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1.
Exp Eye Res ; 242: 109883, 2024 May.
Article in English | MEDLINE | ID: mdl-38561106

ABSTRACT

Corneal transplantation represents the primary therapeutic approach for managing corneal endothelial dysfunction, but corneal donors remain scarce. Anterior chamber cell injection emerges as a highly promising alternative strategy for corneal transplantation, with pluripotent stem cells (PSC) demonstrating considerable potential as an optimal cell source. Nevertheless, only a few studies have explored the differentiation of functional corneal endothelial-like cells originating from PSC. In this investigation, a chemical-defined protocol was successfully developed for the differentiation of functional corneal endothelial-like cells derived from human embryonic stem cells (hESC). The application of nicotinamide (NAM) exhibited a remarkable capability in suppressing the fibrotic phenotype, leading to the generation of more homogeneous and well-distinctive differentiated cells. Furthermore, NAM effectively suppressed the expression of genes implicated in endothelial cell migration and extracellular matrix synthesis. Notably, NAM also facilitated the upregulation of surface marker genes specific to functional corneal endothelial cells (CEC), including CD26 (-) CD44 (-∼+-) CD105 (-) CD133 (-) CD166 (+) CD200 (-). Moreover, in vitro functional assays were performed, revealing intact barrier properties and Na+/K+-ATP pump functionality in the differentiated cells treated with NAM. Consequently, our findings provide robust evidence supporting the capacity of NAM to enhance the differentiation of functional CEC originating from hESC, offering potential seed cells for therapeutic interventions of corneal endothelial dysfunction.


Subject(s)
Cell Differentiation , Endothelium, Corneal , Human Embryonic Stem Cells , Niacinamide , Humans , Cell Differentiation/drug effects , Niacinamide/pharmacology , Endothelium, Corneal/metabolism , Endothelium, Corneal/cytology , Endothelium, Corneal/drug effects , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Cells, Cultured , Vitamin B Complex/pharmacology , Flow Cytometry , Cell Movement/drug effects , Antigens, CD/metabolism , Antigens, CD/genetics
2.
Eye Contact Lens ; 50(6): 276-278, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38661367

ABSTRACT

PURPOSE: To report a case of corneal endothelial damage caused by alcohol-containing chlorhexidine gluconate (CG-A) and its progression over time. METHODS: This was a case report. RESULTS: A 22-year-old man underwent neurosurgery under general anesthesia. CG-A (1%) was used for disinfection after the application of corneal protection tape. Postoperatively, the patient presented with hyperemia and swelling of the left conjunctiva and was referred to our department. Initial examination revealed left corneal epithelial erosion and corneal edema, which improved on postoperative day 14. The corneal endothelial cell density (ECD) was 3,345 cells/mm 2 on day 14, decreased rapidly to 2,090 cells/mm 2 on day 42, and slowly reduced to 1,122 cells/mm 2 on day 168. Thereafter, no decrease in ECD was observed. CONCLUSIONS: CG formulations can lead to a persistent decrease in ECD over several months, even after improvement of acute corneal edema.


Subject(s)
Chlorhexidine , Endothelium, Corneal , Humans , Male , Chlorhexidine/analogs & derivatives , Chlorhexidine/adverse effects , Young Adult , Endothelium, Corneal/pathology , Endothelium, Corneal/drug effects , Corneal Edema/chemically induced , Corneal Edema/etiology , Corneal Edema/diagnosis , Anti-Infective Agents, Local/adverse effects , Disinfection/methods , Ethanol/adverse effects , Corneal Endothelial Cell Loss/pathology , Corneal Endothelial Cell Loss/diagnosis
3.
Medicine (Baltimore) ; 103(17): e37937, 2024 Apr 26.
Article in English | MEDLINE | ID: mdl-38669379

