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Corneal endothelial cells (CECs) are essential for maintaining corneal transparency and hydration through their barrier and pump functions. The COL8A2 gene encodes a component of the extracellular matrix of the cornea, which is crucial for the normal functioning of these cells. Mutations in COL8A2 are linked to corneal dystrophies, emphasizing the gene's importance in corneal health. The purpose of this research is to explore the effects of COL8A2 activation within CECs, to understand its contribution to cellular behavior and health. COL8A2 CRISPR/dCas9 activation system (aCOL8A2) was used to activate the COL8A2. In rats, wound healing and mitochondrial function were assessed after COL8A2 activation. As a result, aCOL8A2 promoted wound healing of rat corneal endothelium by increasing mitochondrial membrane potential. In cultured human CECs, proteomic analysis was performed to screen and identify the differential protein profiles between control and aCOL8A2 cells. Western blot was used to validate the differential proteins from both cells. Mitochondrial function and intracellular distribution were assessed by measuring ATP production and mitochondrial membrane potential. In cultured human CECs, aCOL8A2 increased COL8A2 and phospho-YAP levels. Transendothelial electrical resistance (TEER) was increased and actin cytoskeleton was attenuated by aCOL8A2. Gene ontology analysis revealed that the proteins were mainly involved in the regulation of folate biosynthesis, ECM-receptor interaction, cell differentiation, NADP activity and cytoskeleton. ATP production was increased, mitochondrial membrane potential was polarized and mitochondrial distribution was widespread in the aCOL8A2 group. In conclusion, aCOL8A2 induces a regulatory cascade affecting mitochondrial positioning and efficiency, mediated by alterations in the cytoskeletal architecture and the YAP signaling pathway. This sequence of events serves to bolster the functional capacities of corneal endothelial cells, including their pump and barrier functions, essential for corneal health and transparency., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK-3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of α-SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na + /K + ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed that CHIR99021 induced downregulation of EnMT markers (α-SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the β-catenin and TGFβ pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT, providing a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof. Graziella Pellegrini is in the board of directors of Holostem Terapie Avanzate S.r.l. Holostem Terapie Avanzate S.r.l. owns a patent (n. 102022000020061) filed the 29th of September 2022., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

