HCE Cells Research Articles

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

PurposeBenzalkonium 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. MethodsHuman 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. ResultsIn 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. ConclusionsNecroptosis 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.

Role of Mesenchymal Stem Cell-Derived Conditioned Medium in Modulating the Benzalkonium Chloride-Induced Cytotoxic Effects in Cultured Corneal Epithelial Cells In Vitro

Purpose Benzalkonium chloride (BAK) is a common preservative in ophthalmic formulations that causes cytotoxic damage to the corneal epithelial cells. This study aims to explore the role of mesenchymal stem cell (MSC)-derived conditioned medium in modulating the BAK-induced cytotoxic effects in cultured human corneal epithelial cells (HCECs) as a cell-free therapeutic agent. Methods The in vitro cultured HCECs derived from a HCE cell line were treated with BAK (0.001% and 0.005%, diluted in DMEM/F12, v/v) for 15 min, washed with 1xPBS, and allowed to recover for 24 h in human bone marrow MSC-derived conditioned medium (MSC-CM: undiluted (100%) and diluted (50%, v/v)). On the other hand, HCECs were co-incubated with BAK (0.005%, v/v) and MSC-CM (100% and 50%, v/v) for 24 h. The HCEC-derived conditioned medium (HCE-CM) was used as an optimal control for MSC-CM, whereas HCECs cultured in DMEM/F12 were used as a control. The DMEM/F12 was used as the base medium for the culture of HCECs and preparation of HCE- and MSC-CM. The role of MSC-CM in modulating the metabolic activity, cell death, epithelial repair, and proliferation, in BAK-treated HCECs was evaluated using MTT assay, Propidium iodide staining, scratch assay, and Ki-67 staining, respectively. Results Compared to the control, recovery of BAK-treated (0.001% and 0.005%, for 15 min) HCECs in MSC-CM showed significantly reduced cell death with enhanced metabolic activity, epithelial repair, and proliferation. However, in comparison with HCE-CM, the beneficial effects of MSC-CM were predominantly observed at lower BAK concentration (0.001%, for 15 min). Whereas the co-incubation of BAK (0.005%) and MSC-CM for a longer duration (24 h) was marginally beneficial. Conclusions Our results suggest that the MSC-CM is effective in modulating the BAK-induced cell death, retardation of metabolic activity and proliferation in cultured HCECs, particularly at lower concentration (0.001%) and shorter exposure (15 min) of BAK.

Mucoadhesive Hybrid System of Silk Fibroin Nanoparticles and Thermosensitive In Situ Hydrogel for Amphotericin B Delivery: A Potential Option for Fungal Keratitis Treatment.

The purpose of this work was to investigate the feasibility of a novel ophthalmic formulation of amphotericin B-encapsulated silk fibroin nanoparticles incorporated in situ hydrogel (AmB-FNPs ISG) for fungal keratitis (FK) treatment. AmB-FNPs ISG composites were successfully developed and have shown optimized physicochemical properties for ocular drug delivery. Antifungal effects against Candida albicans and in vitro ocular irritation using corneal epithelial cells were performed to evaluate the efficacy and safety of the composite formulations. The combined system of AmB-FNPs-ISG exhibited effective antifungal activity and showed significantly less toxicity to HCE cells than commercial AmB. In vitro and ex vivo mucoadhesive tests demonstrated that the combination of silk fibroin nanoparticles with in situ hydrogels could enhance the adhesion ability of the particles on the ocular surface for more than 6 h, which would increase the ocular retention time of AmB and reduce the frequency of administration during the treatment. In addition, AmB-FNP-PEG ISG showed good physical and chemical stability under storage condition for 90 days. These findings indicate that AmB-FNP-PEG ISG has a great potential and be used in mucoadhesive AmB eye drops for FK treatment.