ABSTRACT

To observe alterations in corneal morphology caused by repeated intravitreal injections of anti-vascular endothelial growth factor (VEGF). Prospective cohort study. Seventy-seven eyes were treated with intravitreal injection of anti-VEGF from June 2021 to March 2023. There were 25 eyes of neovascular age-related macular degeneration (nAMD), 24 eyes of diabetic macular edema (DME), and 28 eyes of retinal vein occlusion (RVO). Aflibercept was used in 37 eyes and Ranibizumab was used in 40 eyes. 3 + PRN was used. Corneal endothelium and corneal thickness were measured using a corneal endothelial microscope. The data related to central corneal thickness, corneal endothelial cell density (ECD), average cell size, coefficient of variation (CV), proportion of hexagonal cells (Hex%) was collected. A comparison was also made between baseline and the dynamic changes of all indexes 1 year following the last injection. It was observed that in comparison to baseline, ECD and Hex% decreased significantly after the 3rd injection of Aflibercept and Ranibizumab. However, ECD did not decrease further and remained at the same level as after the last injection. Hex% and average cell size increased to a certain extent in comparison to the last injection. All the changes were found to be statistically significant (P < .01). After 3 injections, ECD in DME group was markedly lower than that in nAMD and RVO group, but the CV in DME group was higher than that in nAMD as well as RVO groups, and all the differences were statistically significant (P < .05). Following intravitreal anti-VEGF therapy, DME is more likely than other disorders to result in a decrease in ECD. Repeated intravitreal injections of anti-VEGF drugs can reduce the Hex% and ECD to a certain extent. After the last injection, Hex% can progressively recover, and ECD can remain stable without further declining. After injections, ECD in DME group was found to be significantly lower than that in nAMD and RVO groups, but CV in DME group was significantly higher in comparison to the other 2 groups. In patients with macular edema, repeated intravitreal injections of anti-VEGF may have certain effects on corneal morphology. Patients with diabetes mellitus in particular should pay special attention to corneal safety following repeated intravitreal injections if they have significantly reduced ECD at baseline.


Subject(s)
Angiogenesis Inhibitors , Cornea , Intravitreal Injections , Macular Edema , Ranibizumab , Receptors, Vascular Endothelial Growth Factor , Recombinant Fusion Proteins , Vascular Endothelial Growth Factor A , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/adverse effects , Cornea/pathology , Cornea/drug effects , Endothelium, Corneal/drug effects , Endothelium, Corneal/pathology , Macular Degeneration/drug therapy , Macular Edema/drug therapy , Prospective Studies , Ranibizumab/administration & dosage , Receptors, Vascular Endothelial Growth Factor/administration & dosage , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/therapeutic use , Retinal Vein Occlusion/drug therapy , Vascular Endothelial Growth Factor A/antagonists & inhibitors
4.
Biomed Pharmacother ; 174: 116435, 2024 May.
Article in English | MEDLINE | ID: mdl-38513591

ABSTRACT

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.


Subject(s)
Cell Proliferation , Endothelium, Corneal , Protein Kinase Inhibitors , Regeneration , rho-Associated Kinases , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/metabolism , Cell Proliferation/drug effects , Protein Kinase Inhibitors/pharmacology , Endothelium, Corneal/drug effects , Regeneration/drug effects , Animals , Drug Repositioning , Cell Movement/drug effects , Cell Line , Humans , Endothelial Cells/drug effects
5.
Cutan Ocul Toxicol ; 43(2): 120-123, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38235962

ABSTRACT

PURPOSE: To determine the structure and properties of corneal endothelial cells in children and adolescents with ADHD who received methylphenidate treatment at least six months. METHOD: The prospective, observational study included 33 eyes of 33 patients diagnosed with ADHD who received methylphenidate treatment for at least six months, 33 eyes of 33 patients newly diagnosed with ADHD who did not start medication treatment, and 33 eyes of 33 healthy individuals. Average cell density, coefficient of variation, maximum cell area, normal cell area, minimum cell area, average cell area, and hexagonality ratio values were evaluated by non-contact specular microscopy. The parameters recorded in all three groups were compared. RESULTS: The average age of children in the ADHD + MPH, ADHD, and control groups is 9 ± 1.7, 8.9 ± 2.3, and 8.9 ± 1.8 years, respectively. (p > 0.05) The average MPH treatment dose is 0.94 ± 0.19 mg/kg, the average daily MPH intake is 34.12 ± 14.04 mg, and the average duration of use of MPH is 24.03 ± 12.46 months. Central corneal thickness (CCT) was measured as an average of 540.45 ± 31.23 in the ADHD + MPH group, 540.61 ± 29.69 in the ADHD group, and 546.58 ± 27.72 in the control group. (p = 0.499) The average coefficient of variation (CV) values were measured as 25.48 ± 4.22 in the ADHD + MPH group, 26.12 ± 3.48 in the ADHD group, and 26.12 ± 3.64 in the control group. (p = 0.491) The average hexagonality ratio (%) (HEX) values were measured as 69.45 ± 8.41 in the ADHD + MPH group, 68.21 ± 6.82 in the ADHD group, and 68.91 ± 7.97 in the control group. (p = 0.892) No statistically significant difference was observed between all three groups in terms of all parameters. CONCLUSION: Methylphenidate treatment administered for at least six months with a diagnosis of ADHD did not have a toxic effect on the corneal endothelium.


