Jackson CJ, Myklebust Ernø IT, Ringstad H, Tønseth KA, Dartt DA, Utheim TP. Simple limbal epithelial transplantation: Current status and future perspectives. Stem Cells Transl Med 2020;9(3):316-327.Abstract
Damage to limbal stem cells as a result of injury or disease can lead to limbal stem cell deficiency (LSCD). This disease is characterized by decreased vision that is often painful and may progress to blindness. Clinical features include inflammation, neovascularization, and persistent cornea epithelial defects. Successful strategies for treatment involve transplantation of grafts harvested from the limbus of the alternate healthy eye, called conjunctival-limbal autograft (CLAU) and transplantation of limbal cell sheets cultured from limbal biopsies, termed cultured limbal epithelial transplantation (CLET). In 2012, Sangwan and colleagues presented simple limbal epithelial transplantation (SLET), a novel transplantation technique that combines the benefits of CLAU and CLET and avoids the challenges associated with both. In SLET a small biopsy from the limbus of the healthy eye is divided and distributed over human amniotic membrane, which is placed on the affected cornea. Outgrowth occurs from each small explant and a complete corneal epithelium is typically formed within 2 weeks. Advantages of SLET include reduced risk of iatrogenic LSCD occurring in the healthy cornea at harvest; direct transfer circumventing the need for cell culture; and the opportunity to perform biopsy harvest and transplantation in one operation. Success so far using SLET is comparable with CLAU and CLET. Of note, 336 of 404 (83%) operations using SLET resulted in restoration of the corneal epithelium, whereas visual acuity improved in 258 of the 373 (69%) reported cases. This review summarizes the results of 31 studies published on SLET since 2012. Progress, advantages, challenges, and suggestions for future studies are presented.
Liu C, Miyajima T, Melangath G, Miyai T, Vasanth S, Deshpande N, Kumar V, Ong Tone S, Gupta R, Zhu S, Vojnovic D, Chen Y, Rogan EG, Mondal B, Zahid M, Jurkunas UV. Ultraviolet A light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected. Proc Natl Acad Sci U S A 2020;117(1):573-583.Abstract
Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of -acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.
Miyajima T, Melangath G, Zhu S, Deshpande N, Vasanth S, Mondal B, Kumar V, Chen Y, Price MO, Price FW, Rogan EG, Zahid M, Jurkunas UV. Loss of NQO1 generates genotoxic estrogen-DNA adducts in Fuchs Endothelial Corneal Dystrophy. Free Radic Biol Med 2020;147:69-79.Abstract
Fuchs Endothelial Corneal Dystrophy (FECD) is an age-related genetically complex disease characterized by increased oxidative DNA damage and progressive degeneration of corneal endothelial cells (HCEnCs). FECD has a greater incidence and advanced phenotype in women, suggesting a possible role of hormones in the sex-driven differences seen in the disease pathogenesis. In this study, catechol estrogen (4-OHE), the byproduct of estrogen metabolism, induced genotoxic estrogen-DNA adducts formation, macromolecular DNA damage, and apoptotic cell death in HCEnCs; these findings were potentiated by menadione (MN)-mediated reactive oxygen species (ROS). Expression of NQO1, a key enzyme that neutralizes reactive estrogen metabolites, was downregulated in FECD, indicating HCEnC susceptibility to reactive estrogen metabolism in FECD. NQO1 deficiency in vitro exacerbated the estrogen-DNA adduct formation and loss of cell viability, which was rescued by the supplementation of N-acetylcysteine, a ROS scavenger. Notably, overexpression of NQO1 in HCEnCs treated with MN and 4-OHE quenched the ROS formation, thereby reducing the DNA damage and endothelial cell loss. This study signifies a pivotal role for NQO1 in mitigating the macromolecular oxidative DNA damage arising from the interplay between intracellular ROS and impaired endogenous estrogen metabolism in post-mitotic ocular tissue cells. A dysfunctional Nrf2-NQO1 axis in FECD renders HCEnCs susceptible to catechol estrogens and estrogen-DNA adducts formation. This novel study highlights the potential role of NQO1-mediated estrogen metabolite genotoxicity in explaining the higher incidence of FECD in females.