A comparison between the effects of two liposome-encapsulated bevacizumab formulations on ocular neovascularization inhibition

Bevacizumab (BVZ), an anti-VEGF antibody, has demonstrated reliable outcomes in the treatment of irritating ocular neovascularization. Frequent intravitreal injections are necessitated due to rapid clearance and short local accessibility. We recruited liposome as a highly prevailing drug delivery system to enhance drug availability. Two liposome formulations were characterized and their in vitro stability was analyzed. The toxicity of the formulations on some ocular cell lines was also evaluated. In addition, the anti-angiogenic effects of formulations were examined. Drug permeation was measured across ARPE-19 and HCE cell lines as in vitro cellular barrier models. Results revealed that NLP-DOPE-BVZ acquired high stability at 4 °C, 24 °C, and 37 °C for 45 days. It also showed more capacity to entrap BVZ in NLP-DOPE-BVZ (DEE% 69.1 ± 1.4 and DLE% 55.66 ± 1.15) as compared to NLP-BVZ (DEE% 43.57 ± 14.64, and DLE% 37.72 ± 12.01). Although both formulations inhibited the migration and proliferation of HUVECs, NLP-DOPE-BVZ was more effective at inhibiting angiogenesis. Furthermore, NLP-DOPE-BVZ better crossed our established barrier cellular models. Based on the findings, the inclusion of DOPE in NLPs has significantly enhanced the features of drug carriers. This makes them a potential candidate for treating ocular neovascularization and other related ailments.

EZH2 Promotes Corneal Endothelial Cell Apoptosis by Mediating H3K27me3 and Inhibiting HO-1 Transcription

Purpose This paper aims to explore the molecular mechanism of Enhancer of Zeste Homolog 2 (EZH2)-mediated H3K27me3 in human corneal endothelial cells (HCEC) apoptosis by inhibiting Heme oxygenase-1 (HO-1) transcription to provide a potential target for the treatment of corneal apoptosis. Methods HCECs were cultured in vitro and transfected with si-EZH2, pcDNA3.1-EZH2, pcDNA3.1-HO-1, GSK-J4 (an effective H3K27me3 demethylase inhibitor), and corresponding controls. Western Blot assay was used to detect the levels of EZH2, HO-1, H3K27me3, and apoptosis-related proteins (Bcl-2, Bax, and Cleaved-caspase-3) in HCECs; CCK-8 assay was conducted to detect cell viability and flow cytometry to analyze the apoptosis. HO-1 mRNA levels were detected by RT-qPCR and changes in H3K27me3 levels on the HO-1 promoter were detected by chromatin immunoprecipitation. Results HCECs transfected with si-EZH2 showed significantly lower EZH2 mRNA and protein levels, higher HCEC viability, lower apoptosis rates, higher antiapoptotic protein Bcl-2 expression, lower proapoptotic protein (Bax and Cleaved-caspase-3) levels, and significantly higher HO-1 expression. HCECs transfected with pcDNA3.1-EZH2 showed the opposite results. EZH2 repressed HO-1 transcription by mediating H3K27me3. H3K27me27 was enriched in the HO-1 promoter and overexpression of EZH2 increased H3K27me27 levels. Promotion of H3K27me3 partially reversed the mitigating effect of si-EZH2 on HCEC apoptosis. Overexpression of HO-1 partially reversed the apoptosis-promoting effects of EZH2 and H3K27me3 on HCECs. Conclusions EZH2 promotes HCE cell apoptosis by mediating H3K27me3 to inhibit HO-1 transcription.

Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE.

Dry eye inflammation is a key step in a vicious circle and needs to be better understood in order to break it. The goals of this work were to, first, characterize alarmins and cytokines released by ocular surface cells in the hyperosmolar context and, second, study the role of NFAT5 in this process. Finally, we studied the potential action of these alarmins in ocular surface epithelial cells and macrophages via RAGE pathways. HCE and WKD cell lines were cultured in a NaCl-hyperosmolar medium and the expression of alarmins (S100A4, S100A8, S100A9, and HMGB1), cytokines (IL6, IL8, TNFα, and MCP1), and NFAT5 were assessed using RT-qPCR, ELISA and multiplex, Western blot, immunofluorescence, and luciferase assays. In selected experiments, an inhibitor of RAGE (RAP) or NFAT5 siRNAs were added before the hyperosmolar stimulations. HCE and WKD cells or macrophages were treated with recombinant proteins of alarmins (with or without RAP) and analyzed for cytokine expression and chemotaxis, respectively. Hyperosmolarity induced epithelial cell inflammation depending on cell type. NFAT5, but not RAGE or alarmins, participated in triggering epithelial inflammation. Furthermore, the release of alarmins induced macrophage migration through RAGE. These in vitro results suggest that NFAT5 and RAGE have a role in dry eye inflammation.

