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2017

ZA S, JA T, UV J. Peripheral endothelial cell count is a predictor of disease severity in advanced Fuchs Endothelial Corneal Dystrophy.. Cornea. 2017;36(10):1166–1171.
Publisher's Version

Purpose: In advanced Fuchs endothelial corneal dystrophy (FECD), central endothelial changes do not correlate with disease severity. The peripheral endothelial cell count (ECC) has not been studied as a marker of FECD severity. The goal of this study was to determine the relationship between the peripheral ECC and known clinical markers of FECD in advanced cases.

Methods: Patients with FECD examined between January 1, 2013, and September 1, 2016, by 1 cornea specialist were identified. Medical records from all previous visits were reviewed to include eyes with high-quality central and peripheral in vivo confocal microscopy images performed on the same day as a clinical evaluation. Endothelial photographs were used to perform manual cell counts centrally and peripherally. Clinical grading of FECD from 1 to 4 was performed at the slit-lamp.

Results: We identified 154 eyes of 126 patients that met criteria for inclusion. With higher disease grades, central ECC and peripheral ECC decreased, visual acuity worsened, and central corneal thickness (CCT) increased (all P < 0.05). In patients with advanced disease (defined as either grade 3 or 4, CCT >700, or central ECC <350), the peripheral ECC was the best predictor of disease severity and had the highest number of statistically significant correlations with other clinical markers compared with competing variables.

Conclusions: In advanced FECD, severity is best determined by the peripheral ECC compared with the central ECC, visual acuity, clinical disease grade, and CCT. The peripheral ECC should be added to the clinical parameters used to evaluate FECD severity.

Kim EC, Toyono T, Berlinicke C, Zack D, Jurkunas U, Usui T, Jun A. Screening and Characterization of Drugs That Protect Corneal Endothelial Cells Against Unfolded Protein Response and Oxidative Stress. Invest Ophthalmol Vis Sci. 2017;58(2):892–900. doi:10.1167/iovs.16-20147
Purpose: To screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress. Methods: Bovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected "hits" were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs). Results: Forty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels. Conclusions: Oxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy.

2016

Halilovic A, Schmedt T, Benischke A-S, Hamill C, Chen Y, Santos JH, Jurkunas U. Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy. Antioxid Redox Signal. 2016;24(18):1072–83. doi:10.1089/ars.2015.6532
AIMS: Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. RESULTS: We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. INNOVATION: This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. CONCLUSION: MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072-1083.
Katikireddy KR, Jurkunas U. Limbal Stromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells. Methods Mol Biol. 2016;1341:437–44. doi:10.1007/7651_2015_229
From the derivation of the first human embryonic stem (hES) cell line to the development of induced pluripotent stem (iPS) cells; it has become evident that tissue specific stem cells are able to differentiate into a specific somatic cell types. The understanding of key processes such as the signaling pathways and the role of the microenvironment in epidermal/epithelial development has provided important clues for the derivation of specific epithelial cell types.Various differentiation protocols/methods were used to attain specific epithelial cell types. Here, we describe in detail the procedure to follow for isolation of tissue specific stem cells, mimicking their microenvironment to attain stem cell characteristics, and their potential differentiation to corneal epithelial cells.
Liu C, Vojnovic D, Kochevar I, Jurkunas U. UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells. Invest Ophthalmol Vis Sci. 2016;57(4):2319–27. doi:10.1167/iovs.16-19097
PURPOSE: To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress. METHODS: Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay. RESULTS: Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death. CONCLUSIONS: Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD).
Katikireddy KR, Schmedt T, Price M, Price F, Jurkunas U. Existence of Neural Crest-Derived Progenitor Cells in Normal and Fuchs Endothelial Dystrophy Corneal Endothelium. Am J Pathol. 2016;186(10):2736–50. doi:10.1016/j.ajpath.2016.06.011

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.