Greiner JV, Glonek T, Korb DR, Lindsay ME, Oliver PJ, Olson MCD. Corneal Cryopreservation Using Glycerylphosphorylcholine-Enriched Medium. Cornea 2020;39(3):370-375.Abstract
PURPOSE: To determine the effects of prolonged cryopreservation at subzero-degree temperatures on corneal transparency and histology after treatment with preservation medium containing the phosphodiester glycerylphosphorylcholine (GPC). METHODS: Rabbit corneas (n = 30) were immersed for 3 hours in K-Sol preservation medium containing 30 mM GPC. Three groups with 6 corneas each were refrigerated at -8°C for 2 weeks and liquid nitrogen temperature for 2 and 6 weeks, respectively. Two groups with 6 corneas each immersed in K-Sol preservation medium only were refrigerated at -8°C for 2 weeks and liquid nitrogen temperature for 6 weeks, respectively. Postthawing corneal transparency was measured on a grading scale after which corneas were prepared for and analyzed by light and transmission electron microscopy. RESULTS: All 3 groups of corneas preserved with GPC maintained a greater degree of corneal transparency compared with corneas preserved without GPC. The number of corneas retaining epithelial and endothelial layers increased in all groups where corneas were preserved in medium containing GPC, in contrast to corneas preserved in medium without GPC. Cytoplasmic vacuolization or nuclear damage was greater in corneas preserved without GPC. Similar findings were found in corneas stored at -8°C and liquid nitrogen temperatures. CONCLUSIONS: This study demonstrates a cryoprotective effect of corneas preserved in K-Sol containing the phosphodiester GPC at subzero-degree temperatures. In corneas immersed in preservation medium containing GPC, a higher degree of transparency is maintained and a lesser degree of histopathologic changes is observed with storage at both -8°C and in liquid nitrogen.
Koo EH, Pineda R, Afshari N, Eghrari A. Learning Descemet Membrane Endothelial Keratoplasty: A Survey of U.S. Corneal Surgeons. Cornea 2020;39(5):590-593.Abstract
PURPOSE: The transition to Descemet membrane endothelial keratoplasty (DMEK) is frequently challenging, requiring the adoption of new techniques, skills, and methods. We sought to draw on surgeons' initial experiences with DMEK to characterize the learning curve associated with this procedure and identify factors that could be linked to the frequency of primary graft failure (PGF) in the first 10 cases. METHODS: We invited corneal surgeons based in the United States who started performing the DMEK procedure within the past 2 years to answer a 12-question survey using an online survey platform. We analyzed quantitative and qualitative data. A Fisher exact test was used to determine whether preoperative approaches to preparation were associated with decreased PGF rates. RESULTS: A total of 100 US-based corneal surgeons replied from 34 of 50 states. Of these, 68% reported that DMEK comprised a majority of their endothelial keratoplasty cases. Approximately half of surgeons (52%) had performed more than 20 DMEK cases by the time of the survey, and 51% felt equally comfortable performing DMEK relative to Descemet stripping endothelial keratoplasty. Among the respondents, 37% answered that they had experienced PGF in the first 10 cases. Scrubbing in with an experienced colleague before surgery was associated with a decreased likelihood of at least one case of PGF (31%, P = 0.049), but not participation in a wet lab with an experienced instructor or mentor (38%, P = 0.50), nor having an eye bank representative present in the operating room (43%, P = 0.886). CONCLUSIONS: The collective experience of 100 surgeons beginning DMEK confirms the importance of mentorship and that the accompaniment of an experienced colleague during the learning curve is associated with lower rates of PGF.
Silva RNE, Taniguchi EV, Cruzat A, Paschalis EI, Pasquale LR, Colby KA, Dohlman CH, Chodosh J, Shen LQ. Angle Anatomy and Glaucoma in Patients With Boston Keratoprosthesis. Cornea 2020;39(6):713-719.Abstract
PURPOSE: To quantitatively analyze the angle anatomy in eyes with a Boston type 1 keratoprosthesis (KPro) using anterior segment optical coherence tomography (AS-OCT) and to assess the diagnostic ability of AS-OCT in KPro-associated glaucoma. METHODS: AS-OCT (RTVue) images from KPro eyes with and without glaucoma were reviewed. The angle opening distance at 500 μm from the scleral spur (AOD500), trabecular-iris angle at 500 μm from the scleral spur (TIA500), and trabecular-iris surface area at 500 μm from the scleral spur (TISA500) were measured by 2 observers masked to the diagnosis. The measurements for each visible quadrant were compared between KPro eyes with and without glaucoma. RESULTS: Twenty-two eyes with glaucoma and 17 eyes without glaucoma from 39 patients with KPro were included. Of the 4 quadrants imaged, the temporal angle was the most visible (79.5%) and angle measurements of the temporal quadrant were the only ones that differentiated the 2 groups: the mean AOD500, TIA500, and TISA500 were significantly lower in KPro eyes with glaucoma than without glaucoma (388.2 ± 234.4 μm vs. 624.5 ± 310.5 μm, P = 0.02; 26.1 ± 14.0 degrees vs. 39.1 ± 17.1 degrees, P = 0.03; and 0.15 ± 0.09 mm vs. 0.23 ± 0.12 mm, P = 0.03; respectively). The highest area under the receiver operating characteristic curve for detecting glaucoma was 0.75 for temporal TIA500 (95% confidence interval 0.57-0.94, P = 0.02) with 50% specificity at 80% of sensitivity and a cutoff value of 37 degrees. CONCLUSIONS: The temporal angle was the most visible on AS-OCT in eyes with a KPro. Significant narrowing of the temporal angle detected on AS-OCT was associated with glaucoma in these eyes.