Beneficial Effects of Plasma Rich in Growth Factors (PRGF) Versus Autologous Serum and Topical Insulin in Ocular Surface Cells

Purpose In the last few decades, several blood derived products such as platelet-rich plasma (PRP), plasma rich in growth factors (PRGF) and autologous serum (AS) have been used for the treatment of ocular surface disorders. Recently, insulin has been proposed to be used as an alternative for the treatment of ocular surface diseases. The aim of this study was to evaluate the biological potential of PRGF eye drops in comparison with AS and insulin on ocular surface cells. Methods Blood from three healthy young donors was collected to obtain autologous serum (AS) eye drops and plasma rich in growth factors (PRGF) eye drops. Insulin (Actrapid®) was diluted at 1 and 0.2 IU/mL. The biological potential of PRGF, AS and insulin was assessed by proliferation in HCE, HK and HConF cells. Wound healing assay was performed in HCE cells after incubation with the different treatments. HConF and HK cells were differentiated to myofibroblast after treatment with 2.5 ng/mL of TGF-β1 and then incubated with all treatments. Results PRGF eye drops induced significantly higher proliferation rate compared to AS or insulin in HConF and HK cells, but not in HCE cells. In addition, the percentage of wound healing area was significantly reduced after PRGF treatment in comparison with AS or insulin treatment. Furthermore, PRGF significantly reduced the number of myodifferentiated cells compared to AS and insulin at both concentrations analyzed. Conclusion The results obtained in the present study show that PRGF increases the biological activity of the ocular surface cells and reduces the expression of fibrosis marker compared to insulin or AS. Translational relevance The present study suggests that plasma rich in growth factors eye drops are a more effective therapy than insulin and autologous serum eye drops for the treatment of ocular surface diseases.

Biocompatible Magnetic Conjugated Polymer Nanoparticles for Optical and Lifetime Imaging Applications in the First Biological Window.

Conjugated polymers are organic semiconductors that can be used for fluorescence microscopy of living specimens. Here, we report the encapsulation of the bright-red-emitting conjugated polymer, poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorenylene}-alt-co-{2,5-bis(N,N'-diphenylamino)-1,4-phenylene}] (CN-FO-DPD), and superparamagnetic iron oxide nanoparticles (SPIONs) within poly(styrene-co-maleic anhydride) (PSMA) micelles. The resulting particles exhibited an emission peak at 657 nm, a fluorescence quantum yield of 21%, an average diameter of 65 nm, and a ζ potential of -30 mV. They are taken up by cells, and we describe their use in fluorescence microscopy of living Hela cells and zebrafish embryos and their associated cytotoxicity in HEK, HeLa, and HCE cells.

Improved ocular delivery of quercetin and resveratrol: A comparative study between binary and ternary cyclodextrin complexes

The number of patients affected by Dry Eye Disease (DED) had notably increased worldwide, addressing the need of novel therapeutic approaches. Polyphenols, quercetin (QUE) and resveratrol (RSV) show necessary antioxidant and anti-inflammatory properties to manage DED, but their application as topical eyedrops is restricted by low aqueous solubility and low chemical stability. Cyclodextrins (CD) are widely used to improve physicochemical characteristics of drugs. Consequently, the aim of this study was to make a comparison between binary complexes with quercetin, resveratrol and cyclodextrins and tertiary complexes adding hyaluronic acid (HA). Both complexes were able to enhance solubility and stability of QUE and RSV. AFM imaging and DLS measurements disclose the formation of spherical nanoaggregates within tertiary complexes of both QUE and RSV with mean diameters of 103 and 82 nm. Neither complex demonstrated cytotoxic effect in in vitro studies in corneal (HCE) and conjunctival (IM-ConjEpi) cell lines. In HCE cells, complexes containing QUE or RSV at their highest concentrations were able to scavenge more than 95 % of the ROS that were produced intracellularly (p < 0.005). Similar response was observed with IM-ConjEpi cells. The antioxidant effect was maintained in the complexes with HA. This confirmed their potential as viable topical treatment for DED.