Subject(s)
Attention Deficit Disorder with Hyperactivity , Endothelium, Corneal , Methylphenidate , Humans , Attention Deficit Disorder with Hyperactivity/drug therapy , Child , Methylphenidate/therapeutic use , Methylphenidate/administration & dosage , Male , Female , Adolescent , Endothelium, Corneal/pathology , Endothelium, Corneal/drug effects , Central Nervous System Stimulants/therapeutic use , Central Nervous System Stimulants/administration & dosage , Microscopy , Prospective Studies , Cell Count , Endothelial Cells/drug effects , Endothelial Cells/pathology
6.
Int J Mol Sci ; 22(22)2021 Nov 22.
Article in English | MEDLINE | ID: mdl-34830446

ABSTRACT

Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to -40 °C and 3 °C/min to -120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.


Subject(s)
Cornea/growth & development , Corneal Transplantation/methods , Cryopreservation/standards , Endothelium, Corneal/ultrastructure , Cold Temperature , Cornea/pathology , Cornea/ultrastructure , Corneal Transplantation/adverse effects , Dimethyl Sulfoxide/pharmacology , Endothelium, Corneal/cytology , Endothelium, Corneal/drug effects , Humans , Microscopy, Electron, Scanning , Spain , Tissue Banks
8.
Biomed Pharmacother ; 144: 112306, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34656060

ABSTRACT

BACKGROUND: The pumping function of corneal endothelial cells (CECs) plays a pivotal role in the maintenance of corneal water homeostasis. Corneal endothelial dysfunction (CED) leads to corneal edema and opacity, but with the exception of keratoplasty, no optimal therapeutic strategies have been established for CED. In this study, we aimed to investigate the ameliorative effect of ascorbic acid (AA) on CED and the underlying mechanism of action in the corneal endothelium. METHODS: Rabbit corneal endothelial damage was induced by anterior chamber injection of benzalkonium chloride (BAK). AA was topically administered to the corneal surface, and the transparency and thickness of the cornea were assessed by external eye photography, slit-lamp photography, and ultrasonic pachymetry. To further analyze the mechanism, rabbit CECs and immortalized human CECs (B4G12 cells) were cultured. A ferric reducing/antioxidant and AA (FRASC) assay was performed to measure the AA concentration. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (BrdU) labeling assays, and protein expression was examined by liquid chromatography-mass spectrometry (LC/MS) and immunoblotting. The involvement of glucose transporter 1 (GLUT1) and phospho-ERK was evaluated via GLUT1-siRNA and phospho-ERK inhibitor (PD98059) treatment. INTERPRETATION: We observed that topical AA ameliorates BAK-induced rabbit corneal endothelial damage. Furthermore, we demonstrated that AA is transported into B4G12 cells via GLUT1, and afterward, AA increases ERK phosphorylation and promotes cell proliferation. Our findings indicate that CEC proliferation stimulated via the noncanonical AA-GLUT1-ERK axis contributes to AA-enhanced healing of CED.


Subject(s)
Ascorbic Acid/pharmacology , Cell Proliferation/drug effects , Corneal Endothelial Cell Loss/prevention & control , Endothelial Cells/drug effects , Endothelium, Corneal/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Glucose Transporter Type 1/metabolism , Wound Healing/drug effects , Administration, Ophthalmic , Animals , Ascorbic Acid/administration & dosage , Ascorbic Acid/metabolism , Benzalkonium Compounds , Cell Line , Corneal Endothelial Cell Loss/chemically induced , Corneal Endothelial Cell Loss/metabolism , Corneal Endothelial Cell Loss/pathology , Disease Models, Animal , Endothelial Cells/enzymology , Endothelial Cells/pathology , Endothelium, Corneal/enzymology , Endothelium, Corneal/pathology , Glucose Transporter Type 1/genetics , Humans , Phosphorylation , Rabbits , Signal Transduction
9.
Invest Ophthalmol Vis Sci ; 62(12): 4, 2021 09 02.
Article in English | MEDLINE | ID: mdl-34499705

