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PurposeWe sought to define the role of Wwtr1 in murine ocular structure and function and determine the role of mechanotransduction in Fuchs' endothelial corneal dystrophy (FECD), with emphasis on interactions between corneal endothelial cells (CEnCs) and Descemet's membrane (DM).MethodsA Wwtr1 deficient mouse colony was established, and advanced ocular imaging, atomic force microscope (AFM), and histology/immunofluorescence were performed. Corneal endothelial wound healing was assessed using cryoinjury and phototherapeutic keratectomy in Wwtr1 deficient mice. Expression of WWTR1/TAZ was determined in the corneal endothelium from normal and FECD-affected patients; WWTR1 was screened for coding sequence variants in this FECD cohort.ResultsMice deficient in Wwtr1 had reduced CEnC density, abnormal CEnC morphology, softer DM, and thinner corneas versus wildtype controls by 2 months of age. Additionally, CEnCs had altered expression and localization of Na/K-ATPase and ZO-1. Further, Wwtr1 deficient mice had impaired CEnC wound healing. The WWTR1 transcript was highly expressed in healthy human CEnCs comparable to other genes implicated in FECD pathogenesis. Although WWTR1 mRNA expression was comparable between healthy and FECD-affected patients, WWTR1/TAZ protein concentrations were higher and localized to the nucleus surrounding guttae. No genetic associations were found in WWTR1 and FECD in a patient cohort compared to controls.ConclusionsThere are common phenotypic abnormalities seen between Wwtr1 deficient and FECD-affected patients, suggesting that Wwtr1 deficient mice could function as a murine model of late-onset FECD. Despite the lack of a genetic association between FECD and WWTR1, aberrant WWTR1/TAZ protein subcellular localization and degradation may play critical roles in the pathogenesis of FECD.

Fuchs endothelial corneal dystrophy (FECD), a progressive polygenic disease that causes degeneration of the corneal endothelium, is the leading indication for corneal transplantation in the U.S. Characteristically, loss of corneal endothelial cells and formation of degenerative extracellular “guttae” result in failure to maintain appropriate corneal hydration through active ion pumping to counter the passive leakage of aqueous humor. FECD pathogenesis is linked to oxidative stress and environmental exposure to ultraviolet A (UVA), and impaired endogenous response to oxidative stress is common to the disease across multiple genotypes. UV irradiation is also known to cause cellular damage by ferroptosis, a nonapoptotic oxidative cell death resulting from iron-mediated lipid peroxidation. Although a possible role for ferroptosis in FECD has been postulated to account for the increased susceptibility to oxidative damage and lipid peroxidation, this has not been explored systematically. In this study, we investigated the roles of genetic background and UVA exposure in causing lipid membrane damage and endothelial cell degeneration in FECD. We first sought clinical evidence of iron-mediated lipid peroxidation in surgical samples obtained from FECD patients undergoing endothelial keratoplasty, and found increased levels of cytosolic ferrous iron (Fe2+) and evidence of lipid peroxidation were present in end-stage diseased tissues compared with healthy human donor corneas. Next, using immortalized (F35T) and primary cell cultures modeling the TCF4 intronic trinucleotide repeat expansion genotype, we found that alterations in gene and protein expression involved in ferroptosis were conserved in both cell culture and tissue models of FECD disease compared to controls, including elevated levels of the ferroptosis-specific marker transferrin receptor 1. F35T immortalized cells showed significantly higher basal lipid peroxidation than control HCEC-B4G12 cells, indicating higher susceptibility to ferroptosis attributable to genetic background. Then, we tested the impact of physiologically relevant doses of UVA irradiation on F35T cell cultures and found increased cytosolic Fe2+ iron levels indicating a role for ferroptosis in FECD disease progression. Finally, we tested the effects of inhibitory ferroptosis molecules. We found that F35T cells were more prone to RSL3 induced ferroptosis than healthy controls with deferoxamine chelation which indicated increased susceptibility in FECD cells, and cell death could be prevented after experimentally-induced ferroptosis using solubilized antioxidant ubiquinol indicating a role for anti-ferroptosis therapies. This investigation demonstrates that FECD genetic background and UVA exposure contribute to basal and incidental iron-mediated lipid peroxidation and cell death in FECD, and provides the basis for future investigations of ferroptosis-mediated oxidative damage and disease progression in FECD.