We developed a mathematical model to describe healing processes in bovine corneal endothelial (BCE) cells in culture, triggered by mechanical wounds with parallel edges. Previous findings from our laboratory show that, in these cases, BCE monolayers exhibit an approximately constant healing velocity. Also, that caspase-dependent apoptosis occurs, with the fraction of apoptotic cells increasing with the distance traveled by the healing edge. In addition, in this study we report the novel findings that, for wound scratch assays performed preserving the basal extracellular matrix: i) the healing cells increase their en face surface area in a characteristic fashion, and ii) the average length of the segments of the cell columns actively participating in the healing process increases linearly with time. These latter observations preclude the utilization of standard traveling wave formalisms to model wound healing in BCE cells. Instead, we developed and studied a simple phenomenological model based on a plausible formula for the spreading dynamics of the individual healing cells, that incorporates original evidence about the process in BCE cells. The model can be simulated to: i) obtain an approximately constant healing velocity; ii) reproduce the profile of the healing cell areas, and iii) obtain approximately linear time dependences of the mean cell area and average length of the front active segments per column. In view of its accuracy to account for the experimental observations, the model can also be acceptably employed to quantify the appearance of apoptotic cells during BCE wound healing. The strategy utilized here could offer a novel formal framework to represent modifications undergone by some epithelial cell lines during wound healing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Hyperlipidemia impacts on various diseases, such as atherosclerosis, hypertension, and diabetes mellitus. However, its influence, if any, on ocular tissues is largely unknown. Herein, we developed hyperlipidemic murine models by feeding 4-week-old male wild-type mice with a high-fat diet and apolipoprotein E knockout (ApoE -/- ) mice with a high-fat diet or standard diet to investigate the corneal endothelial change under hyperlipidemic conditions. Oil Red O staining showed an accumulation of lipid droplets in corneal endothelial cells (CECs) of hyperlipidemic mice. Other manifestations included a reduced cell density and distorted cell morphology, a disruption of the endothelial cell tight junctions and adhesion junctions, a reduced number of surface microvilli, down-regulation of Na + -K + -ATPase expression and function, activation of oxidative stress, changes in mitochondrial ultrastructure, and increased apoptosis. CEC recovery after injury, moreover, was diminished in hyperlipidemic mice; and high palmitate levels were found in the aqueous humor. In vitro hyperlipemia model, moreover, was found to be associated with dose-dependent CEC cytotoxicity, altered cell morphology, reduced pump function, and an induction of oxidative stress, leading to functional and pathologic changes in the corneal endothelium., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Corneal endothelium (CE) is a monolayer of mitochondria-rich cells, critical for maintaining corneal transparency compatible with clear vision. Fuchs endothelial corneal dystrophy (FECD) is a heterogeneous, genetically complex disorder, where oxidative stress plays a key role in the rosette formation during the degenerative loss of CE. Increased mitochondrial fragmentation along with excessive mitophagy activation has been detected in FECD; however, the mechanism of aberrant mitochondrial dynamics in CE cell loss is poorly understood. Here, the role of oxidative stress in mitophagy activation in FECD is investigated. Immunoblotting of FECD ex vivo specimens revealed an accumulation of PINK1 and phospho-Parkin (Ser65) along with loss of total Parkin and total Drp1. Similarly, modeling of rosette formation with menadione (MN), led to phospho-Parkin accumulation in fragmented mitochondria resulting in mitophagy-induced mitochondrial clearance, albeit possibly in a PINK1-independent manner. Loss of PINK1, phospho-Drp1, and total Drp1 was prominent after MN-induced oxidative stress, but not after mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone. Moreover, MN-induced mitophagy led to degradation of Parkin along with sequestration of Drp1 and PINK1 that was rescued by mitophagy inhibition. This study shows that in FECD, intracellular oxidative stress induces Parkin-mediated mitochondrial fragmentation where endogenous Drp1 and PINK1 are sequestered and degraded by mitophagy during degenerative loss of post-mitotic cells of ocular tissue., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Fuchs endothelial corneal dystrophy (FECD) is a corneal pathology that affects the endothelial cell's ability to maintain deturgescence, resulting in a progressive loss of corneal transparency. In this study, we investigated the expression of function-related proteins in corneal endothelial cells using FECD or healthy corneal endothelial cells, either in a cell culture two-dimensional model or in an engineered corneal endothelium three-dimensional tissue model. No statistically significant difference in gene regulation was observed for the function-related families ATP1, SLC4, SLC16, AQP, TJP, and CDH between the FECD and the healthy cell models. Similarly, no difference in barrier integrity (transendothelial electrical resistance measurements and permeability assays) was observed in vitro between FECD and healthy cultured cells. Protein expression of the key function-related families was decreased for Na + /K + -ATPase α1 subunit, monocarboxylate transporters 1 and 4 in native ex vivo end-stage FECD specimens, whereas it returned to levels comparable to that of healthy tissues in the engineered FECD model. These results indicate that cell expansion and tissue engineering culture conditions can generate a corneal endothelium from pathologic FECD cells, with levels of function-related proteins similar to that of healthy tissues. Overall, these results explain why it is possible to reform a functional endothelium using corneal endothelial cells isolated from nonfunctional FECD pathologic specimens., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma, aging, and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein, we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging, propensity to form spheres, and increased colony forming efficacy compared with the primary cells. The collective expression of stem cell-related genes SOX2, OCT4, LGR5, TP63 (p63), as well as neural crest marker genes PSIP1 (p75(NTR)), PAX3, SOX9, AP2B1 (AP-2β), and NES, generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2, β-III tubulin, and glial fibrillary acidic protein positive neurons and into p75(NTR)-positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion, we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The C-terminal Eps15 homology domain-containing (EHD) proteins play a key role in endocytic recycling, a fundamental cellular process that ensures the return of endocytosed membrane components and receptors back to the cell surface. To define the in vivo biological functions of EHD1, we have generated Ehd1 knockout mice and previously reported a requirement of EHD1 for spermatogenesis. Here, we show that approximately 56% of the Ehd1-null mice displayed gross ocular abnormalities, including anophthalmia, aphakia, microphthalmia and congenital cataracts. Histological characterization of ocular abnormalities showed pleiotropic defects that include a smaller or absent lens, persistence of lens stalk and hyaloid vasculature, and deformed optic cups. To test whether these profound ocular defects resulted from the loss of EHD1 in the lens or in non-lenticular tissues, we deleted the Ehd1 gene selectively in the presumptive lens ectoderm using Le-Cre. Conditional Ehd1 deletion in the lens resulted in developmental defects that included thin epithelial layers, small lenses and absence of corneal endothelium. Ehd1 deletion in the lens also resulted in reduced lens epithelial proliferation, survival and expression of junctional proteins E-cadherin and ZO-1. Finally, Le-Cre-mediated deletion of Ehd1 in the lens led to defects in corneal endothelial differentiation. Taken together, these data reveal a unique role for EHD1 in early lens development and suggest a previously unknown link between the endocytic recycling pathway and regulation of key developmental processes including proliferation, differentiation and morphogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ex vivo culture or regeneration of corneal endothelial cells often is subjected to gradual endothelial-mesenchymal transition and loss of function. Here, we found that during ex vivo culture, bovine corneal endothelial cells underwent endothelial-mesenchymal transition and had an up-regulated expression and activity of matrix metalloproteinases. Inhibition of matrix metalloproteinase activity in confluent bovine corneal endothelial cells decreased the level of endothelial-mesenchymal transition regulators: snail and slug. The phosphorylation and degradation of the key Wnt signaling pathway modulator active β-catenin also were accelerated with the broad-spectrum matrix metalloproteinase inhibitor Marimastat, which may result from decreased N-cadherin shedding and increased intact N-cadherin molecules on the cell membrane. Intracameral injection of Marimastat also suppressed basic fibroblast growth factor-induced endothelial-mesenchymal transition in a rat corneal endothelium cryo-injury model and significantly diminished the corneal edema. Our study indicated that inhibition of matrix metalloproteinase activity can reverse endothelial-mesenchymal transition and preserve the function of corneal endothelial cells both during ex vivo culture and in vivo. This may offer a potential therapeutic target in regenerative medicine for the treatment of corneal endothelial dysfunctions., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: Acromegaly has various impacts on many organs. The ophthalmologic effects of acromegaly have not yet been investigated in detail. The aim of the current study was to evaluate qualitative and quantitative changes in corneal endothelial cells and central corneal thickness (CCT) of the patients with acromegaly., Design: In this prospective, cross-sectional study, 128 eyes of 64 patients with acromegaly (female/male=40/24) and 208 eyes of 104 age and gender-matched healthy volunteers (female/male=69/35) were included. Endothelial cell density (ECD), cellular area (CA), coefficient of variation (CV) in cell size, percentage of hexagonal cells, and CCT were measured in patients with acromegaly and in healthy volunteers using the noncontact specular microscopy (SP-3000P: Topcon Corporation, Tokyo, Japan)., Results: ECD and CA were lower in cases with acromegaly than in controls (ECD in acromegaly: 2615.65 cell/mm(2) and in controls: 2700.35 cell/mm(2); p=0.002. CA in acromegaly: 382.30μm(2) and in controls: 400.30μm(2); p=0.02). In the entire group with acromegaly, the time elapsed since diagnosis was positively correlated with CA and was negatively correlated with ECD (r=+0.39, p=0.001 and r=-0.42, p=0.001)., Conclusions: The endothelial layer of the cornea may be under risk of impairment with prolonged disease duration in acromegaly. Consistency of the corneal endothelium should be also sought during long-term follow-up of the cases with acromegaly., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The purpose of this work was to investigate the relationship between the feed composition of 2-hydroxyethyl methacrylate (HEMA)/acrylic acid (AAc) and hydrogel material compatibility towards ocular anterior segment tissues, particularly the corneal endothelium. The monomer solutions of HEMA and AAc were mixed at varying volume ratios of 92:0, 87:5, 82:10, 77:15, and 72:20, and were subjected to UV irradiation. Then, the 7-mm-diameter membrane implants made from photopolymerized materials were placed into the ocular anterior chamber for 4days and assessed by biomicroscopic examinations, corneal thickness measurements, and quantitative real-time reverse transcription polymerase chain reaction analyses. The poly(HEMA-co-AAc) implants prepared from the solution mixture containing 0-10vol.% AAc displayed good biocompatibility. However, with increasing volume ratio of AAc and HEMA from 15:77 to 20:72, the enhanced inflammatory response, decreased endothelial cell density, and increased ocular score and corneal thickness were observed, probably due to the influence of surface charge of copolymer membranes. On the other hand, the ionic pump function of corneal endothelium exposed to photopolymerized membranes was examined by analyzing the Na(+),K(+)-ATPase alpha 1 subunit (ATP1A1) expression level. The presence of the implants having higher amount of AAc incorporated in the copolymers (i.e., 15.1 to 24.7μmol) and zeta potential (i.e., -38.6 to -56.5mV) may lead to abnormal transmembrane transport. It is concluded that the chemical composition of HEMA/AAc has an important influence on the corneal tissue responses to polymeric biomaterials., (© 2013.)