ABSTRACT

Purpose: SLC4A11, an electrogenic H+ transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown. Methods: The presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses. Thiol crosslinked peptide binding to SLC4A11 was screened by untargeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. Direct protein interactions between SLC4A11 and chaperones were examined using coimmunoprecipitation analysis and proximity ligation assay. Knockdown or pharmacologic inhibition of chaperones in human corneal endothelial cells (HCECs) or mouse corneal endothelial cells (MCECs), ex vivo kidney, or HA-SLC4A11-transfected fibroblasts was performed to investigate the functional consequences of interfering with mitochondrial SLC4A11 trafficking. Results: SLC4A11 does not contain canonical N-terminal mitochondrial targeting sequences. LC-MS/MS analysis showed that HSC70 and/or HSP90 are bound to HA-SLC4A11-transfected PS120 fibroblast whole-cell lysates or isolated mitochondria, suggesting trafficking through the chaperone-mediated carrier pathway. SLC4A11 and either HSP90 or HSC70 complexes are directly bound to the mitochondrial surface receptor, TOM70. Interference with this trafficking leads to dysfunctional mitochondrial glutamine catabolism and increased reactive oxygen species production. In addition, glutamine (Gln) use upregulated SLC4A11, HSP70, and HSP90 expression in whole-cell lysates or purified mitochondria of HCECs and HA-SLC4A11-transfected fibroblasts. Conclusions: HSP90 and HSC70 are critical in mediating mitochondrial SLC4A11 translocation in corneal endothelial cells and kidney. Gln promotes SLC4A11 import to the mitochondria, and the continuous oxidative stress derived from Gln catabolism induced HSP70 and HSP90, protecting cells against oxidative stress.


Subject(s)
Ammonia/pharmacology , Anion Transport Proteins/genetics , Corneal Dystrophies, Hereditary/genetics , Endothelium, Corneal/metabolism , Mitochondria/pathology , Proteomics/methods , Symporters/genetics , Animals , Anion Transport Proteins/metabolism , Cells, Cultured , Corneal Dystrophies, Hereditary/metabolism , Corneal Dystrophies, Hereditary/pathology , Endothelium, Corneal/drug effects , Endothelium, Corneal/pathology , Mice , Mice, Knockout , Mitochondria/metabolism , Molecular Chaperones/drug effects , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein Transport , Symporters/metabolism
10.
Sci Rep ; 11(1): 18514, 2021 09 16.
Article in English | MEDLINE | ID: mdl-34531501

ABSTRACT

Amantadine hydrochloride (HCl) is commonly prescribed for treating influenza A virus infection and Parkinson's disease. Recently, several studies have indicated that the use of amantadine HCl is associated with corneal edema; however, the cytotoxic effect of amantadine HCl has not been investigated. In the present study, the effects of amantadine HCl on cell growth, proliferation, and apoptosis in bovine cornea endothelial cells, and in vitro endothelial permeability were examined. Results showed that lower doses of amantadine HCl do not affect cell growth (≤ 20 µΜ), whereas higher doses of amantadine HCl inhibits cell growth (≥ 50 µΜ), induces apoptosis (2000 µΜ), increases sub-G1 phase growth arrest (2000 µΜ), causes DNA damage (≥ 1000 µΜ), and induces endothelial hyperpermeability (≥ 1000 µΜ) in bovine cornea endothelial cells; additionally, we also found that amantadine HCl attenuates the proliferation (≥ 200 µΜ) and arrests cell cycle at G1 phase (≥ 200 µΜ) in bovine cornea endothelial cells. In the present study, we measured the cytotoxic doses of amantadine HCl on cornea endothelial cells, which might be applied in evaluating the association of corneal edema.


