The Neuropeptide α-Melanocyte-Stimulating Hormone Prevents Persistent Corneal Edema following Injury.

Corneal endothelial cells (CEnCs) regulate corneal hydration and maintain tissue transparency through their barrier and pump function. However, these cells exhibit limited regenerative capacity following injury. Currently, corneal transplantation is the only established therapy for restoring endothelial function, and there are no pharmacologic interventions available for restoring endothelial function. This study investigated the efficacy of the neuropeptide α-melanocyte-stimulating hormone (α-MSH) in promoting endothelial regeneration during the critical window between ocular injury and the onset of endothelial decompensation using an established murine model of injury using transcorneal freezing. Local administration of α-MSH following injury prevented corneal edema and opacity, reduced leukocyte infiltration, and limited CEnC apoptosis while promoting their proliferation. These results suggest that α-MSH has a proregenerative and cytoprotective function on CEnCs and shows promise as a therapy for the prevention and management of corneal endothelial dysfunction.

Investigating the Role of TGF-ß Signaling Pathways in Human Corneal Endothelial Cell Primary Culture.

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial-mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-ßs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-ß signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-ß1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-ß-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-ß1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-ß receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-ß1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD.

Anti-inflammatory and wound healing potential of medicinal maggot excretions/secretions at the ocular surface.

PURPOSE: In the skin, Lucilia sericata maggot excretions/secretions (ES) accelerate wound healing and limit inflammation. This study aimed to determine whether ES have similar beneficial effects at the ocular surface. METHODS: Human corneal epithelial cells (HCEC) were cultured with ES and cell viability was determined by the MTT assay. Additionally, mRNA expression of growth factors, antimicrobial peptides (AMPs) and cytokines was assessed by qPCR. ES ability to modulate TLR-induced IL-6 and IL-8 expression was determined by qPCR and ELISA. ES potential to promote corneal healing was evaluated in vitro by a migration assay in HCEC, and in vivo using a mouse model. RESULTS: ES did not impair HCEC viability up to 25 µg/ml. Among the factors evaluated, only hBD-2 was upregulated (2.5-fold) by 1.5 µg/ml ES after 6 hrs (P = 0.04). In HCEC, ES reduced Poly I:C-induced IL-6 and IL-8 mRNA (P ≤ 0.001) and protein (P ≤ 0.0001) expression. A similar effect was observed with Flagellin (TLR5 agonist) but it was less robust for FSL-1 (TLR2/6 agonist) and Pam3CSK4 (TLR1/2 agonist). The greatest in vitro migration effect was observed with 6.2 µg/ml ES after 44 hrs where gap area compared to vehicle was 53.3 ± 3.7% vs. 72.6 ± 5.4% (P = 0.001). In the mouse model, the maximum healing effect was present with 1.5 µg/ml ES after 12 hrs with a wound area of 19.0 ± 2.7% vs. 60.1 ± 21.6% (P = 0.003) or 77% reduction of the wound area compared to the negative control. CONCLUSIONS: ES significantly reduce in vitro TLR-induced production of inflammatory cytokines and promote corneal wound healing.

In vitro evaluation of platelet extracellular vesicles (PEVs) for corneal endothelial regeneration.