Purpose: To assess the reproducibility of manual graft preparation and evaluate the incidence rate and nature of structural anomalies of Descemet's membrane (DM) preventing successful graft preparation in DM endothelial keratoplasty (DMEK)., Design: Prospective, single-center, nonrandomized, consecutive case series., Participants: We analyzed 350 corneoscleral buttons from donors aged 18-95 years stored in Optisol-GS or Dulbecco's modified Eagle's medium and used for DMEK surgery in 343 consecutive patients with Fuchs' endothelial dystrophy or pseudophakic bullous keratopathy., Methods: Residual endothelial cell-DM complexes obtained after successful DM stripping for DMEK and whole donor corneas obtained after unsuccessful DM stripping were examined by transmission electron microscopy and immunohistochemistry., Main Outcome Measures: Accuracy of the cleavage plane between DM and corneal stroma and structural abnormalities of the DM-stroma interface., Results: Uneventful manual separation without any disruption of DM was achieved in 335 of 350 donor corneas (95.7%) by use of a previously established bimanual submerged preparation technique. Correspondingly, the peeled DM specimens revealed a regular and smooth cleavage plane exposing the amorphous interfacial matrix on their anterior surface. Although 8 of 350 donor corneas (2.3%) showed focal adhesions of DM to the corneal stroma and developed isolated tears during stripping, preparation of the graft could be successfully completed. However, 7 of the 350 donor corneas (2.0%) showed extremely strong adhesion and multiple tears of DM, preventing successful preparation of the graft. These specimens revealed either ultrastructural (peg-like interlockings) or biochemical abnormalities (increased staining intensities for adhesive glycoproteins) along the DM-stroma interface., Conclusions: Using an appropriate technique, manual preparation of grafts for DMEK with reproducible tissue qualities is possible in the vast majority (98%) of donor corneas. Although a relatively rare phenomenon, interindividual variations in DM structure and composition may be responsible for failure of graft preparation in about 2% of donor corneas., Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article., (Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Endothelial-to-mesenchymal transition (EnMT) is a cell transformation process involved in both morphogenesis and pathogenesis. EnMT of corneal endothelial cells happens after endothelial injury and during ex vivo culture. Previous studies have shown that the transforming growth factor-β signaling pathway is involved in this transition. In this study, we found that rat corneal endothelial cells could spontaneously undergo EnMT during ex vivo culture. This change in rat corneal endothelial cells was associated with Notch signaling pathway activation after the first passage, which was blocked by the Notch inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). This inhibitor also prevented transforming growth factor β1-, β2-, and β3-induced EnMT and reversed transformed rat corneal endothelial cells to a normal phenotype. Furthermore, DAPT treatment blocked retrocorneal membrane formation in a rat corneal endothelium damage model. Our study indicates that the Notch signaling pathway is involved in the corneal EnMT process, which may be a novel therapeutic target for treating corneal endothelial fibrogenic disorders., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: To describe the clinicopathologic features of congenital ectropion uvea associated with glaucoma in neurofibromatosis-1 (NF-1)., Design: Retrospective case series., Participants and Controls: Five cases of NF-1 associated with glaucoma, from which enucleated eyes were available, and 2 eye bank eyes used as controls., Methods: The clinical features and courses of these patients were reviewed. Formalin-fixed, paraffin-embedded eyes were examined by light and electron microscopy. Immunohistochemistry using antineurofibromin, anti-glial fibrillary acidic protein, and antivimentin was performed in 3 patients. Gene expression of the mitogen-activated protein kinase (MAPK) signaling pathway was examined in corneal endothelial cells in 1 patient., Main Outcome Measures: Cause of glaucoma in patients with ectropion uvea and NF-1., Results: The age of patients at the time of glaucoma diagnosis ranged from birth to 13 years. Four of the 5 patients had megalocornea and buphthalmos at presentation. Ectropion uvea was noted clinically in 2 patients, but was demonstrated histopathologically in all 5 patients. On histopathologic examination, all patients had varying degrees of angle closure secondary to endothelialization of the anterior chamber angle. Uveal neurofibromas were noted in all patients; anteriorly displaced ciliary processes were noted in 4 of 5 patients who demonstrated ciliary body involvement with neurofibromas. Absence of Schlemm's canal was observed. The endothelial cells lining the closed angle demonstrated positive stain results with the vimentin antibody. Positive antineurofibromin immunolabeling was detected in normal control corneal endothelium, but was absent in corneal endothelium in patients with endothelialization of the angle. Upregulation of genes from the MAPK signaling pathway was demonstrated in the corneal endothelial cells isolated from the NF-1 eyes., Conclusions: Ectropion uvea in NF-1 glaucoma is secondary to endothelialization of the anterior chamber angle and is associated commonly with severe pediatric glaucoma in NF-1 patients. The endothelial cell proliferation may be related to overexpression of the Ras (Rat sarcoma)-MAPK genes in these eyes., (Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To define the cleavage plane between Descemet's membrane (DM) and posterior corneal stroma in Descemet's membrane endothelial keratoplasty (DMEK) concerning its ultrastructural and immunohistochemical characteristics., Design: Observational, consecutive case series., Participants: Fifteen corneoscleral buttons from donors 71.5±4.3 years of age stored in Optisol-GS and used for DMEK surgery in 15 consecutive patients., Methods: Endothelial cell-DM complexes (EDMs) and corresponding corneoscleral rims were investigated by transmission electron microscopy and immunohistochemistry using a panel of antibodies against adhesive matrix proteins., Main Outcome Measures: Ultrastructural and immunohistochemical characteristics of interface and cleavage plane between DM and posterior stroma., Results: Connection between DM and corneal stroma was mediated predominantly by amorphous material of the interfacial matrix and projecting stromal collagen fibers. After DM stripping, the cleavage plane was located consistently between interfacial matrix and posterior stromal collagen lamellae, providing a largely smooth anterior EDM surface exposing the interfacial zone. Interindividual variations in amount and composition of the interfacial matrix resulted in variable degrees of EDM surface irregularities and variable staining patterns for adhesive matrix proteins such as fibronectin, vitronectin, amyloid P, osteonectin/secreted protein acidic and rich in cysteine (SPARC), fibulin-1, fibulin-2, fibulin-3, fibrillin-1, and keratoepithelin., Conclusions: The findings provide evidence for the existence of a physiologic cleavage plane between the interfacial matrix, the anteriormost adhesive zone of DM, and the corneal stroma, suggesting a relatively weak attachment that can be disconnected by mechanical forces. Interindividual variations in structure and composition of the interfacial matrix may provide an explanation for the variable attachment of EDM grafts to the recipients' corneal stroma and thus may affect the postoperative clinical outcome., Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article., (Copyright © 2011 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

