Colocalization of increased transforming growth factor-β-induced protein (TGFBIp) and Clusterin in Fuchs endothelial corneal dystrophy

PURPOSE: To investigate the differential expression of TGFBIp in normal human and Fuchs endothelial corneal dystrophy (FECD) endothelial cell-Descemet’s membrane (HCEC-DM) complex, and to asses the structural role of TGFBIp and clusterin (CLU) in guttae formation. METHODS: HCEC-DM complex was dissected from stroma in normal and FECD samples. Proteins were separated by 2-D gel electrophoresis and subjected to proteomic analysis. N-terminal processing of TGFBIp was detected by Western blot analysis with two separate antibodies against the N- and C-terminal regions of TGFBIp. Expression of TGFBI mRNA was compared by using real-time PCR. Subcellular localization of TGFBIp and CLU in corneal guttae was assessed by fluorescence confocal microscopy. RESULTS: A major 68-kDa fragment and a minor 39-kDa fragment of TGFBIp were identified on 2-D gels. Western blot analysis revealed an age-dependent proteolytic processing of the TGFBIp N terminus resulting in the increased formation of 57-kDa (P = 0.04) and 39-kDa (P = 0.03) fragments in older donors. FECD HCEC-DM showed a significant increase in the 68-kDa (P = 0.04), 57-kDa (P = 0.01), and 39- kDa (P = 0.03) fragments of TGFBIp. Real-time PCR analysis revealed that TGFBI mRNA was significantly increased (P = 0.04) in FECD samples. TGFBIp formed aggregates at the lower portions of guttae, next to Descemet’s membrane, whereas CLU localized mostly on top of the TGFBIp-stained areas at the level of the endothelial cell nuclear plane. CONCLUSIONS: The overexpression of proaggregative protein CLU, and proadhesive protein TGFBIp, have been colocalized in the guttae. Such findings provide us with a better understanding of the major contributors involved in the aberrant cell-extracellular matrix interactions seen in the guttae of patients with FECD

Telomerase Immortalization of Human Corneal Endothelial Cells Yields Functional Hexagonal Monolayers

Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness–the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine

Increased Clusterin Expression in Fuchs’ Endothelial Dystrophy

Purpose: To compare the relative expression of peroxiredoxin (Prx) proteins in normal human corneal endothelium with endothelium in corneas affected by Fuchs’ endothelial dystrophy (FED) and between normal human endothelium and epithelial/stromal tissue. Methods: Human corneal endothelial cell-Descemet’s membrane (HCEC-DM) complexes from normal and FED corneal buttons were dissected from the epithelium/stroma. For proteomic analysis, HCEC-DM protein extracts were separated by using two-dimensional gel electrophoresis. Relative differences in protein spot density was analyzed. Proteins of interest, including Prx isoforms, were identified by MALDI-TOF (matrix-assisted desorption ionization-time of flight) mass spectrometry. Western blot analysis compared the relative expression of Prx isoforms in normal and FED endothelium and between normal endothelium and normal epithelium/stroma. Expression of Prx-2 mRNA was compared by using real-time PCR. Results: Proteomic analysis identified differences in the relative expression of Prx isoforms between normal and FED endothelium. Western blot analysis confirmed that expression of Prx-2, −3, and −5 was significantly decreased (P \u3c 0.05) in FED cells. Normal HCECs expressed significantly (P \u3c 0.05) higher levels of Prx-2 and −3 than did the epithelium/stroma. Expression of Prx-5 was not significantly different (P \u3e 0.05) in the endothelium versus the epithelium/stroma. Real-time PCR analysis revealed that Prx-2 mRNA was significantly decreased (P = 0.027) in FED samples. Conclusions: Prx proteins were identified in human corneal endothelium. The fact that Prx-2 and −3 were expressed at significantly higher levels in HCEC-DM compared with the epithelium/stroma reflects the different physiologic activities of individual corneal cell types. Significantly decreased expression of Prx-2, −3, and −5 in FED may suggest an alteration in the ability of endothelial cells to withstand oxidant-induced damage and may be closely related to the pathogenesis of this disease