Hydroxypropyl methacrylamide-based copolymeric nanoparticles loaded with moxifloxacin as a mucoadhesive, cornea-penetrating nanomedicine eye drop with enhanced therapeutic benefits in bacterial keratitis

Bacterial keratitis (BK) is a leading cause of visual impairment. The fluoroquinolone antibiotic moxifloxacin (Mox), being highly water-soluble, suffers from poor corneal penetration leading to unsatisfactory therapeutic outcomes in BK. Here, we prepared Mox-loaded co-polymeric nanoparticles (NPs) by entrapping the drug in co-polymeric NPs constituted by the self-assembly of a water-soluble copolymer, poly(ethylene glycol)-b-p(hydroxypropyl) methacrylamide (mPH). The polymer (mPH) was prepared using a radical polymerization technique at different mPEG: HPMA ratios of 1:70/100/150. The polymer/nanoparticles were characterized by GPC, CAC, DLS, SEM, XRD, DSC, FTIR, % DL, % EE, and release studies. The ex vivo muco-adhesiveness and corneal permeation ability were judged using a texture analyzer and Franz Diffusion Cells. In vitro cellular uptake, cytotoxicity, and safety assessment were performed using HCE cells in monolayers, spheroids, and multilayers in transwells. The DOE-optimized colloidal solution of Mox-mPH NPs (1:150) displayed a particle size of ~116 nm, superior drug loading (8.3%), entrapment (83.2%), robust mucoadhesion ex vivo, and ocular retention in vivo (~6 h) (judged by in vivo image analysis). The non-irritant formulation, Mox-mPH NPs (1:150) (proven by HET-CAM test) exhibited intense antimicrobial activity against P. aeruginosa, S. pneumoniae, and S. aureus in vitro analyzed by live-dead cells assay, zone of inhibition studies, and by determining the minimum inhibitory and bactericidal concentrations. The polymeric nanoparticles, mPH (1:150), decreased the opacity and the bacterial load compared to the other treatment groups. The studies warrant the safe and effective topical application of the Mox-mPH NPs solution in bacterial keratitis.

Inulin-Based Polymeric Micelles Functionalized with Ocular Permeation Enhancers: Improvement of Dexamethasone Permeation/Penetration through Bovine Corneas.

Ophthalmic drug delivery is still a challenge due to the protective barriers of the eye. A common strategy to promote drug absorption is the use of ocular permeation enhancers, while an innovative approach is the use of polymeric micelles. In the present work, the two mentioned approaches were coupled by conjugating ocular permeation enhancers (PEG2000, carnitine, creatine, taurine) to an inulin-based co-polymer (INU-EDA-RA) in order to obtain self-assembling biopolymers with permeation enhancer properties for the hydrophobic drug dexamethasone (DEX). Inulin derivatives were properly synthetized, were found to expose about 2% mol/mol of enhancer molecules in the side chain, and resulted able to self-assemble at various concentrations by varying the pH and the ionic strength of the medium. Moreover, the ability of polymeric micelles to load dexamethasone was demonstrated, and size, mucoadhesiveness, and cytocompatibility against HCE cells were evaluated. Furthermore, the efficacy of the permeation enhancer was evaluated by ex vivo permeation studies to determine the performance of the used enhancers, which resulted in PEG2000 > CAR > TAU > CRE, while entrapment ability studies resulted in CAR > TAU > PEG2000 > CRE, both for fluorescent-labelled and DEX-loaded micelles. Finally, an increase in terms of calculated Kp and Ac parameters was demonstrated, compared with the values calculated for DEX suspension.

MiR-205-3p protects human corneal epithelial cells from ultraviolet damage by inhibiting autophagy via targeting TLR4/NF-κB signaling.

MiRNA has been found to have therapeutic effect on corneal damage. This paper aimed to study the effect of miR-205-3p on corneal damage induced by ultraviolet (UV) radiation. HCE cells were exposed to UV light and transfected. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot were used to determine miRNA/mRNA and protein expression. CCK-8 assay, Edu incorporation experiment, and flow cytometry were used to separately measure cell activity, proliferation and apoptosis. LC3 puncta were researched by immunofluorescence experiment. TNF-α, IL-6 and IL-1β levels in cells were detected by enzyme-linked immunosorbent assay (ELISA) kit. MDA, SOD, and GSH-PX levels were measured using detection kits. Reactive oxygen species (ROS) level was reflected by detecting DCFH-DA density. Luciferase activity assay was performed to verify the regulating relationship between miR-205-3p and TLR4. UV radiation decreased HCE cell viability, proliferation, and increased HCE cell apoptosis and autophagy (all p < 0.01). When exposed UV radiation, the overexpression of miR-205-3p group elevated HCE cells viability, proliferation and weakened HCE cells apoptosis and autophagy (all p < 0.01). MiR-205-3p inhibited inflammation and oxidative stress in HCE cells induced by UV radiation (p < 0.01). MiR-205-3p directly inhibited TLR4 expression. The upregulation of TLR4 significantly reversed the effects of miR-205-3p on HCE cells phenotypes induced by UV radiation (p < 0.01). MiR-205-3p protected HCE cells from UV damage by inhibiting autophagy via targeting TLR4.