One of the most important steps during Descemet stripping automated endothelial keratoplasty (DSAEK) is attaching the donor Descemet lenticule to the host's cornea. This is usually carried out by injecting air into the anterior chamber with a syringe and needle. We present an alternative technique using an air fluid exchange system connected to an anterior chamber maintainer used in 22 cases in our institute. This technique has proven to be simple and fast, thus facilitating the successful performance of DSAEK surgery., (Copyright © 2011 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4°C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4-8h). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: To report the clinical and histologic findings of a new subset of idiopathic corneal edema: zipper cell endotheliopathy., Design: Observational case report., Participant: A 55-year-old woman with unilateral bullous keratopathy., Methods: Clinical observation consisted of slit-lamp examination and in vivo confocal microscopy (IVCM). Aqueous humor samples and the excised corneal button were analyzed for the presence of herpes viruses. The excised cornea was subjected to detailed immunohistochemistry (IHC) and scanning and transmission electron microscopy., Main Outcome Measures: Clinical and pathologic characteristics of zipper cell endotheliopathy., Results: In vivo confocal microscopy revealed unique morphologic alterations of the corneal endothelial layer. Focal areas of denudation were surrounded by endothelial cells with zipper-like cell borders and intercellular structures. Besides central corneal edema, no other signs of corneal inflammation were detected. A herpes virus origin for the bullous keratopathy was excluded. The IHC analysis disclosed positive staining for cytokeratin (CK) 7, CK8/18, and CK19, suggesting epithelial metaplasia of the endothelial cells. Ultrastructural examination confirmed the IVCM findings by showing large areas of endothelial denudation and vacuolated endothelial cells with large, broad-based extensions that partially overlapped neighboring cells. Despite extensive complementary research and review of the literature, the endothelial alterations could not be attributed to any known corneal disorder., Conclusions: To the authors' knowledge, zipper cell endotheliopathy is a new subset of idiopathic corneal edema. The case report presented illustrates the potential use of IVCM to differentiate the spectrum of corneal disorders and to discover new corneal diseases., (Copyright © 2010 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