Corneal endothelial cells (CECs) slowly decrease in number with increasing age, which is a clinical issue as these cells have very limited regenerative ability. Therapeutic platelet biomaterials are increasingly used in regenerative medicine and cell therapy because of their safety, cost-effective manufacture, and global availability from collected platelet concentrates (PCs). Platelet extracellular vesicles (PEVs) are a complex mixture of potent bioactive vesicles rich in molecules believed to be instrumental in tissue repair and regeneration. In this study we investigated the feasibility of using a PEVs preparation as an innovative regenerative biotherapy for corneal endothelial dysfunction. The PEVs were isolated from clinical-grade human PC supernatants by 20,000 × g ultracentrifugation and resuspension. PEVs exhibited a regular, fairly rounded shape, with an average size of <200 nm and were present at a concentration of approximately 1011 /mL. PEVs expressed cluster of differentiation 41 (CD41) and CD61, characteristic platelets membrane markers, and CD9 and CD63. ELISA and LC-MS/MS proteomic analyses revealed that the PEVs contained mixtures of growth factors and multiple other trophic factors, as well as proteins related to extracellular exosomes with functional activities associated with cell cadherin and adherens pathways. CECs treated with PEVs showed increased viability, an enhanced wound-healing rate, stronger proliferation markers, and an improved adhesion rate. PEVs did not exert cellular toxicity as evidenced by the maintenance of cellular morphology and preservation of corneal endothelial proteins. These findings clearly support further investigations of PEV biomaterials in animal models for translation as a new CEC regeneration biotherapy.

The NLRP1 Inflammasome Induces Pyroptosis in Human Corneal Epithelial Cells.

PURPOSE: Inflammasomes are multiprotein complexes that detect danger-associated signals and trigger an immunostimulatory form of cell death called pyroptosis. NLRP1 is an innate immune receptor that assembles into an inflammasome, but the primary cell types in which NLRP1 is functional have not yet been fully established. Mutations in NLRP1 are associated with diseases of barrier epithelial tissues, including skin lesions and corneal intraepithelial dyskeratosis, suggesting that NLRP1 functions within the eye. Here, we investigated the expression and activity of the NLRP1 inflammasome in primary human corneal epithelial (pHCE) cells. METHODS: The small molecule Val-boroPro (VbP) activates the NLRP1 inflammasome. Proteasome (bortezomib, MG132) and caspase-1 (VX-765, Z-VAD-FMK) inhibitors block NLRP1 activation and downstream pyroptosis, respectively. Here, we treated pHCE cells with VbP alone or in combination proteasome inhibitors and caspase-1 inhibitors. We assessed NLRP1 expression and hallmarks of pyroptosis, including lytic cell rupture, cytokine processing and release, and gasdermin D (GSDMD) processing. RESULTS: VbP triggered pyroptosis in pHCE cells, as determined by cytokine secretion, GSDMD processing, and lactate dehydrogenase (LDH) release. Proteasome and caspase-1 inhibitors completely blocked this pyroptotic cell death. In contrast, other primary ocular epithelial cells did not undergo NLRP1-dependent pyroptosis. CONCLUSIONS: Our findings demonstrate that NLRP1 forms a functional inflammasome in pHCE cells. Importantly, these data reveal that NLRP1 is a key innate immune sensor of the corneal epithelium, and moreover indicate how aberrant inflammasome activation causes corneal damage. Blockade of NLRP1 signaling may benefit patients with hyperactive NLRP1 mutations and warrants further investigation.

Expression of immune response genes in human corneal epithelial cells interacting with Aspergillus flavus conidia.

BACKGROUND: Aspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics. METHODS: Human corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes. RESULTS: Different morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells. CONCLUSIONS: Combining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.

Corneal Densitometry Assessed With Scheimpflug Camera in Healthy Corneas and Correlation With Specular Microscopy Values and Age.

PURPOSE: To investigate correlations between specular microscopy endothelial parameters and age with corneal densitometry values, as they are presented from a Scheimpflug device, in different levels of the cornea. METHODS: Two hundred eighty-four eyes of 142 healthy subjects were included in this observational, prospective study. Corneal densitometry was evaluated with Scheimpflug imaging system in the central 0- to 2-mm annular zone of the cornea, whereas the endothelial cell properties were assessed with the use of a noncontact specular microscope. RESULTS: Corneal densitometry values of all corneal layers were statistically significant and positively correlated with age. In univariate linear regression analysis among corneal densitometry values and the endothelial parameters, only endothelial cell density (CD) was statistically significant and inversely correlated with densitometry values in all corneal layers. In stepwise multivariate linear regression analysis, after adjustment for age, hexagonality was statistically significant and inversely correlated with posterior densitometry values, whereas coefficient of variation was positively and significantly correlated with the anterior densitometry values. When repeating stepwise multivariate linear regression analysis without adjusting for age, CD was negatively and significantly correlated with corneal densitometry values of all layers, whereas coefficient of variation was positively and significantly correlated with anterior and total corneal densitometry values. CONCLUSIONS: Corneal densitometry increases with age. It is also inversely correlated with CD, and this might be used as an indirect way to assess the status of the corneal endothelium.