Qazi Y, Hurwitz S, Khan S, Jurkunas U, Dana R, Hamrah P. Validity and Reliability of a Novel Ocular Pain Assessment Survey (OPAS) in Quantifying and Monitoring Corneal and Ocular Surface Pain. Ophthalmology. 2016;123(7):1458–68. doi:10.1016/j.ophtha.2016.03.006
PURPOSE: To validate the Ocular Pain Assessment Survey (OPAS), specifically designed to measure ocular pain and quality of life for use by eye care practitioners and researchers. DESIGN: A single-center cohort study was conducted among patients with and without corneal and ocular surface pain at initial and follow-up visits over a 6-month period. The content of the OPAS was guided by literature review, a body of experts, and incorporating conceptual frameworks from existing pain questionnaires. The Wong-Baker FACES Pain Rating Scale served as the gold standard for measuring the intensity of ocular pain. PARTICIPANTS: A total of 102 patients aged 18 to 80 years completed the OPAS at the initial visit. A total of 21 patients were followed up after treatment. METHODS: Indices of validity and internal consistency (Spearman's rank-order, rs, or Pearson's correlation coefficients, rp), and coefficient of reliability (Cronbach's α) were determined in addition to equivalence testing, exploratory factor analysis (EFA), and diagnostic analysis. MAIN OUTCOME MEASURES: Eye pain intensity was the primary outcome measure, and interference with quality of life (QoL), aggravating factors, associated factors, associated non-eye pain intensity, and self-reported symptomatic relief were the secondary outcome measures. RESULTS: The OPAS had criterion validity at both initial (rs = 0.71; n = 102; P 0.01) and follow-up visits (rs = 0.97; n = 21; P 0.01). Equivalence tests yielded OPAS and gold standard equivalence for both the initial and follow-up visits. The EFA supported 6 subscales (eye pain intensity at 24 hours and 2 weeks, non-eye pain intensity, QoL, aggravating factors, and associated factors) confirming multidimensionality. Cronbach's α >0.83 for all subscales established strong internal consistency, which correlated with the gold standard, including 24-hour eye pain intensity and QoL interference scores (rp = 0.81, 0.64, respectively P 0.001). At follow-up, reduction in pain scores was accompanied by improvement in all dimensions of the OPAS. Percentage change in QoL correlated to percentage change in the gold standard (rp = 0.53; P  0.05). The OPAS was sensitive (94%), specific (81%), and accurate (91%), with a diagnostic odds ratio >50. CONCLUSIONS: The OPAS is a valid, reliable, and responsive tool with strong psychometric and diagnostic properties in the multidimensional quantification of corneal and ocular surface pain intensity, and QoL.