Bakshi SK, Graney J, Paschalis EI, Agarwal S, Basu S, Iyer G, Liu C, Srinivasan B, Chodosh J. Design and Outcomes of a Novel Keratoprosthesis: Addressing Unmet Needs in End-Stage Cicatricial Corneal Blindness. Cornea 2020;39(4):484-490.Abstract
PURPOSE: The most commonly applied prosthetic devices for corneal blindness in the setting of severe cicatricial keratoconjunctivitis are the Boston keratoprosthesis type II and the modified osteo-odonto-keratoprosthesis, with these requiring either normal eyelid skin or a healthy cuspid tooth, respectively. For patients with neither attribute, we developed a new keratoprosthesis device combining positive aspects of both Boston keratoprosthesis type II and modified osteo-odonto-keratoprosthesis, which we have named the "Lux." METHODS: Short-term postoperative outcomes for the Lux keratoprosthesis, best-corrected visual acuity (BCVA), device retention, and complications, were examined in a retrospective case series of 9 eyes of 9 patients implanted at 4 centers. RESULTS: Seven of 9 (77.8%) eyes had cicatricial corneal blindness due to autoimmune disease and 2 (22.2%) from severe burns. Preoperative BCVA was ≤hand motions in all patients. Three (33.3%) had previously received at least 1 keratoprosthesis in the affected eye, and 4 (44.4%) had previously undergone ≥1 therapeutic keratoplasty. One patient had 19 previous eye surgeries. The mean duration of postoperative follow-up was 18.7 months (range 7-28 months). BCVA of ≥20/200 was achieved in all 9 patients, with 2 (22.2%) reaching 20/20 at the last examination, and all 9 (100%) of the devices were retained. One recipient developed a retinal detachment 2 months after implantation. Two (22.2%) patients required placement of a glaucoma drainage device. CONCLUSIONS: The Lux keratoprosthesis was developed for patients with severe cicatricial keratoconjunctivitis who were otherwise not candidates for existing keratoprosthesis designs. Short-term outcomes after implantation of the Lux keratoprosthesis were encouraging.
Coco G, Foulsham W, Nakao T, Yin J, Amouzegar A, Taketani Y, Chauhan SK, Dana R. Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin-10. Am J Transplant 2020;20(2):389-398.Abstract
The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft-attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating the host's alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using 2 well-established models of corneal transplantation (low-risk and high-risk models), we show that Tregs derived from low-risk graft recipients have a superior capacity in protecting CEnCs against effector T cell-mediated and interferon-γ and tumor necrosis factor-α-induced cell death compared to Tregs derived from high-risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low-risk hosts occurs independently of direct cell-cell contact and is mediated by the immunoregulatory cytokine IL-10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL-10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.
Xiao J, Adil MY, Chen X, Utheim ØA, Ræder S, Tønseth KA, Lagali NS, Dartt DA, Utheim TP. Functional and Morphological Evaluation of Meibomian Glands in the Assessment of Meibomian Gland Dysfunction Subtype and Severity. Am J Ophthalmol 2020;209:160-167.Abstract
PURPOSE: To classify subtypes of meibomian gland dysfunction (MGD) and evaluate the dependency of dry eye signs, symptoms, and parameters on MGD subtype. DESIGN: Cross-sectional study. STUDY POPULATION: the right eyes of 447 patients with MGD of various subtypes and 20 healthy volunteers. METHODS: Patients were divided into 4 subtypes of MGD based on meibum expression, meibum quality, and MG loss on meibography images (meibograde of 0-6). Subtypes were patients with high meibum delivery (hypersecretory and nonobvious MGD) and those with low meibum delivery (hyposecretory and obstructive MGD). Additional clinical tests included tear film break-up time (TFBUT), ocular staining, osmolarity, Schirmer I, blink interval timing and the Ocular Surface Disease Index (OSDI) questionnaire. RESULTS: A total of 78 eyes had hypersecretory MGD; 49 eyes had nonobvious MGD; 66 eyes had hyposecretory MGD; and 254 eyes had obstructive MGD. Increased tear film osmolarity and lower TFBUT were found in the low-delivery groups; hyposecretory (P = 0.006, P = 0.016) and obstructive MGD (P = 0.008, P = 0.006) relative to high-delivery MGD (hypersecretory and nonobvious groups, respectively). Worse ocular symptoms and ocular staining were also found in low-delivery MGD groups than the high delivery MGD groups (P < 0.01 and P < 0.006, respectively). CONCLUSIONS: Patients with low-delivery MGD had worse dry eye parameters and ocular symptoms than those with high meibum delivery, indicating the pivotal role of meibum secretion in ocular surface health that should be targeted in MGD therapy. Furthermore, nonobvious MGD cannot be diagnosed using conventional dry eye tests and requires morphologic assessment of meibography images to confirm MG loss.