The lens in the vertebrate eye has been shown to be critical for proper differentiation of the surrounding ocular tissues including the cornea, iris and ciliary body. In mice, previous investigators have assayed the consequences of molecular ablation of the lens. However, in these studies, lens ablation was initiated (and completed) after the cornea, retina, iris and ciliary body had initiated their differentiation programs thereby precluding analysis of the early role of the lens in fate determination of these tissues. In the present study, we have ablated the lens precursor cells of the surface ectoderm by generation of transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens ectodermal precursors. In these mice, lens precursor cells fail to express Sox2, Prox1 and alphaA-crystallin and die before the formation of a lens placode. The Tox176 mice also showed profound alterations in the corneal differentiation program. The corneal epithelium displayed histological features of the skin, and expressed markers of skin differentiation such as Keratin 1 and 10 instead of Keratin 12, a marker of corneal epithelial differentiation. In the Tox176 mice, in the absence of the lens, extensive folding of the retina was seen. However, differentiation of the major cell types in the retina including the ganglion, amacrine, bipolar and horizontal cells was not affected. Unexpectedly, ectopic placement of the retinal pigmented epithelium was seen between the folds of the retina. Initial specification of the presumptive ciliary body and iris at the anterior margins of the retina was not altered in the Tox176 mice but their subsequent differentiation was blocked. Lacrimal and Harderian glands, which are derived from the Pax6-expressing surface ectodermal precursors, also failed to differentiate. These results suggest that, in mice, specification of the retina, ciliary body and iris occurs at the very outset of eye development and independent of the lens. In addition, our results also suggest that the lens cells of the surface ectoderm may be critical for the proper differentiation of the corneal epithelium.

Purpose: To describe the histopathologic features of Descemet's membrane (DM) obtained from Fuchs' endothelial corneal dystrophy (FECD) corneas undergoing Descemet's stripping with endothelial keratoplasty (DSEK) and to assess the presence of advanced glycation end products (AGEs) and their receptors in FECD endothelium and DM., Design: Prospective observational case series., Participants: Five eyes of 5 patients undergoing DSEK for FECD and 4 normal control eyebank corneas., Methods: Descemet's membrane and corneal endothelium from FECD patients undergoing DSEK were assessed with hematoxylin-eosin staining and immunohistochemistry for AGEs, receptor of AGEs (RAGE), and galectin 3 (AGE-R3)., Main Outcome Measures: Histopathologic abnormalities and presence of AGEs, RAGE, and AGE-R3 in DSEK specimens., Results: Histopathologic assessment of DSEK specimens from FECD patients disclosed thickening and nodularity of DM and loss of endothelial cells. Immunohistochemical staining of FECD DM for AGE, RAGE, and AGE-R3 showed an abundance of AGEs in the anterior portion of DM, mild positivity for RAGE, and moderate positivity for AGE-R3., Conclusions: Tissue quality after DSEK is sufficient to allow detailed histopathologic analysis. The presence of AGEs, RAGE, and AGE-R3 in DM and corneal endothelium of FECD patients supports a link between accumulation of AGEs, oxidative stress, and corneal endothelial cell apoptosis in the pathogenesis of FECD.

The loss of corneal endothelial cells plays a critical role in many corneal diseases and is a common phenomenon following cornea transplantation. In addition, the non-regenerative capacity of human corneal endothelial cells (HCEC) ultimately requires appropriate protection of corneal tissues during ex vivo storage to ensure vitality of the cells. However, only 70% of donor corneas can be used for grafting because of endothelial deficiencies. Corneal endothelial cell loss during storage is mainly induced by apoptotic cell death. This study was undertaken, for proof of principle, to investigate whether over-expression of cyto-protective molecules Bcl-x(L), Bag-1, and HO-1 prevents the loss of corneal endothelial cells both in vitro and in vivo. We demonstrate that gene transfer of both Bcl-x(L) and HO-1 has cyto-protective effects on HCEC in vitro. However, gene transfer of a single cyto-protective molecule does not prevent its rejection upon transplantation in a MHC class I/II disparate rat model.

A human corneal equivalent is under development with potential applications in pharmaceutical testing, biomedical research, and transplantation, but the ability to distribute this engineered tissue, depends on successful cryopreservation. Tissue recovery after exposure to conditions during cryopreservation depends on the response of its constituent cells to the changing environment as ice forms and solutes concentrate. This study defines the osmotic properties that define the rate of water movement across the plasma membrane of isolated human corneal endothelial, stroma, and epithelial cells. Cells were transferred from an isotonic (300 mosm/kg) to an anisotonic (150-1500 mosm/kg) solution at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after anisosmotic exposure allowed calculation of hydraulic conductivity (L(p)) and osmotically inactive volume fraction (V(b)). Experimental values for L(p) at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (E(a)). The L(p) for endothelial, stroma, and epithelial cells at 37 degrees C was 1.98+/-0.32,1.50+/-0.30, and 1.19+/-0.14 microm/min/atm, and the V(b) was 0.28, 0.27, and 0.41, respectively. The E(a) for endothelial, stroma, and epithelial cells was 14.8, 12.0, and 14.1 kcal/mol, respectively, suggesting the absence of aqueous pores. These osmotic parameters and temperature dependencies allow simulation of osmotic responses of human corneal cells to cryopreservation conditions, allowing amount of supercooling to be calculated to indicate the likelihood of intracellular freezing. Simulations show that differences in the osmotic parameters for the constituent cells in the bioengineered cornea result in significant implications for cryopreservation of the engineered corneal equivalent.