2015

Chauhan, Jurkunas, Funaki, Dastjerdi, Dana. Quantification of allospecific and nonspecific corneal endothelial cell damage after corneal transplantation. Eye (Lond). 2015;29(1):136–44. doi:10.1038/eye.2014.248
PURPOSE: To investigate the effect of host immunity (allospecific) and surgical manipulation (non-allospecific) on corneal endothelial cells (CECs) in corneal transplantation. METHODS: Draining lymph nodes and grafted C57BL/6 corneas were harvested from syngeneic recipients, allograft acceptors, and allograft rejectors (BALB/c) 1, 3, and 8 weeks after transplantation. We analyzed CEC apoptosis using an ex vivo cornea-in-the-cup assay, and visualized cell-to-cell junctions using immunohistochemical staining (ZO-1). Automatic cell analysis using Confoscan software was used to measure CEC density as well as changes in CEC morphology by quantifying the coefficient of variation in cell size (polymegethism) and shape (pleomorphism). RESULTS: The cornea-in-the-cup assay showed that allogeneic acceptor T cells and to an even greater extent rejector T cells (but not syngeneic T cells) induced CEC apoptosis. CEC density after corneal transplantation was significantly reduced in allogeneic acceptors compared with syngeneic grafts (P0.001), and CEC density was even further reduced in the allo-rejector group compared with the allo-acceptor group. Allogeneic grafts showed a greater increase in the coefficient of variation in cell size (polymegethism) when compared with syngeneic grafts 1 week after transplantation (P=P0.001). However, pleomorphism was not significantly different between syngeneic and allo-acceptor grafts, indicating that polymegethism (but not pleomorphism or cell density) is a sensitive indicator of the effect of alloimmunity on CECs. CONCLUSIONS: Our data demonstrate that host alloimmunity rather than surgical manipulation alone is the major cause of CEC damage in corneal transplantation, and such morphologic changes of CECs can be detected before the clinically visible onset of allograft rejection.
Saboo U, Amparo F, Shikari H, Jurkunas U, Dana R. Outcomes of phacoemulsification in patients with chronic ocular graft-versus-host disease. Graefes Arch Clin Exp Ophthalmol. 2015;253(6):901–7. doi:10.1007/s00417-015-2940-3
PURPOSE: The purpose of this study was to evaluate the outcomes of phacoemulsification in patients with ocular graft-versus-host disease (GVHD). METHODS: The occurrence of cataracts, cataract surgery, and its outcomes were analyzed in the medical records of 229 patients (458 eyes) with ocular GVHD. Outcome measures included pre- and postoperative corrected distance visual acuity (CDVA) and the rate of postoperative complications. RESULTS: Of the 458 eyes evaluated, 58 were pseudophakic; from the 400 phakic eyes, 238 (59 %) presented with cataracts and 62 (26 %) underwent cataract surgery. Analysis of postoperative complications and visual outcomes at 1 month was performed in 51 eyes in which detailed surgical and immediate postoperative records were available. Preoperatively, the mean CDVA was 0.67 ± 0.57 LogMAR (Snellen 20/93), improving postoperatively to 0.17 ± 0.18 (Snellen 20/29) at 1 month (P  0.0001), and to 0.13 ± 0.14 (Snellen 20/26) by the final follow-up visit (P  0.0001). Postoperative complications included corneal epithelial defects (8 %), filamentary keratitis (6 %), worsening of corneal epitheliopathy (16 %), posterior capsular opacification (18 %), and cystoid macular edema (4 %). A corrected distance visual acuity of 20/30 or better was achieved in 87 % of the eyes; suboptimal CDVA improvement was attributable to severe ocular surface disease, pre-existing advanced glaucoma, and prior macular surgery. CONCLUSIONS: Phacoemulsification in patients with chronic ocular GVHD is a safe and efficacious procedure resulting in significant visual improvement. Overall, postoperative adverse events responded well to timely management.

2014

Liu C, Chen Y, Kochevar I, Jurkunas U. Decreased DJ-1 leads to impaired Nrf2-regulated antioxidant defense and increased UV-A-induced apoptosis in corneal endothelial cells. Invest Ophthalmol Vis Sci. 2014;55(9):5551–60. doi:10.1167/iovs.14-14580
PURPOSE: To investigate the role of DJ-1 in Nrf2-regulated antioxidant defense in corneal endothelial cells (CECs) at baseline and in response to ultraviolet A (UV-A)-induced oxidative stress. METHODS: DJ-1-deficient CECs were obtained by transfection of an immortalized normal human corneal endothelial cell line (HCECi) with DJ-1 small interfering RNA (siRNA) or by isolation of CECs from ex vivo corneas of DJ-1 knockout mice. Levels of reactive oxygen species (ROS), protein carbonyls, Nrf2 subcellular localization, Nrf2 target genes, and protein interaction between Keap1/Nrf2 and Cul3/Nrf2 were compared between normal and DJ-1-deficient CECs. Oxidative stress was induced by irradiating HCECi cells with UV-A, and cell death and levels of activated caspase3 and phospho-p53 were determined. RESULTS: DJ-1 siRNA-treated cells exhibited increased levels of ROS production and protein carbonyls as well as a 2.2-fold decrease in nuclear Nrf2 protein when compared to controls. DJ-1 downregulation led to attenuated gene expression of Nrf2 and its target genes HO-1 and NQO1. Similar levels of Nrf2 inhibitor, Keap1, and Cul3/Nrf2 and Keap1/Nrf2 were observed in DJ-1 siRNA-treated cells as compared to controls. Ultraviolet A irradiation resulted in a 3.0-fold increase in cell death and elevated levels of activated caspase3 and phospho-p53 in DJ-1 siRNA-treated cells compared to controls. CONCLUSIONS: Downregulation of DJ-1 impairs nuclear translocation of Nrf2, causing decreased antioxidant gene expression and increased oxidative damage. The decline in DJ-1 levels leads to heightened CEC susceptibility to UV-A light by activating p53-dependent apoptosis. Targeting the DJ-1-Nrf2 axis may provide a potential therapeutic approach for enhancing antioxidant defense in corneal endothelial disorders.