Apoptotic effects of norfloxacin on corneal endothelial cells.

Norfloxacin, a frequently used ocular antibiotic, might have cytotoxic effect on human corneal endothelial cells (HCECs), subsequently damage the cornea and finally impair human vision. However, the possible mechanisms of cytotoxicity of norfloxacin to HCEC line are unclear. Herein, we investigated the cytotoxicity of norfloxacin and its underlying cellular and molecular mechanisms using in vitro cultured non-transfected HCECs and verified the cytotoxicity with cat corneal endothelium in vivo. In the present study, the cytotoxicity of norfloxacin in the in vitro cultured HCECs was recognized by causing abnormal morphology such as cell shrinkage and detachment from plate bottom, and decline of viability of in vitro cultured HCECs. Then, its cytotoxicity was verified by inducing reduction of cell density and morphological abnormality of in vivo cat corneal endothelial cells. Furthermore, the cytotoxicity of norfloxacin in HCECs was corroborated as apoptosis by elevation of plasma membrane permeability, S phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in in vitro cultured HCECs and apoptosis-like swollen cells in the in vivo model. Moreover, norfloxacin induced extrinsic death receptor-mediated apoptosis pathway by activating caspase-2/-8/-3 and intrinsic mitochondrion-dependent apoptosis pathway by downregulating anti-apoptotic Bcl-2 and upregulating of pro-apoptotic Bad, which disrupted mitochondrial transmembrane potential, subsequently upregulated cytoplasmic cytochrome c and apoptosis-inducing factor and finally activated caspase-9/-3. Generally, norfloxacin induces HCE cell apoptosis via a death receptor-mediated and mitochondrion-dependent signaling pathway.

Multi-laboratory Validation Study of the Vitrigel-Eye Irritancy Test Method as an Alternative to In Vivo Eye Irritation Testing.

Collagen vitrigel membranes (CVMs) comprising high-density collagen fibrils equivalent to in vivo connective tissues have been widely used in cell culture applications. A human corneal epithelium (hCE) model was previously developed by the Takezawa group, by culturing HCE-T cells (derived from hCE cells) on a CVM scaffold in a chamber that provided an air-liquid interface culture system. This hCE model was used to establish a new test method, known as the Vitrigel-Eye Irritancy Test (Vitrigel-EIT) method, which can be used to estimate the ocular irritation potential of test chemicals by analysing relative changes in transepithelial electrical resistance (TEER) over time. The current study was conducted in order to assess the reliability and relevance of the Vitrigel-EIT method at three participating laboratories by determining the method's within-laboratory reproducibility and between-laboratory reproducibility, as well as its capacity for distinguishing non-irritants from irritants in a bottom-up approach. The initial test sample size was found to be too low to evaluate the predictive capacity of the test method, and so it was evaluated with additional in-house data for a total of 93 test chemicals. The results showed 80-100% within-laboratory reproducibility and an excellent between-laboratory reproducibility that met the acceptance criteria of 80%. However, the method's predictive capacity for distinguishing non-irritants (test chemicals not requiring classification and labelling for eye irritation or serious eye damage, i.e. United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS) No Category) from irritants (GHS Categories 1 and 2) in a bottom-up approach was unacceptable because of false negative rates as high as 16.7%. After considerable review of the data with a view to using the method for regulatory purposes, it was determined that a more defined applicability domain, excluding test chemical solutions with a pH of 5 or less and solid test chemicals, improved the false negative rate to 4.2%. These results suggested that, within this carefully defined applicability domain, the Vitrigel-EIT method could be a useful alternative for distinguishing test chemicals that are ocular non-irritants from those that are irritants as part of a bottom-up approach.