Objective: To evaluate tear production, corneal topography, accuracy of refractive correction, and best spectacle-corrected visual acuity in eyes that had moderate to severe rose bengal staining develop on the flap compared with eyes with little or no staining on the flap, the first few months after laser in situ keratomileusis (LASIK). None of the eyes in this study had significant preoperative dry eye disease., Design: Retrospective case control study., Participants: Individual eyes of 19 consecutive patients with moderate to severe punctate epithelial erosions and rose bengal staining on the flap 1 to 3 months after LASIK were compared with eyes of 19 concurrent patients who did not have punctate epithelial erosions or more than trace staining on the flap develop., Methods: Nonparametric statistical analyses were used to compare tear secretion, corneal topographic irregularity, spherical equivalent, and visual acuity 3 and 6 months after surgery. Some eyes in both groups also had analysis of tear secretion 1 month after surgery., Main Outcome Measures: Schirmer's test without anesthesia, the topographic corneal irregularity measurement (CIM), the difference between attempted and achieved spherical equivalent, and the loss of best spectacle-corrected visual acuity., Results: There was no difference in tear production 1, 3, or 6 months after LASIK in patients who had punctate epithelial erosions and rose bengal staining on the flap develop and those who did not. There was no significant difference in the CIM or mean accuracy of the refractive correction in the two groups, but some patients had a transient decrease in best spectacle-corrected visual acuity. Flap rose bengal staining resolved by 6 months after LASIK in most affected patients., Conclusions: LASIK-induced rose bengal staining in patients without preexisting dry eye is likely neurotrophic epitheliopathy, because there is no difference in mean tear production between patients who have significant punctate epithelial erosions and rose bengal staining develop on the flap and those who do not. The signs and symptoms of LASIK-induced (presumed) neurotrophic epitheliopathy tend to resolve approximately 6 months after surgery. This disorder tends to be more common and severe in patients with pre-existing dry eye disease.

Evidence suggests the involvement of growth hormone (GH), insulin-like growth factor I (IGF-I) and somatostatin in the pathology associated with diabetic retinopathy. We examined the effect of IGF-I on human retinal endothelial cell (HREC) survival following high glucose exposure and serum starvation, examined the signalling pathways mediating the protective effect of IGF-I on HREC, and characterized somatostatin receptor-induced retinal endothelial cell death. IGF-I (10 ng/ml) protected HREC from apoptosis induced by high glucose and serum starvation. Wortmannin, a specific inhibitor of phosphotidylinositol-3-kinase, blocks the ability of IGF-I to protect HREC from apoptosis. Incubation of HREC in serum-free medium caused a time-dependent increase in c-Jun N-terminal kinase (JNK) activity, and continuous culture of HREC in the presence of IGF-I or vascular endothelial growth factor (VEGF) prevented JNK activation and arrested apoptosis. Activation of tyrosine kinase receptors results in extracellular signal-related kinase (ERK) activation and activation of ERK is required for proliferation. Both IGF-I and VEGF produced a time- and concentration-dependent increase in the activation of ERK. Type 2 and type 3 somatostatin receptors have been implicated in cell-cycle arrest and apoptosis. Activation of the type 3 receptor in HREC resulted in cell death. These studies suggest that IGF-I is critical for HREC survival, and that somatostatin analogues acting through the type 3 receptor have direct effects on retinal endothelial cells. Furthermore, it appears that the therapeutic efficacy of somatostatin analogues lies not only in systemic inhibition of GH, but also in modulating local growth factor effects.

The cornea is a simple, nonvascularized structure, advantageous for studying the molecular components of epithelial and stromal wound repair. Fibronectin (Fn), of uncertain source and composition, accumulates in healing corneas. We postulated that local synthesis of Fn occurs, as exogenous plasma/tear-derived Fn, which lack the embryonic EIIIA and EIIIB segments, have no consistent beneficial effect on healing. Two contrasting corneal wounds were examined by in situ hybridization: a wound of the anterior stroma, basement membrane, and epithelium (anterior excimer laser keratectomy) and a superficial wound restricted to the epithelium that preserved the basement membrane (mechanical scrape). Both wounds heal without scarring. In normal corneas, only the endothelium had detectable Fn mRNA, containing the V and EIIIB domains, sporadically and at low levels. After anterior keratectomies, extensive expression of Fn mRNA occurred in a specific distribution that changed during the phases of healing. Before re-epithelialization (days 1 and 2) V+, EIIIA+, and EIIIB+ isoforms were diffusely found in stromal cells under and adjacent to the wound. After re-epithelialization (days 3 to 42) and reconstitution of laminin in the regenerating basement membrane zone, V+, EIIIA+, and EIIIB+ isoform synthesis was largely restricted to subepithelial stromal cells at the epithelial/stromal interface. In addition, the corneal epithelial cells focally expressed Fn mRNA. The endothelium showed increased levels of V+, EIIIA+, and EIIIB+ Fn mRNA in open and recently re-epithelialized wounds. At 12 weeks after keratectomy, Fn mRNA expression returned to control levels. In contrast, scrape wounds had only a modest increase of stromal and endothelial Fn mRNA (EIIIA+, EIIIB+, and V+) during the first 7 days and no evidence of epithelial Fn synthesis. Embryonic Fn isoforms are synthesized transiently by the cornea in response to even the most superficial wounds and are likely to be relevant to corneal healing and restoration of structure without scar formation.