Subject(s)
Amantadine/toxicity , Antiviral Agents/toxicity , Cornea/drug effects , Endothelial Cells/drug effects , Endothelium, Corneal/drug effects , Animals , Apoptosis/drug effects , Cattle , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cells, Cultured
11.
Invest Ophthalmol Vis Sci ; 62(12): 15, 2021 09 02.
Article in English | MEDLINE | ID: mdl-34533563

ABSTRACT

Purpose: The Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice. Methods: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay. Autophagy and lysosome functions were examined in wild type (WT) and KO cells as well as animals treated with the mitochondrial ROS quencher MitoQ. Results: Even though autophagy activation was evident, autophagy flux was aberrant in Slc4a11 KO cells and corneal endothelium. Expression of lysosomal proteins and lysosomal mass were decreased along with reduced nuclear translocation of lysosomal master regulator, transcription factor EB (TFEB). MitoQ reversed aberrant lysosomal functions and TFEB nuclear localization in KO cells. MitoQ injections in KO animals reduced corneal edema and decreased the rate of endothelial cell loss. Conclusions: Mitochondrial ROS disrupts TFEB signaling causing lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction in an animal model of CHED, and to identify a potential therapeutic approach.


Subject(s)
Autophagy/physiology , Corneal Dystrophies, Hereditary/metabolism , Disease Models, Animal , Lysosomes/metabolism , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Animals , Anion Transport Proteins/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Blotting, Western , Cathepsin L/metabolism , Cells, Cultured , Corneal Dystrophies, Hereditary/genetics , Corneal Dystrophies, Hereditary/pathology , Endothelium, Corneal/drug effects , Endothelium, Corneal/metabolism , Gene Expression Regulation , Immunohistochemistry , Injections, Intraperitoneal , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Organophosphorus Compounds/pharmacology , Real-Time Polymerase Chain Reaction , Symporters/genetics , Transfection , Ubiquinone/analogs & derivatives , Ubiquinone/pharmacology
12.
Cutan Ocul Toxicol ; 40(4): 332-337, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34342246

ABSTRACT

PURPOSE: In the present clinical study, it was aimed to investigate the possible effects of Trypan blue (TB) use on the corneal endothelium during cataract surgery in eyes with pseudoexfoliation syndrome (PEX) during a three-month follow-up period using the contralateral eye control design. METHODS: This prospective, randomised controlled, individual cohort study included 92 eyes of 46 patients with bilateral PEX and cataracts. While 1% TB was applied to one eye of the patients before capsulorhexis (study group), it was not applied to the other eye (control group). Both groups were compared preoperatively and postoperatively in terms of endothelial cell density (ECD), endothelial cell loss (%), pleomorphism, polymegathism and central corneal thickness (CCT) using specular microscopy. RESULTS: Preoperative corneal ECD was measured as 2362.56 ± 253.27 in the study group, 2380.84 ± 220.54 in the control group, and 2145.58 ± 221.71 in the study group and 2184.97 ± 200.94 cells/mm2 in the control group in the postoperative 3rd-month follow-up (p = 0.71 and = 0.37, respectively). In addition, there were no significant differences between the two groups in terms of the percentage of hexagonal cells, coefficient of variation (CV), and CCT both preoperatively and postoperatively 3 months later (p = 0.78, =0.39, =0.95 preoperatively and p = 0.31, =0.26, =0.83 postoperatively, respectively). CONCLUSION: This study demonstrated that the injection of 1% TB into the anterior chamber for staining the anterior capsule during cataract surgery did not cause significant corneal endothelial changes at postoperative 3rd months, despite the increased fragility of corneal endothelial cells in patients with PEX.


Subject(s)
Cataract Extraction/adverse effects , Cataract/pathology , Endothelium, Corneal/drug effects , Exfoliation Syndrome/surgery , Trypan Blue/adverse effects , Adult , Cataract/etiology , Cataract Extraction/methods , Endothelium, Corneal/pathology , Exfoliation Syndrome/complications , Exfoliation Syndrome/pathology , Female , Follow-Up Studies , Humans , Injections, Intraocular , Male , Middle Aged , Prospective Studies , Trypan Blue/administration & dosage
13.
Cells ; 10(6)2021 06 11.
Article in English | MEDLINE | ID: mdl-34207965