Voriconazole-Cyclodextrin Supramolecular Ternary Complex-Loaded Ocular Films for Management of Fungal Keratitis.

Fungal keratitis is one of the leading causes of ophthalmic mycosis affecting the vision due to corneal scarring. Voriconazole (VRC) is the most preferred azole antifungal agent for treating ocular mycotic infections. Ocular drug delivery is challenging due to the shorter corneal residence time of the formulation requiring frequent administration, leading to poor patient compliance. The present study aimed at improving the solubility, transcorneal permeation, and efficacy of voriconazole via the formation of cyclodextrin-based ternary complexes and incorporation of the complex into mucoadhesive films. A phase solubility study suggested a ∼14-fold improvement in VRC solubility, whereas physicochemical characterization confirmed the inclusion of VRC in the cyclodextrin inner cavity. In silico docking studies were performed to predict the docking conformation and stability of the inclusion complex. Complex-loaded films showed sustained release of voriconazole from the films and improved transcorneal permeation by ∼4-fold with an improved flux of 8.36 µg/(cm2 h) for ternary complex-loaded films compared to 1.86 µg/(cm2 h) for the pure VRC film. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and hen's egg-chorioallantoic membrane test (HET-CAM) assays confirmed that the complexes and ocular films were nonirritant and safe for ocular administration. The antifungal study performed using Aspergillus fumigatus and Fusarium oxysporum suggested improved antifungal activity compared to the pure drug film. In conclusion, the supramolecular cyclodextrin ternary complex proved to be a promising strategy for enhancing the solubility and permeability and augmenting the antifungal activity of voriconazole in the management of fungal keratitis.

An ex vivo model of human corneal rim perfusion organ culture.

The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 µl/min, the outflow facility was 0.359 ± 0.216 µl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.

Human Adenovirus Species D Interactions with Corneal Stromal Cells.

Notable among the many communicable agents known to infect the human cornea is the human adenovirus, with less than ten adenoviruses having corneal tropism out of more than 100 known types. The syndrome of epidemic keratoconjunctivitis (EKC), caused principally by human adenovirus, presents acutely with epithelial keratitis, and later with stromal keratitis that can be chronic and recurrent. In this review, we discuss the current state of knowledge regarding the molecular biology of adenovirus infection of corneal stromal cells, among which the fibroblast-like keratocyte is the most predominant, in order to elucidate basic pathophysiologic mechanisms of stromal keratitis in the human patient with EKC.

Membranes Prepared from Recombinant RGD-Silk Fibroin as Substrates for Human Corneal Cells.

A recombinant formulation of silk fibroin containing the arginine-glycine-aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring Bombyx mori silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal-epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.

[The in vitro effect of antiseptics on epithelial cells of human cornea and conjunctiva]. / Vozdeistvie antiseptikov na epitelial'nye kletki rogovitsy i kon"yunktivy cheloveka in vitro.