Studies on the toxic effects of dehydro-L-ascorbic acid (DHAA) have been extended to include evaluations over time periods up to 3 hr. and to test for specific effects on a membrane transport protein, a membrane-bound enzyme and a soluble intracellular enzyme. In studies on cultured corneal endothelial cells, DHAA concentrations of 1, 2, and 5 mM over 3 hr. had an inhibitory effect on subsequent uptake of DHAA present at a tracer level. Surviving fragments of human placenta and alkaline phosphatase activity of the placental brush-border membrane were susceptible to the effect of DHAA at a high concentration (10 mM). Because intracellular metabolism of DHAA was not affected, and an increase in membrane permeability was not detected, it is concluded that a specific membrane transport protein might be the site of DHAA-induced damage. These studies support the concept that the oxidized form of ascorbic acid (vitamin C) has potential toxic effects on biological systems and suggests that proteins that mediate transport and metabolism may be sites where DHAA causes damage.

In a prospective, open, pilot study, the authors evaluated the effects of intraocular injection of streptokinase on intraocular fibrin. The study was composed of nine patients in whom fibrinous exudate developed after pars plana vitrectomy. There were seven male and two female patients with a mean age of 44 years (range, 27 to 65 years). Fibrinous exudates were seen 1 to 22 days after surgery with obstruction of the view of the posterior segment. Each patient received a single intraocular injection of 1000 IU of purified streptokinase. Four hours after the streptokinase injection, complete dissolution of the fibrin was noted in each patient. There were no clinically detectable adverse effects from the intraocular administration of streptokinase. Streptokinase may be considered in the dissolution of intractable intraocular fibrinous exudates after vitrectomy.

The clinical and specular microscopic results of 40 cases (39 patients) of penetrating keratoplasty during which a Kelman-style anterior chamber intraocular lens was implanted were reviewed retrospectively. Thirty-one pseudophakic eyes received an intraocular lens exchange and nine aphakic eyes received a secondary intraocular lens. Postoperative follow-up averaged 24.5 months (range, 3 to 51 months). At 1, 2, and 3 years after keratoplasty, 39.3%, 63.2%, and 63.6% of eyes, respectively, had visual acuities of 20/40 or better. Ninety-five percent of the grafts remained clear. Causes of poor postoperative visual acuity included cystoid macular edema (32.5%), new glaucoma (22.5%), and age-related macular degeneration (10.0%). Other causes were endothelial rejection leading to graft failure, corneal ulceration, and retinal detachment. Corneal endothelial cell loss by specular microscopy was 11.5% at 1 years, 21.3% at 2 years, and 25.0% at 3 years. These results were compared with cell loss associated with iris-sutured posterior chamber lenses in penetrating keratoplasty. Visual outcomes and complication rates were similar between these two methods; however, the endothelial attrition at 1 and 2 years for the sutured posterior chamber lens was greater than that of the Kelman anterior chamber lens.

The endothelium lining the posterior corneal surface performs physiologic pump functions essential to corneal clarity and integrity. A hallmark of keratitis, anterior ocular inflammation, and corneal allograft rejection is leukocyte adherence to the corneal endothelium (CE) forming keratitic precipitates. To elucidate mechanisms governing cornea-leukocyte interactions, cultured human CE cells and intact corneas were examined for expression of intercellular adhesion molecule-1 (ICAM-1), which binds the lymphocyte function-associated antigen-1 (LFA-1) on all leukocytes and enhances delayed-type hypersensitivity mediated by class II major histocompatibility complex antigens. Immunohistochemistry on culture CE cells using monoclonal anti-ICAM-1 antibody yield positive staining that increased after exposure to interleukin-1-beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (gamma-IFN). Standard leukocyte adherence assays demonstrated ICAM-1-mediated CE-neutrophil binding, which was specifically blocked by antibody to ICAM-1 or antibodies to LFA-1 on neutrophils. In whole human corneas, gamma-IFN increased CE and stromal keratocyte ICAM-1 immunoreactivity and enhanced CE-neutrophil adherence. As in CE cell cultures, antibody to ICAM-1 effectively blocked neutrophil binding to the CE cells of whole corneas. These results are the first to demonstrate ICAM-1 in ocular tissue. They indicate that CE cells express functional ICAM-1, which may be modulated by inflammatory cytokines, ICAM-1 provides mechanisms for keratitic precipitate formation, regulation of corneal leukocyte trafficking and the generation of immune responses that may be crucial to allograft rejection.