ABSTRACT

This study aims to obtain sufficient corneal endothelial cells for regenerative application. We examined the combinatory effects of Rho-associated kinase (ROCK) inhibitor Y-27632 and mesenchymal stem cell-derived conditioned medium (MSC-CM) on the proliferation and senescence of rabbit corneal endothelial cells (rCECs). rCECs were cultured in a control medium, a control medium mixed with either Y-27632 or MSC-CM, and a combinatory medium containing Y-27632 and MSC-CM. Cells were analyzed for morphology, cell size, nuclei/cytoplasmic ratio, proliferation capacity and gene expression. rCECs cultured in a combinatory culture medium showed a higher passage number, cell proliferation, and low senescence. rCECs on collagen type I film showed high expression of tight junction. The cell proliferation marker Ki-67 was positively stained either in Y-27632 or MSC-CM-containing media. Genes related to cell proliferation resulted in negligible changes in MKI67, CIP2A, and PCNA in the combinatory medium, suggesting proliferative capacity was maintained. In contrast, all of these genes were significantly downregulated in the other groups. Senescence marker ß-galactosidase-positive cells significantly decreased in either MSC-CM and/or Y-27632 mixed media. Senescence-related genes downregulated LMNB1 and MAP2K6, and upregulated MMP2. Cell cycle checkpoint genes such as CDC25C, CDCA2, and CIP2A did not vary in the combinatory medium but were significantly downregulated in either ROCK inhibitor or MSC-CM alone. These results imply the synergistic effect of combinatory culture medium on corneal endothelial cell proliferation and high cell number. This study supports high potential for translation to the development of human corneal endothelial tissue regeneration.


Subject(s)
Cell Proliferation , Cellular Senescence , Culture Media, Conditioned/pharmacology , Endothelium, Corneal/cytology , Mesenchymal Stem Cells/cytology , Protein Kinase Inhibitors/pharmacology , rho-Associated Kinases/antagonists & inhibitors , Amides/pharmacology , Animals , Cell Differentiation , Cell Movement , Cells, Cultured , Endothelium, Corneal/drug effects , Endothelium, Corneal/enzymology , Enzyme Inhibitors/pharmacology , Humans , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/enzymology , Pyridines/pharmacology , Rabbits
14.
Invest Ophthalmol Vis Sci ; 62(9): 2, 2021 07 01.
Article in English | MEDLINE | ID: mdl-34196654

ABSTRACT

Purpose: Previous work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H2) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism. Methods: MNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H2. The effect of H2 was examined using morphological and functional assays. Results: It was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na+/K+-ATPase, whereas H2 improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H2 could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway. Conclusions: This study suggests that topical application of H2 could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.


Subject(s)
Apoptosis/drug effects , Corneal Injuries/chemically induced , Endothelium, Corneal/metabolism , Hydrogen/pharmacology , Oxidative Stress , Up-Regulation , Animals , Cell Count , Cells, Cultured , Corneal Injuries/metabolism , Corneal Injuries/pathology , Disease Models, Animal , Endothelium, Corneal/drug effects , Endothelium, Corneal/pathology , Male , Methylnitrosourea/toxicity , Rabbits , Rats , Transcriptional Activation
15.
Cutan Ocul Toxicol ; 40(3): 252-256, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34074199

ABSTRACT

PURPOSE: This study aimed to determine if the corneal endothelium was affected by chemotherapy. METHODS: Chemotherapy patients were recruited to undergo specular microscopy before treatment and again at 1- and 2-year follow-up visits. One eye per patient, per follow-up, was selected for comparison to baseline. RESULTS: Forty-six volunteers completed baseline and at least one follow-up assessment. From 51 eyes, there was no significant change in endothelial cell density for 41 eyes assessed at one year (MD = 0.73%, 95% CI -1.33 to 2.78%) and 18 eyes at two years (MD = 0.31%, 95% CI -3.53 to 4.15%). CONCLUSION: Although other studies have shown that chemotherapy can adversely affect the corneal epithelium, this study showed no measurable change in endothelial cell density.


Subject(s)
Antineoplastic Agents/adverse effects , Endothelium, Corneal/drug effects , Neoplasms/drug therapy , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Cell Count , Endothelial Cells/drug effects , Endothelium, Corneal/diagnostic imaging , Endothelium, Corneal/transplantation , Female , Follow-Up Studies , Humans , Male , Middle Aged , Photography , Prospective Studies , Slit Lamp Microscopy
16.
Exp Eye Res ; 207: 108574, 2021 06.
Article in English | MEDLINE | ID: mdl-33848524

