation and promote the apoptosis of corneal epithelial cells under hypertonic condition.

The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration.

Diabetic keratopathy (DK) is an important diabetic complication at the ocular surface. Chronic low-grade inflammation mediated by the NLRP3 inflammasome promotes pathogenesis of diabetes and its complications. However, the effect of the NLRP3 inflammasome on DK pathogenesis remains elusive. Wild-type (WT) and Nlrp3 knockout (KO) C57BL/6 mice were used to establish a type I diabetes model by intraperitoneal injection of streptozotocin. The effect of the NLRP3 inflammasome on diabetic corneal wound healing and never regeneration was examined by a corneal epithelial abrasion model. Western blot, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and pharmacological treatment were performed to investigate the regulatory mechanism of advanced glycation end products (AGEs) on NLRP3 inflammasome activation and corneal wound healing in vivo. The cultured mouse corneal epithelial cells (TKE2) were used to evaluate the effect and mechanism of AGEs on NLRP3 inflammasome activation in vitro. We revealed that NLRP3 inflammasome-mediated inflammation and pyroptosis contributed to DK pathogenesis. Under physiological conditions, the NLRP3 inflammasome was required for corneal wound healing and nerve regeneration. However, under a diabetic scenario, sustained activation of the NLRP3 inflammasome resulted in postponed corneal wound healing and impaired nerve regeneration. Mechanistically, the accumulated AGEs promoted hyperactivation of the NLRP3 inflammasome through ROS production. Moreover, genetically and pharmacologically blocking the AGEs/ROS/NLRP3 inflammasome axis significantly expedited diabetic corneal epithelial wound closure and nerve regeneration. Our results revealed that AGEs-induced hyperactivation of the NLRP3 inflammasome resulted in delayed diabetic corneal wound healing and impaired nerve regeneration, which further highlighted the NLRP3 inflammasome as a promising target for DK treatment.

Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo.

It has been a long-standing challenge to obtain from cell cultures adequate amounts of mouse corneal epithelial cells (mCEC) to perform transplantation surgery. This limitation is attributable to the passage dependent declines in their proliferative activity. We describe here development of a novel 6C medium that contains six different modulators of different signaling pathways, which control proliferative mCEC activity. Its usage shortens the time and effort required to obtain epithelial sheets for hastening healing of an epithelial wound in an experimental animal model. This serum-free 6C medium contains:Y27632, forskolin, SB431542, DAPT, IWP-2, LDN-193189 and also DermaLife K keratinocyte calcium. Their inclusion inhibits rises in four specific markers of epithelial mesenchymal transdifferentiation:ZEB1/2, Snail, β-catenin and α-SMA. This medium is applied in a feeder-free air-lifted system to obtain sufficient populations of epithelial progenitor cells whose procurement is facilitated due to suppression of progenitor epithelial cell transdifferentiation into epithelial-mesenchymal cells. Diminution of this decline in transdifferentiation was confirmed based on the invariance of P63, K14, Pax6, and K12 gene expression levels. This cell culture technique is expected to facilitate ex vivo characterization of mechanisms underlying cell fate determination. Furthermore, its implementation will improve yields of progenitor mouse corneal epithelial cells, which increases the likelihood of using these cells as a source to generate epithelial sheets for performing transplantation surgery to treat limbal stem cell deficiency in a clinical setting. In addition, the novel insight obtainable from such studies is expected to improve the outcomes of corneal regenerative medicine.

Silk films with nanotopography and extracellular proteins enhance corneal epithelial wound healing

Corneal wound healing depends on extracellular matrix (ECM) and topographical cues that modulate migration and proliferation of regenerating cells. In our study, silk films with either flat or nanotopography patterned parallel ridge widths of 2000, 1000, 800 nm surfaces were combined with ECMs which include collagen type I (collagen I), fibronectin, laminin, and Poly-d-Lysine to accelerate corneal wound healing. Silk films with 800 nm ridge width provided better cell spreading and wound recovery than other size topographies. Coating 800 nm patterned silk films with collagen I proves to optimally further increased mouse and rabbit corneal epithelial cells growth and wound recovery. This enhanced cellular response correlated with redistribution and increase in size and total amount of focal adhesion. Transcriptomics and signaling pathway analysis suggested that silk topography regulates cell behaviors via actin nucleation ARP-WASP complex pathway, which regulate filopodia formation. This mechanism was further explored and inhibition of Cdc42, a key protein in this pathway, delayed wound healing and decreased the length, density, and alignment of filopodia. Inhibition of Cdc42 in vivo resulted in delayed re-epithelization of injured corneas. We conclude that silk film nanotopography in combination with collagen I constitutes a better substrate for corneal wound repair than either nanotopography or ECM alone.