Effective and safe antiseptic eye preparations are necessary for prevention and treatment of infectious and inflammatory eye diseases. PURPOSE: in vitro evaluation of the effect of antiseptic eye drops on corneal and conjunctival epithelial cells. MATERIAL AND METHODS: Antiseptic eye drops «Bactavit¼, «Vitabact¼ and «Ocomistin¼ were the object of the study. Immortalized human corneal epithelial cell lines (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) were used as the test systems. The viability of the cells was assessed by their metabolic activity and morphology using the MTT test and phase-contrast microscopy. RESULTS: Antiseptic eye drops belonging to different groups of chemical compounds induced cytotoxic effects on the cells of corneal epithelium (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) of varying degrees, leading to morphological and functional changes in those cells. CONCLUSION: The study confirms the possibility of using cultured cells for the in vitro comparative assessment of the cytotoxic effect of antiseptic ophthalmic agents.

USP10 Promotes Fibronectin Recycling, Secretion, and Organization.

Purpose: Integrins play a central role in myofibroblast pathological adhesion, over-contraction, and TGFß activation. Previously, we demonstrated that after corneal wounding, αv integrins are protected from intracellular degradation by upregulation of the deubiquitinase USP10, leading to cell-surface integrin accumulation. Because integrins bind to and internalize extracellular matrix (ECM), we tested whether extracellular fibronectin (FN) accumulation can result from an increase in integrin and matrix recycling in primary human corneal fibroblasts (HCFs). Methods: Primary HCFs were isolated from cadaver eyes. HCFs were transfected with either USP10 cDNA or control cDNA by nucleofection. Internalized FN was quantified with a FN ELISA. Recycled extracellular integrin and FN were detected with streptavidin-488 by live cell confocal microscopy (Zeiss LSM 780). Endogenous FN extra domain A was detected by immunocytochemistry. Cell size and removal of FN from the cell surface was determined by flow cytometry. Results: USP10 overexpression increased α5ß1 (1.9-fold; P < 0.001) and αv (1.7-fold; P < 0.05) integrin recycling, with a concomitant increase in biotinylated FN internalization (2.1-fold; P < 0.05) and recycling over 4 days (1.7-2.2-fold; P < 0.05). The dependence of FN recycling on integrins was demonstrated by α5ß1 and αv integrin blocking antibodies, which, compared with control IgG, decreased biotinylated FN recycling (62% and 84%, respectively; P < 0.05). Overall, we established that extracellular FN was composed of approximately 1/3 recycled biotinylated FN and 2/3 endogenously secreted FN. Conclusions: Our data suggest that reduced integrin degradation with a subsequent increase in integrin/FN recycling after wounding may be a newly identified mechanism for the characteristic accumulation of ECM in corneal scar tissue.

Olive Pomace Phenolic Compounds Stability and Safety Evaluation: From Raw Material to Future Ophthalmic Applications.

Nowadays, increasing interest in olive pomace (OP) valorization aims to improve olive's industry sustainability. Interestingly, several studies propose a high-value application for OP extracts containing its main phenolic compounds, hydroxytyrosol and oleuropein, as therapy for ocular surface diseases. In this work, the stability and accessibility of OP total phenolic and flavonoid content, main representative compounds, and antioxidant activity were assessed under different pretreatment conditions. Among them, lyophilization and supercritical CO2 extraction were found to increase significantly most responses measured in the produced extracts. Two selected extracts (CONV and OPT3) were obtained by different techniques (conventional and pressurized liquid extraction); Their aqueous solutions were characterized by HPLC-DAD-MS/MS. Additionally, their safety and stability were evaluated according to EMA requirements towards their approval as ophthalmic products: their genotoxic effect on ocular surface cells and their 6-months storage stability at 4 different temperature/moisture conditions (CPMP/ICH/2736/99), together with pure hydroxytyrosol and oleuropein solutions. The concentration of hydroxytyrosol and oleuropein in pure or extract solutions was tracked, and possible degradation products were putatively identified by HPLC-DAD-MS/MS. Hydroxytyrosol and oleuropein had different stability as standard or extract solutions, with oleuropein also showing different degradation profile. All compounds/extracts were safe for ophthalmic use at the concentrations tested.

Thermo-Gelling Dendronized Chitosans as Biomimetic Scaffolds for Corneal Tissue Engineering.