ABSTRACT

PURPOSE: Chronic corneal endothelial cell (CEC) loss results in corneal edema and vision loss in conditions such as pseudophakic bullous keratopathy (PBK), Fuchs' dystrophy, and corneal graft failure. Low CEC density has been associated with an elevation of intraocular pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. These cytokines are capable of triggering pyroptosis, a programmed cell death mechanism mediated by the inflammasome, prompting the activation of the pro-inflammatory cytokine interleukin (IL)-1ß, the perpetuation of inflammation, and subsequent damage of corneal endothelial tissue. Therefore, the purpose of this study was to determine the deleterious contribution of the inflammasome and pyroptosis to CEC loss. METHODS: CECs from human donor corneas were treated ex vivo with TNF-α and IFN-γ for 48 h. Levels of caspase-1 and IL-1ß were then assayed by ELISA, and the expression of caspase-1 and gasdermin-D (GSDM-D) were confirmed by immunofluorescence. Endothelial cell damage was analyzed by a lactate dehydrogenase (LDH) release assay, and oxidative stress was determined by measuring the levels of reactive oxygen species (ROS) in the culture media. RESULTS: Inflammasome activation and oxidative stress were elevated in CECs following exposure to TNF-α and IFN-γ, which resulted in cell death by pyroptosis as determined by LDH release which was inhibited by the caspase-1 inhibitor Ac-YVAD-cmk. CONCLUSION: CEC death is induced by the pro-inflammatory cytokines TNF-α and IFN-γ, which contribute to inflammasome activation. Moreover, the inflammasome is a promising therapeutic target for the treatment of chronic CEC loss.


Subject(s)
Endothelium, Corneal/drug effects , Endothelium, Corneal/pathology , Inflammasomes/metabolism , Interferon-gamma/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Adult , Aged , Caspase 1/metabolism , Cell Death , Endothelium, Corneal/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Humans , Interleukin-1beta/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Male , Microscopy, Fluorescence , Middle Aged , Oxidative Stress , Phosphate-Binding Proteins/metabolism , Reactive Oxygen Species/metabolism , Tissue Donors , Young Adult
17.
Biochemistry (Mosc) ; 86(3): 382-388, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33838637

ABSTRACT

Diseases of the cornea are a frequent cause of blindness worldwide. Keratoplasty is an efficient method for treating severely damaged cornea. The functional competence of corneal endothelial cells is crucial for successful grafting, which requires improving the media for the hypothermic cornea preservation, as well as developing the methods for the evaluation of the corneal functional properties. The transport of water and ions by the corneal endothelium is important for the viability and optic properties of the cornea. We studied the impact of SkQ1 on the equilibrium sodium concentration in the endothelial cells after hypothermic preservation of pig cornea at 4°C for 1, 5, and 10 days in standard Eusol-C solution. The intracellular sodium concentration in the endothelial cells was assayed using the fluorescent dye Sodium Green; the images were analyzed with the custom-designed CytoDynamics computer program. The concentrations of sodium in the pig corneal endothelium significantly increased after 10 days of hypothermic preservation, while addition of 1.0 nM SkQ1 to the preservation medium decreased the equilibrium concentration of intracellular sodium (at 37°C). After 10 days of hypothermic preservation, the permeability of the plasma membrane for sodium decreased in the control cells, but not in the cells preserved in the presence of 1 nM SkQ1. Therefore, SkQ1 increased the ability of endothelial cells to restore the intracellular sodium concentration, which makes SkQ1 a promising agent for facilitating retention of the functional competence of endothelial cells during cold preservation.


Subject(s)
Endothelium, Corneal/metabolism , Hypothermia, Induced , Plastoquinone/analogs & derivatives , Sodium/analysis , Tissue Preservation/methods , Animals , Cold Temperature , Cornea/chemistry , Cornea/drug effects , Cornea/metabolism , Endothelium, Corneal/chemistry , Endothelium, Corneal/drug effects , Plastoquinone/pharmacology , Sodium/metabolism , Sus scrofa/metabolism , Sus scrofa/physiology
18.
BMJ Case Rep ; 14(2)2021 Feb 19.
Article in English | MEDLINE | ID: mdl-33608341

ABSTRACT

A 61-year-old male patient presented with decreased vision and recurrent redness in his right eye since the past 4 years. He had been diagnosed elsewhere as HLA-B27 positive anterior uveitis and was on oral methotrexate and topical corticosteroids for recurrent disease. He was on maximal medical therapy for glaucoma. Examination showed prominent inferior corneal oedema with pigmented keratic precipitates and elevated intraocular pressure. He underwent combined trabeculectomy with mitomycin C and cataract surgery. The aqueous sample tested positive for cytomegalovirus. He responded well to oral valganciclovir with resolution of uveitis, the intraocular pressure was well controlled and the corneal oedema resolved completely.