A folic acid-sensitive polyfluorene based “turn-off” fluorescence nanoprobe for folate receptor overexpressed cancer cell imaging

Herein, we have developed an efficient approach for folate receptor (FR) over expressed cancer cell targeting and imaging through non-covalent conjugation between folic acid (FA) and a polyfluorene based amphiphilic, fluorescent polymer nanoprobes (CPF) by fluorescence “turn off” mechanism. The pH dependent supramolecular complexation/decomplexation tendency of CPF nanoprobes with FA provides the effective reversible fluorescence “turn off/turn on” of CPF fluorescence. The complete pH dependent complexation/decomplexation phenomena is characterized by photophysical and morphological characterization. The FESEM image study reveals the alteration of nanosphere structure of CPF to interconnected network after complexation with FA at pH 9 and consequently restoration of nanosphere morphology by decomplexation at pH 2. Again, the CPF nanoprobes exhibit 91% fluorescence quenching even in physiological pH 7.4 to reveal that the nanoprobes can be a suitable candidate for FA targeted turn-off fluorescent nanoprobes for FA pre-treated FR-positive cancer cells in vivo study. The fluorescence of CPF nanoprobes is significantly quenched in FA pre-treated FR-positive B16F10 cancer cell whereas it remains unchanged in FR-non overexpressed normal human corneal cell (HCE cells) even after FA pre-treatment. Furthermore, the present study can provide a dissimilar protocol to use a “turn off” fluorescent nanoprobe for FR-positive cancer cell imaging using free FA and fluorescent polymer nanoparticle rather than the conventional direct FA-fluorescent conjugated polymer complex.

Narrow Spectrum Kinase Inhibitors Demonstrate Promise for the Treatment of Dry Eye Disease and Other Ocular Inflammatory Disorders.

The purpose of this study is to determine the potential of narrow spectrum kinase inhibitors (NSKIs) to treat inflammatory eye disorders. Human conjunctival epithelial (HCE) cells were retrieved from subjects via impression cytology. Real-time quantitative PCR (qPCR) was performed on HCE cells to determine gene expression of NSKI kinase targets and proinflammatory cytokines in dry eye disease (DED) patients versus healthy controls. qPCR also assessed p38α expression in hyperosmolar-treated Chang conjunctival epithelial cells. Interaction of NSKI TOP1362 with the kinases was evaluated in ATP-dependent Z-LYTE and competition binding assays. Anti-inflammatory activity was assessed in human peripheral blood mononuclear cells and primary macrophages. In an endotoxin-induced uveitis (EIU) study, lipopolysaccharide (LPS) was administered intravitreally to Lewis rats. TOP1362, dexamethasone, or vehicle was administered topically, and inflammatory cytokine levels were measured 6 hours after LPS injection. HCE cells from DED patients showed significantly increased expression of p38α, spleen tyrosine kinase (Syk), Src, lymphocyte-specific protein tyrosine kinase (Lck), interleukin one beta (IL-1β), interleukin eight (IL-8), monocyte chemotactic protein-1 (MCP-1), and matrix metalloproteinase-9 (MMP-9). TOP1362 strongly inhibited the kinase targets p38α, Syk, Src, and Lck, blocked the rise in p38α expression in hyperosmolar Chang cells, and potently reduced inflammatory cytokine release in cellular models of innate and adaptive immunities. In the EIU model, TOP1362 dose-dependently attenuated the LPS-induced rise in inflammatory cell infiltration and ocular cytokine levels with efficacy comparable to that of dexamethasone. TOP1362 is a potent inhibitor of kinases upregulated in DED and markedly attenuates proinflammatory cytokine release in vitro and in vivo, highlighting the therapeutic potential of NSKIs for treating ocular inflammation, such as that observed in DED.

The expression of matrix metalloproteinases and their inhibitors in corneal fibroblasts by alarmins from necrotic corneal epithelial cells.