Biomimetic scaffolds with transparent, biocompatible, and in situ-forming properties are highly desirable for corneal tissue engineering, which can deeply fill corneal stromal defects with irregular shapes and support tissue regeneration. We here engineer a novel class of corneal scaffolds from oligoethylene glycol (OEG)-based dendronized chitosans (DCs), whose aqueous solutions show intriguing sol-gel transitions triggered by physiological temperature, resulting in highly transparent hydrogels. Gelling points of these hydrogels can be easily tuned, and furthermore, their mechanical strengths can be significantly enhanced when injected into PBS at 37 °C instead of pure water. In vitro tests indicate that these DC hydrogels exhibit excellent biocompatibility and can promote proliferation and migration of keratocyte. When applied in the rabbit eyes with corneal stromal defects, in situ formed DC hydrogels play a positive effect for new tissue regeneration. Overall, this thermo-gelling DCs possess appealing features as corneal tissue substitutes with their excellent biocompatibility and unprecedented thermoresponsiveness.

Tear miRNA expression analysis reveals miR-203 as a potential regulator of corneal epithelial cells.

BACKGROUND: microRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. They are found within cells and in body fluids. Extracellular miRNAs have been shown to associate with the surrounding tissues. Therefore, we predicted that miRNAs in tears may contribute to regulate corneal epithelial cell function. However, information on the miRNA expression profile of tears is limited and the specific functions of tear miRNAs for corneal epithelial cells are still unknown. To study the role of tear miRNAs, we determined which miRNAs are highly expressed in tears and examined the involvement of miRNAs in corneal epithelial cell viability. METHODS: miRNAs extracted from monkey tears and sera were subjected to microarray analysis. miRNAs of which expression levels were higher in tears than in sera were selected, and their expression levels were quantified by quantitative polymerase chain reaction (qPCR). To examine miRNA function, mimics and inhibitors of miRNAs were transfected into human corneal epithelial (HCE-T) cells and incubated for 24 or 48 h. After transfection of miRNA mimics and inhibitors, the viability of HCE-T cells was measured using the water soluble tetrazolium salt (WST) assay, and microarray analysis and qPCR were performed using total RNA extracted from HCE-T cells. siRNAs of the candidate targets for miR-203 were transfected into HCE-T cells and the WST assay was performed. To determine a direct target gene for miR-203, a dual luciferase reporter assay was performed in HCE-T cells using a luciferase reporter plasmid containing 3'-UTR of human IGFBP5. RESULTS: Microarray and qPCR analyses showed that miR-184 and miR-203 were expressed significantly more highly in tears than in sera (165,542.8- and 567.8-fold, respectively, p < 0.05). Of these two miRNAs, transfection of a miR-203 mimic significantly reduced the viability of HCE-T cells (p < 0.05), while a miR-203 inhibitor significantly increased this viability (p < 0.05). miR-203 mimic downregulated insulin-like growth factor-binding protein 5 (IGFBP5) and nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1), while miR-203 inhibitor upregulated these two genes. Transfection of IGFBP5-siRNA decreased the viability of HCE-T cells. miR-203 mimic significantly diminished the luciferase reporter activity. CONCLUSIONS: In this study, we identified miRNAs that are highly expressed in tears, and the inhibition of miR-203 increases the viability of corneal epithelial cells. Our results suggest that miR-203 contributes to regulating the homeostasis of corneal epithelial cells.

Dynamic changes in and time sequence of ultraviolet B-induced apoptosis in rat corneal epithelial cells.