One of six silicone oils, differing in both viscosity and manufacture, was infused into the anterior chambers of rabbit eyes. Polydimethylsiloxane oil, 5000 cps, caused an increased corneal endothelial permeability to inulin and dextran at 24, 96, and 168 hours after placement into the eye. Intraocular pressures were slightly elevated in the experimental eyes, compared with contralateral controls, at 24 and 144 hours after infusion. The effects of five other oils on corneal endothelial permeability were examined 168 hours after infusion. All oils increased permeability and caused thinning of endothelial cells, together with the appearance of a retrocorneal membrane, except Dow Corning Medical Fluid 360. The results indicated that contact of most silicone oils with corneal endothelium rapidly induces physiologic and morphologic changes.

Thrombospondin is a cell adhesion molecule which interacts via specific domains with a wide array of extracellular matrix components, including fibrinogen, fibrin, fibronectin, collagen, and heparan sulfate proteoglycan. Although this protein has been localized in several human tissues, its presence in corneal tissues had not been previously established. In the present study, we have demonstrated that cultured bovine corneal endothelial cells synthesize thrombospondin and incorporate it into their extracellular matrix. We have also shown immunofluorescently the presence and distribution of thrombospondin in these cultured cells and in the noninjured and injured corneal endothelium in situ. Ultrastructural immunoperoxidase cytochemistry revealed that thrombospondin could be displaced from the cell surface by heparin, but not by keratan sulfate. Confluent cultures of corneal endothelium synthesize and secrete the three cell adhesion proteins laminin, thrombospondin, and fibronectin in the ratios 1:8.2:51.8. Only the laminin B chains were detected in immunoprecipitates. Immunofluorescent studies of these cultured cells, using a polyclonal antiserum raised against purified thrombospondin, revealed a low level of fluorescence associated with the cell layer but a punctate fluorescent pattern at the level of the extracellular matrix. Noninjured corneal endothelium in situ also demonstrated a low level of fluorescence throughout the cell layer. However, this dramatically changed after a circular freeze injury to the tissue. By 24 h after wounding, cells surrounding the injury zone displayed a prominent fluorescence that was still observed at 48 h post-injury. In addition to its increased intracellular fluorescence, thrombospondin was also localized as migration tracks, oriented in the direction of cellular migration into the wound site. Thus, in corneal endothelium, thrombospondin appears to play a major role in injury-induced cell migration in situ along a natural basement membrane.

Studies on the intact avascular cornea reveal two types of lactate effluxes: exogenous glucose-elicited and spontaneous. The former type exhibits characteristics resembling the proton-lactate symport system previously found in tumor cells and erythrocytes, including an enhanced lactate efflux at a higher extracellular pH and in the presence of H+ and K+ ionophores, and an inhibition by mersalyl with subsequent lactate accumulation in the tissue and cessation of glycolytic activity. The latter type occurs immediately following the incubation of freshly isolated cornea in a medium containing no exogenous glucose, with a rate about 10 times that of exogenous glucose-elicited lactate efflux. It is insensitive to 10 mM iodoacetate and lacks the characteristics of the proton-lactate symport system. Findings reveal that about 50% of corneal glucose utilization occurs in the epithelium, with the stroma and endothelium sharing the other 50% approximately equally. Of the glucose utilized, the lactate formation to pyruvate oxidation rate ratios are approximately 1:1 in the epithelium, 2:1 in the stroma, and 1:2 in the endothelium. About 79% of total tissue lactate is formed in the epithelium and stroma, and in vivo, this is probably pumped into the stromal extracellular space (about 90% of total tissue volume) via the proton-lactate symport system, with spontaneous release into the aqueous humor via a simple diffusion process. The H+ and K+ ionophores facilitate lactate efflux at the expense of the cellular pyruvate pool, without significant effect on the glucose uptake and glycolytic activity. These findings suggest that the ionophore-mediated lactate efflux favors the reduction of low pyruvate concentration in the tissue, rather than parallel increases in glycolytic activity.

The carbonic anhydrase inhibitor, 6-amino-2-benzothiazolesulfonamide, formulated as a 3% suspension in a gel vehicle was instilled in one eye of 21 human subjects in a single dose study to determine its effect on aqueous dynamics. A small but statistically significant effect on aqueous humor flow was observed 2 to 7 hours after application. By 8 hours, the effect had disappeared, and intraocular pressure (IOP) measured 8 hours after application of a single dose was unchanged in these normal volunteers. The drug and its vehicle caused local side effects including irritation, hyperemia, and blurred vision. The authors wondered if multiple doses would produce a greater effect. Four subjects received up to four doses of the drug over 2 days and were restudied. Marked bulbar injection and follicular conjunctivitis, attributable to either the drug or the vehicle, developed in two of the subjects, both contact lens wearers. A milder form of bulbar injection and follicular conjunctivitis developed in a third subject, who received three doses of the drug and was not a contact lens wearer. These side effects precluded additional multiple-dose testing of this formulation of the drug, and no conclusions about the effect of the drug on aqueous flow can be drawn from this portion of the study.

Peters' anomaly is a congenital abnormality that affects mainly the posterior cornea. The pathogenesis is unclear. By immunohistochemical methods, the authors determined the presence of extracellular matrix proteins, including fibronectin, laminin, and collagen types I, III, IV, V, and VI, in seven corneal buttons from five patients with Peters' anomaly. Type III collagen was the only protein absent in either the pathologic or normal human corneas. Compared with normal controls, the stromal lamellae in Peters' anomaly corneas were more irregular and more closely packed. In addition, the fibronectin staining was markedly enhanced in all seven Peters' anomaly corneas. Staining with collagen type VI was also mildly increased. These results suggest that extracellular matrix elements such as fibronectin may be important factors in this developmental disorder.