Sterile ulceration is frequently observed in the cornea following persistent corneal epithelial damage. We examined the effect of alarmins released by necrotic corneal epithelial cells (HCE) on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by corneal fibroblasts. IL-1α and high-mobility group box 1 protein (HMGB1) released into the supernatant derived from necrotic HCE cells were measured with enzyme-linked immunosorbent assay (ELISA). MMPs and TIMPs produced by corneal fibroblasts, stimulated with the supernatant from necrotic HCE cells, were analyzed and measured with protein array and ELISA. To investigate dynamic expression of alarmins in the corneal epithelium, we used immunohistochemistry to observe the expression of human IL-1α in the corneal epithelium of human IL-1α Tg mice with or without cryopexy. We also investigated the expression of MMPs in corneal stroma of the mice treated with cryopexy, using RT-PCR. We detected IL-1α and HMGB-1 in the supernatant of necrotic HCE cells. These supernatants increased the expression of MMP-3 and MMP-1, and decreased that of TIMP-1 and TIMP-2 in human corneal fibroblasts. Almost always these were inhibited by IL-1 receptor antagonist. Recombinant IL-1α increased the production MMP-3 and MMP-1 in corneal fibroblasts. After cryopexy of the epithelium of human IL-1α Tg mice, the expression of human IL-1α was recognized in the cytoplasm but not nucleus of epithelial cells. The level of MMP-3 and MMP-1 mRNAs was elevated in the corneal stroma in mice treated with cryopexy. Alarmins, especially IL-1α, released from necrotic HCE cells may play an important role in the expression of MMPs and TIMPs by corneal fibroblast, resulting in sterile ulceration.

Use of sodium hyaluronate in combination with a blood derivative in the re‐epithelialization of rabbit corneas

PurposeTo analyze if using a bioadhesive (sodium hyaluronate or HaNa) in combination with a blood derivative (s‐PRGF) improves the healing rate and quality of corneal epithelial ulcers.MethodsRabbit corneas removed 7 and 30 days after an in vivo re‐epithelialization assay were include in paraffin and processed for haematoxylin–eosin staining or cryopreserved for immunofluorescent staining. In vitro proliferation and wound healing experiments were performed using the human corneal epithelial HCE cell line and rabbit primary corneal epithelial cultures. We studied the following treatments: 1) 90% s‐PRGF 2) 0.22% NaHa 3) s‐PRGF + NaHa 4) PBS as control treatment for the in vivo assay. All components in treatments 1, 2 and 3 were half the concentration for the in vitro assays, being 1% BSA the control treatment (4). To manufacture s‐PRGF whole blood was collected by venipuncture from healthy volunteers or New Zealand rabbits, respectively.ResultsHealing rate was higher in cultures and in eyes under s‐PRGF treatment than in those treated with HaNa or the combination of both. H‐E sections at 30 days showed more cells in the anterior stroma in eyes under HaNa treatments. In agreement with it, Ki67 staining as well as in vitro proliferation assays demonstrated that HaNa stimulated cell proliferation. All treatments produced stratified and mature epithelia (CK3 positiveness) with barrier function (ZO‐1 staining) and activation of limbal stem cells (CK15 staining), although the HaNa treated epithelia were the less organized. Moreover, eyes treated with only s‐PRGF showed the best integrin β4 staining.ConclusionsHaNa bioadhesive promotes epithelial and stromal cell proliferation, but does not improve the corneal healing capacity of s‐PRGF. s‐PRGF shows a better healing quality in terms of epithelial‐stromal adhesion.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing.

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Roles of TRIM32 in Corneal Epithelial Cells After Infection with Herpes Simplex Virus

Background: Epithelial cells play important roles as a critical barrier in protecting the cornea from microbial pathogens infection. Methods: In this study, we were aiming to investigate the role of E3 ubiquitin ligase tripartite motif protein 32 (TRIM32) in corneal epithelial cells in response to Herpes Simplex Virus type 1 (HSV-1) infection and to elucidate the underlying mechanisms. Results: We found the expression of TRIM32 was increased after infected with HSV-1 both in murine corneas and cultured human epithelial (HCE) cells. Furthermore, knockdown of the expression of TRIM32 significantly aggravated HSV-1 induced herpetic stromal keratitis (HSK) in mice and promoted the replication of HSV-1 in cultured HCE cells. We also observed that silencing of TRIM32 resulted in the decreased expression of IFN-β and suppressed activation of interferon regulatory factor 3 (IRF3) both in vivo and in vitro. Finally, we found TRIM32 positively regulate IFN-β production in corneal epithelial cells through promoting K63-linked polyubiquitination of stimulator of interferon genes (STING). Conclusion: In conclusion, our data suggested that TRIM32 as a crucial positive regulator of HSV-1 induced IFN-β production in corneal epithelial cells, and it played a predominant role in clearing HSV-1 from the cornea.