PURPOSE: To systematically examine the dynamic changes and time sequence in corneal epithelial cell apoptosis after excessive ultraviolet B irradiation. METHODS: Ultraviolet B (144 mJ/cm2) was used to irradiate rat corneal epithelial cells for 2 h. Cell morphology was observed on differential interference contrast microscopy, and the numbers of the different kinds of apoptotic cells were counted using the ImageJ software. Cell viability was measured with the 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide method. Cell apoptotic rate and loss of mitochondrial membrane potential were detected using flow cytometric analyses. The expression levels of 3 apoptotic genes were measured with real-time quantitative polymerase chain reaction at different time points within 0-24 h after irradiation. RESULTS: After 144-mJ/cm2 ultraviolet B irradiation for 2 h, the expression levels of caspase-8 and Bax were highest at 0 h; furthermore, the mitochondrial membrane potential decreased at 0 h and remained constant for 6 h in a subsequent culture. At 6 h, caspase-3 was activated. The decrease in cell viability and increase in apoptotic rate peaked at 6 h. The caspase-3 expression level decreased within 12-24 h, which led to a decline in apoptotic rate and change in apoptotic stage. CONCLUSIONS: The corneal epithelial cells exhibited rapid apoptosis after ultraviolet B irradiation, which was associated with both extrinsic and intrinsic pathways.

Cigarette smoke extract and heated tobacco products promote ferritin cleavage and iron accumulation in human corneal epithelial cells.

The cornea is directly exposed to cigarette smoke, and smoking is a risk factor for several corneal diseases including dry eye syndrome. Currently, heated tobacco products (HTPs) are widely used as substitutes for cigarette smoking around the world. In the present study, we investigated the molecular mechanism(s) leading to cellular injury induced by cigarette smoke extract (CSE) or HTPs. Exposure to CSE perturbed the formation of tight junctions, leading to an increase in cell volume, a decrease in transepithelial electrical resistance (TER) in the human corneal epithelial cell-transformed (HCE-T) cell line. Moreover, CSE exposure induced both lipid peroxidation and ferrous [Fe(II)] ion accumulation in autolysosomal compartments. Interestingly, a cleaved form of ferritin appeared when HCE-T cells were incubated with CSE. This aberrant ferritin processing was suppressed by treatment with autophagy inhibitors. Furthermore, the CSE-induced cell death was suppressed by either ferrostatin-1 or deferoxamine (DFO). CSE exposure also promoted the expression of cytokines whereas DFO treatment inhibited the CSE-induced expression of these cytokines. Exposure to HTPs also induced both HCE-T cell death and cleaved ferritin accumulation in a concentration- and time-dependent manner. These results indicated that CSE or HTPs activated the ferroptosis signaling pathway, which contributed to corneal epithelial cell injury.

Protease, Growth Factor, and Heparanase-Mediated Syndecan-1 Shedding Leads to Enhanced HSV-1 Egress.

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are considered important for the entry of many different viruses. Previously, we demonstrated that heparanase (HPSE), the host enzyme responsible for cleaving HS chains, is upregulated by herpes simplex virus-1 (HSV-1) infection. Higher levels of HPSE accelerate HS removal from the cell surface, facilitating viral release from infected cells. Here, we study the effects of overexpressing HPSE on viral entry, cell-to-cell fusion, plaque formation, and viral egress. We provide new information that higher levels of HPSE reduce syncytial plaque formation while promoting egress and extracellular release of the virions. We also found that transiently enhanced expression of HPSE did not affect HSV-1 entry into host cells or HSV-1-induced cell-to-cell fusion, suggesting that HPSE activation is tightly regulated and facilitates extracellular release of the maturing virions. We demonstrate that an HSPG-shedding agonist, PMA; a protease, thrombin; and a growth factor, EGF as well as bacterially produced recombinant heparinases resulted in enhanced HSV-1 release from HeLa and human corneal epithelial (HCE) cells. Our findings here underscore the significance of syndecan-1 functions in the HSV-1 lifecycle, provide evidence that the shedding of syndecan-1 ectodomain is another way HPSE works to facilitate HSV-1 release, and add new evidence on the significance of various HSPG shedding agonists in HSV-1 release from infected cells.

Arginine Supplementation Promotes Extracellular Matrix and Metabolic Changes in Keratoconus.

Keratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.