Effects of Glycyrrhizin Treatment on Diabetic Cornea.

Purpose: To test how glycyrrhizin (GLY) affects mouse corneal epithelial cells (MCEC) and the diabetic murine cornea. Methods: Viability of MCEC grown under normal or high glucose (HG) with/without GLY was tested by an MTT assay. In addition, C57BL/6 mice were injected with streptozotocin and a subset of control and diabetic mice received GLY in their drinking water. mRNA and protein levels of proinflammatory and oxidative stress molecules were tested by reverse transcription-polymerase chain reaction (RT-PCR) in both models. Ex vivo studies using human diabetic versus control corneas analyzed proinflammatory and oxidative stress markers using RT-PCR and enzyme-linked immunosorbent assay. Results: GLY protected against loss of cell viability induced by HG and significantly reduced HMGB1, IL-1β, TLR2, TLR4, NLRP3, COX2, SOD2, HO-1, GPX2, and GR1. In vivo, corneas of GLY-treated diabetic mice showed significantly decreased mRNA expression for CXCL2, iNOS, and all molecules listed above; GLY also lowered HMGB1 and IL-1β proteins (in vitro and in vivo). Ex vivo studies using diabetic human corneas revealed elevated mRNA levels of inflammatory and oxidative stress molecules (as listed above for in vivo) versus normal age-matched controls. Protein levels for HMGB1 and IL-1β also were elevated in diabetic human versus control corneas. Conclusions: The data provide evidence that GLY treatment attenuates inflammation and oxidative stress in vitro in MCEC and in vivo in the cornea of diabetic mice. Ex vivo data support the similarities of proinflammatory and oxidative stress data in mouse compared to human, suggesting that GLY treatment would have relevancy to patient care.

Silencing TLR4/MyD88/NF-κB Signaling Pathway Alleviated Inflammation of Corneal Epithelial Cells Infected by ISE.

The regulatory role of toll-like receptor 4 (TLR4) in the inactivate staphylococcus epidermidis (ISE)-induced cornea inflammation is not well investigated. Here, TLR4 silence could decrease inflammatory cytokines in corneal epithelial cells treated with ISE. The mouse corneal epithelial cells were exposed to ISE for 24 h, either alone or with the NF-κB inhibitor, TLR4 lentivirus to bilaterally (knock-down or and overexpression). The expression of TLR4 in mouse corneal epithelial cells was investigated using western blot and qRT-PCR assay. The inflammatory cytokine levels were evaluated by qRT-PCR and ELISA, respectively. The relative impact factors of TLR4-mediated NF-κB signaling detected using western blot assay. Results show the expression levels of TLR4 and some inflammatory cytokines were significantly increased in corneal epithelial cells treated with ISE. TLR4 Silence markedly decreased ISE-induced production of IL12, TNF-α, CCL5, and CCL9 in corneal epithelial cells. Furthermore, the nuclear translocation of NF-κB p65 and myeloid differentiation protein 88 (MyD88) in the cells treated with ISE were further reduced by silencing TLR4. Inhibition of TLR4-mediated NF-κB signaling by using BAY11-7082 also alleviated ISE-induced inflammation. In the rescue experiment, transfected the stable TLR4 silenced corneal epithelial cells with TLR4 overexpression lentivirus, we found that TLR4 overexpression can restore the down-regulation of TLR4 and inflammatory cytokines (IL12, TNF-α, CCL9) caused by TLR4 knocked down. Therefore, ISE-induced cornea inflammation was due to the activation of the TLR4/MyD88/NF-κB signaling pathway, and dramatically stimulated IL12, TNF-α, CCL9 secretion. TLR4 silence presented mitigates damage in corneal epithelial cells treated with ISE.