Wang J, Chen D, Sullivan DA, Xie H, Li Y, Liu Y. Expression of Lubricin in the Human Amniotic Membrane. Cornea 2020;39(1):118-121.Abstract
PURPOSE: Lubricin, a boundary lubricant, is the body's unique antiadhesive, antifibrotic, antifriction, and antiinflammatory glycoprotein. This amphiphile is produced by numerous tissues and acts to regulate a number of processes, such as homeostasis, shear stress, tissue development, innate immunity, inflammation, and wound healing. We hypothesize that lubricin is also synthesized and expressed by the amniotic membrane (AM), which also possesses antiadhesive, antifibrotic, and antiinflammatory properties. We also hypothesize that lubricin, at least in part, mediates these AM capabilities. Our goal was to test our hypothesis. METHODS: We obtained multiple samples of fresh, cryopreserved (CP), and freeze-dried (FD) human AMs, as well as fresh placental tissue as positive controls, and processed them for light microscopy, immunofluorescence, and western blot analyses. We also evaluated the ability of recombinant human lubricin to associate with FD-AMs. RESULTS: Our results demonstrate that all fresh placental, fresh AM, and CP-AM samples contained lubricin. Lubricin was expressed in placental chorionic villi, AM epithelial and stromal cells, and CP-AM epithelia. No lubricin could be detected in FD-AMs but could be restored in FD-AMs after overnight incubation with recombinant human lubricin. CONCLUSIONS: This study supports our hypothesis that lubricin is expressed in human AMs. In addition, our data show that preservation methods influence the extent of this expression. Indeed, the disappearance of lubricin in FD-AMs may explain why dried AM reportedly loses its antiinflammatory and antiscarring abilities. It is possible that lubricin may mediate, at least in part, many of the biological properties of AMs.
Argüeso P. Disrupted Glycocalyx as a Source of Ocular Surface Biomarkers. Eye Contact Lens 2020;46 Suppl 2:S53-S56.Abstract
The glycocalyx is a dense and diverse coat of glycans and glycoconjugates responsible for maintaining cell surface integrity and regulating the interaction of cells with the external environment. Transmembrane mucins such as MUC1 and MUC16 comprise a major component of the epithelial glycocalyx and are currently used to monitor disease progression in cancer. At the ocular surface, multiple lines of evidence indicate that abnormal expression of the enzymes responsible for glycan biosynthesis during pathological conditions impairs the glycosylation of transmembrane mucins. It is now becoming clear that these changes contribute to modify the interaction of mucins with galectin-3, a multimeric lectin crucial for preserving the ocular surface epithelial barrier. This review highlights the potential of using the epithelial glycocalyx as a reliable source for the generation of biomarkers to diagnose and monitor ocular surface disease.
Zieske JD, Hutcheon AEK, Guo X. Extracellular Vesicles and Cell-Cell Communication in the Cornea. Anat Rec (Hoboken) 2020;303(6):1727-1734.Abstract
One question that has intrigued cell biologists for many years is, "How do cells interact to influence one another's activity?" The discovery of extracellular vesicles (EVs) and the fact that they carry cargo, which directs cells to undergo changes in morphology and gene expression, has revolutionized this field of research. Little is known regarding the role of EVs in the cornea; however, we have demonstrated that EVs isolated from corneal epithelial cells direct corneal keratocytes to initiate fibrosis. Intriguingly, our data suggest that EVs do not penetrate epithelial basement membrane (BM), perhaps providing a mechanism explaining the importance of BM in the lack of scarring in scrape wounds. Since over 100-million people worldwide suffer from visual impairment as a result of corneal scarring, the role of EVs may be vital to understanding the mechanisms of wound repair. Therefore, we investigated EVs in ex vivo and in vivo-like three-dimensional cultures of human corneal cells using transmission electron microscopy. Some of the major findings were all three major cell types (epithelial, fibroblast, and endothelial cells) appear to release EVs, EVs can be identified using TEM, and EVs appeared to be involved in cell-cell communication. Interestingly, while our previous publication suggests that EVs do not penetrate the epithelial BM, it appears that EVs penetrate the much thicker endothelial BM (Descemet's membrane). These findings indicate the huge potential of EV research in the cornea and wound healing, and suggest that during homeostasis the endothelium and stromal cells are in communication. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.