Decreased Expression of Peroxiredoxins in Fuchs’ Endothelial Dystrophy

Purpose: To compare the relative expression of peroxiredoxin (Prx) proteins in normal human corneal endothelium with endothelium in corneas affected by Fuchs’ endothelial dystrophy (FED) and between normal human endothelium and epithelial/stromal tissue. Methods: Human corneal endothelial cell-Descemet’s membrane (HCEC-DM) complexes from normal and FED corneal buttons were dissected from the epithelium/stroma. For proteomic analysis, HCEC-DM protein extracts were separated by using two-dimensional gel electrophoresis. Relative differences in protein spot density was analyzed. Proteins of interest, including Prx isoforms, were identified by MALDI-TOF (matrix-assisted desorption ionization-time of flight) mass spectrometry. Western blot analysis compared the relative expression of Prx isoforms in normal and FED endothelium and between normal endothelium and normal epithelium/stroma. Expression of Prx-2 mRNA was compared by using real-time PCR. Results: Proteomic analysis identified differences in the relative expression of Prx isoforms between normal and FED endothelium. Western blot analysis confirmed that expression of Prx-2, −3, and −5 was significantly decreased (P \u3c 0.05) in FED cells. Normal HCECs expressed significantly (P \u3c 0.05) higher levels of Prx-2 and −3 than did the epithelium/stroma. Expression of Prx-5 was not significantly different (P \u3e 0.05) in the endothelium versus the epithelium/stroma. Real-time PCR analysis revealed that Prx-2 mRNA was significantly decreased (P = 0.027) in FED samples. Conclusions: Prx proteins were identified in human corneal endothelium. The fact that Prx-2 and −3 were expressed at significantly higher levels in HCEC-DM compared with the epithelium/stroma reflects the different physiologic activities of individual corneal cell types. Significantly decreased expression of Prx-2, −3, and −5 in FED may suggest an alteration in the ability of endothelial cells to withstand oxidant-induced damage and may be closely related to the pathogenesis of this disease

Outcomes of phacoemulsification in patients with chronic ocular graft-versus-host disease

Purpose To evaluate the outcomes of phacoemulsification in patients with ocular graft-versus-host disease (GVHD). Methods The occurrence of cataract, 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 From 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 one 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) and improved postoperatively to 0.17±0.18 (Snellen 20/29) at one 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 accounted by 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

Topical Bevacizumab in the Treatment of Corneal Neovascularization

Objectives To study the safety and efficacy of topical bevacizumab in the treatment of corneal neovascularization (NV). Design In a prospective, open-label, non-comparative study, 10 eyes from 10 patients with stable corneal NV were treated with topical bevacizumab 1.0% for 3 weeks and followed up to 24 weeks. Main Outcome Measures The primary safety variables were the occurrence of ocular and systemic adverse events throughout the course of the study. The primary efficacy variables were neovascular area (NA), measuring the area of the corneal vessels themselves; vessel caliber (VC), measuring the mean diameter of the corneal vessels; and invasion area (IA), measuring the fraction of the total corneal area covered by the vessels. Results From baseline visit to the last follow-up visit, the mean reduction was 47.1% ± 36.7% for NA, 54.1% ± 28.1 for VC, and 12.2% ± 42.0% for IA. The decreases in NA and VC were statistically significant (p = 0.0014 and p = 0.00009, respectively). However, changes in IA did not achieve statistical significance (p = 0.19). Visual acuity and central corneal thickness showed no significant changes. Topical bevacizumab was well-tolerated with no adverse events. Conclusions Short-term topical bevacizumab therapy reduces the severity of corneal NV without local or systemic side-effects. Application to Clinical Practice Topical bevacizumab provides an alternative therapy in the treatment of stable corneal neovascularization

Inflammation and the Nervous System: The Connection in the Cornea in Patients with Infectious Keratitis