Subject(s)
Cytomegalovirus Infections/complications , Cytomegalovirus Infections/diagnosis , Diagnostic Errors , Endothelium, Corneal/virology , Glaucoma/complications , Uveitis/complications , Antiviral Agents/therapeutic use , Cytomegalovirus/isolation & purification , Cytomegalovirus Infections/drug therapy , Endothelium, Corneal/drug effects , Endothelium, Corneal/surgery , HLA-B27 Antigen , Humans , Male , Middle Aged , Uveitis/diagnosis , Uveitis/surgery , Valganciclovir/therapeutic use
19.
Exp Eye Res ; 205: 108517, 2021 04.
Article in English | MEDLINE | ID: mdl-33617851

ABSTRACT

Corneal endothelial dysfunction usually induces corneal haze and oedema, which seriously affect visual function. The main therapeutic strategy for this condition is corneal transplantation, but the use of this strategy is limited by the shortage of healthy donor corneas. Compared with corneal transplantation, drug intervention is less invasive and more accessible; thus, finding an effective pharmaceutical alternative for cornea transplantation is critical for the treatment of corneal endothelial dysfunction. In this study, we established a rabbit scratch model to investigate the effect of fibroblast growth factor 10 (FGF10) on corneal endothelial wound healing. Results showed that FGF10 injection accelerated the recovery of corneal transparency and increased the protein expression levels of ZO1, Na+/K+-ATPase and AQP-1. Moreover, FGF10 significantly inhibited the expression levels of endothelial-to-mesenchymal transition proteins and reduced the expression levels of the proinflammatory factors IL-1ß and TNF-α in the anterior chamber aqueous humour. FGF10 also enhanced the Na+/K+-ATPase activity by enhancing mitochondrial function as a result of its direct interaction with its conjugate receptor. Thus, FGF10 could be a new pharmaceutical preparation as treatment for corneal endothelial dysfunction.


Subject(s)
Corneal Injuries/drug therapy , Endothelium, Corneal/drug effects , Fibroblast Growth Factor 10/pharmacology , Wound Healing/drug effects , Animals , Aquaporin 1/metabolism , Aqueous Humor/metabolism , Blotting, Western , Cell Line , Cells, Cultured , Corneal Injuries/metabolism , Cytokines/metabolism , Endothelium, Corneal/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Male , Microscopy, Confocal , Rabbits , Sodium-Potassium-Exchanging ATPase/metabolism , Zonula Occludens-1 Protein/metabolism
20.
Cutan Ocul Toxicol ; 40(2): 66-69, 2021 Jun.
Article in English | MEDLINE | ID: mdl-33599552

ABSTRACT

PURPOSE: To evaluate the characteristics of corneal parameters in patients with diabetic macular oedema (DME) treated with intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections. METHODS: Participants in this study were 36 patients with DME, treated with either intravitreal ranibizumab (n = 16) or aflibercept (n = 20). All participants underwent best-corrected visual acuity (BCVA) measurement, optical coherence tomography and non-contact specular microscopy to evaluate corneal endothelium parameters (endothelial cell density-ECD, hexagonality, coefficient of variation of the cell size and central corneal thickness-CCT), at baseline and at months 6 and 12 after the first intravitreal injection. Comparisons between baseline and months 6 and 12 were performed. RESULTS: There was no statistically significant difference regarding ECD, hexagonality, coefficient of variation of the cell size and CCT at month 6 and 12 post initial injection compared to baseline in patients with DME. BCVA improved significantly at month 6 and 12 compared to baseline (p < 0.001 for both comparisons). Central retinal thickness was significantly reduced at month 6 and 12 compared to baseline (p < 0.001 for both comparisons). CONCLUSION: Intravitreal anti-VEGF injections in patients with DME were found not to affect corneal parameters, namely ECD, hexagonality, coefficient of variation of the cell size and CCT at the long-term follow-up of 12 months.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Diabetes Complications/drug therapy , Endothelium, Corneal/drug effects , Macular Edema/drug therapy , Ranibizumab/therapeutic use , Receptors, Vascular Endothelial Growth Factor/therapeutic use , Recombinant Fusion Proteins/therapeutic use , Aged , Female , Humans , Intravitreal Injections , Male , Middle Aged , Tomography, Optical Coherence , Vascular Endothelial Growth Factors/antagonists & inhibitors , Visual Acuity
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