Purpose. To study the density and morphologic characteristics of epithelial dendritic cells, as correlated to subbasal corneal nerve alterations in acute infectious keratitis (IK) by in vivo confocal microscopy (IVCM). Methods. IVCM of the central cornea was performed prospectively in 53 eyes with acute bacterial (n = 23), fungal (n = 13), and Acanthamoeba (n = 17) keratitis, and in 20 normal eyes, by using laser in vivo confocal microscopy. Density and morphology of dendritic-shaped cells (DCs) of the central cornea, corneal nerve density, nerve numbers, branching, and tortuosity were assessed and correlated. It should be noted that due to the “in vivo” nature of the study, the exact identity of these DCs cannot be specified, as they could be monocytes or tissue macrophages, but most likely dendritic cells. Results. IVCM revealed the presence of central corneal DCs in all patients and controls. The mean DC density was significantly higher in patients with bacterial (441.1 ± 320.5 cells/mm2; P < 0.0001), fungal (608.9 ± 812.5 cells/mm2; P < 0.0001), and Acanthamoeba keratitis (1000.2 ± 1090.3 cells/mm2; P < 0.0001) compared with controls (49.3 ± 39.6 cells/mm2). DCs had an increased size and dendrites in patients with IK. Corneal nerves were significantly reduced in eyes with IK compared with controls across all subgroups, including nerve density (674.2 ± 976.1 vs. 3913.9 ± 507.4 μm/frame), total nerve numbers (2.7 ± 3.9 vs. 20.2 ± 3.3), main trunks (1.5 ± 2.2 vs. 6.9 ± 1.1), and branching (1.2 ± 2.0 vs. 13.5 ± 3.1; P < 0.0001). A strong association between the diminishment of corneal nerves and the increase of DC density was observed (r = −0.44; P < 0.0005). Conclusions. IVCM reveals an increased density and morphologic changes of central epithelial DCs in infectious keratitis. There is a strong and significant correlation between the increase in DC numbers and the decreased subbasal corneal nerves, suggesting a potential interaction between the immune and nervous system in the cornea

Safety and Efficacy of the Multitargeted Receptor Kinase Inhibitor Pazopanib in the Treatment of Corneal Neovascularization

Purpose. To evaluate the safety and efficacy of topical pazopanib in the treatment of corneal neovascularization (CNV). Methods. Twenty eyes of 20 patients with stable CNV were enrolled in a prospective, open label, noncomparative study and treated with topical pazopanib 0.5% for 3 weeks, and followed for 12 weeks. The primary endpoint was to determine the tolerability and safety of topical pazopanib in the treatment of CNV defined by the occurrence of ocular and systemic adverse events during the study. The secondary endpoint was to evaluate the effect of topical pazopanib on the reduction of (1) neovascular area (NA), defined as the area of the corneal vessels themselves, (2) invasion area (IA), defined as the fraction of the total cornea into which the vessels extend, (3) vessel length (VL), defined as the mean measurement of the extent of vessels from end to end, and (4) vessel caliber (VC), defined as the mean diameter of the corneal vessels. Results. There were no severe adverse events following the use of topical pazopanib. Compared with the baseline visit, NA and VL showed a statistically significant decrease at week 3 (P = 0.02 and 0.01, respectively); and NA, IA, and VL statistically significantly decreased at week 12 (P = 0.03, 0.04, and <0.01, respectively). Visual acuity maintained without changes after the 12 week follow-up. Conclusions. This preliminary study suggests that topical treatment with pazopanib 0.5% is safe, well tolerated, and may have a role as an alternative for the treatment of CNV (ClinicalTrials.gov number, NCT01257750)

Cultivated Autologous Limbal Epithelial Cell (CALEC) Transplantation: Development of Manufacturing Process and Clinical Evaluation of Feasibility and Safety

To treat unilateral limbal stem cell (LSC) deficiency, we developed cultivated autologous limbal epithelial cells (CALEC) using an innovative xenobiotic-free, serum-free, antibiotic-free, two-step manufacturing process for LSC isolation and expansion onto human amniotic membrane with rigorous quality control in a good manufacturing practices facility. Limbal biopsies were used to generate CALEC constructs, and final grafts were evaluated by noninvasive scanning microscopy and tested for viability and sterility. Cultivated cells maintained epithelial cell phenotype with colony-forming and proliferative capacities. Analysis of LSC biomarkers showed preservation of stemness. After preclinical development, a phase 1 clinical trial enrolled five patients with unilateral LSC deficiency. Four of these patients received CALEC transplants, establishing preliminary feasibility. Clinical case histories are reported, with no primary safety events. On the basis of these results, a second recruitment phase of the trial was opened to provide longer term safety and efficacy data on more patients