The corneal endothelium plays a critical role in maintaining corneal clarity. There is an expected decline in cell density with age and disease, and maintaining the health of this cell layer is important as corneal endothelial cells generally are amitotic in vivo. Diabetes mellitus is a highly prevalent disease that damages the corneal endothelium. Diabetes causes structural and functional impairments in the corneal endothelium that decrease cellular reserve in response to stress. These effects have implications to consider for diabetic patients undergoing anterior segment surgery, and for corneal surgeons who use diabetic donor tissue and treat diabetic patients. In this review, we discuss the specifics of how diabetes mellitus impacts the corneal endothelium including alterations in cell morphology, cell density, ultrastructure, pump and barrier function, cataract surgery outcomes, and corneal transplant outcomes with attention to the use of diabetic donor tissue and diabetic transplant recipients.

Purpose To determine whether ubiquinol improves mitochondrial function and cell viability in human donor corneal endothelial cells during hypothermic corneal tissue storage. Methods Endothelial cell Descemet membrane tissues were treated with 10 μM ubiquinol, the reduced form of the antioxidant coenzyme Q10, for 5 days in Optisol-GS storage media before assaying for mitochondrial activity using extracellular flux analysis of oxygen consumption. In addition, endothelial cell Descemet membrane tissues were analyzed for cell viability using apoptosis and necrosis assays. Control tissues from mate corneas were treated with diluent only, and comparisons were analyzed for differences. Results A total of 13 donor corneal tissues with a mean (SEM) preservation time of 11.8 days (0.4) were included for the analysis. Treatment with 10 μM ubiquinol increased spare respiratory capacity by 174% (P = 0.001), maximal respiration by 93% (P = 0.003), and proton leak by 80% (P = 0.047) compared with controls. Cells treated with ubiquinol had no significant change in cell necrosis or apoptosis. Conclusions Preliminary testing in donor corneal tissue at specified doses indicates that ubiquinol may be a useful biocompatible additive to hypothermic corneal storage media that increases corneal endothelial cell mitochondrial function. Additional investigations are indicated to further study and optimize the dose and formulation of ubiquinol for use in preserving donor corneal tissue function during hypothermic storage.

Purpose To determine the concentration of amphotericin B that would be both effective against Candida albicans contamination and safe for corneal endothelial cells (CECs) in cold storage conditions. Methods Triplicate media cultures were inoculated with 10 colony-forming units (CFUs)/mL of C. albicans (American Type Culture Collection 10231), supplemented with amphotericin B (0-20 μg/mL), stored in cold conditions (2°C-8°C) for 72 hours, and analyzed quantitatively for CFUs. C. albicans concentration in each sample was determined initially and after 6, 24, 48, and 72 hours of storage. CEC mitochondrial function (oxygen consumption rate), apoptosis, and necrosis were examined in donor corneas after 7 days of amphotericin B exposure and compared with untreated controls. CEC viability was also examined by calcein-AM staining and Fiji segmentation after 72 hours or 2 weeks of amphotericin B exposure to mimic potential eye bank practices. Results Amphotericin B concentrations of 1.25, 2.5, and 5.0 μg/mL resulted in 0.47, 1.11, and 1.21 log10 CFU reduction after only 6 hours of cold storage and continued to decrease to 3.50, 3.86, and 4.49 log10 reductions after 72 hours, respectively. By contrast, amphotericin B 0.255 µg/mL showed only 1.01 log10 CFU reduction after 72 hours of incubation. CEC mitochondrial function and viability did not differ in donor corneas exposed to amphotericin B ≤2.59 μg/mL compared with the controls. Conclusions Optimal efficacy of amphotericin B against C. albicans is achieved in cold storage conditions at concentrations ≥1.25 μg/mL, and 2.5 μg/mL reduces Candida contamination by >90% after 6 hours of cold storage without sacrificing CEC health.

To determine how the Rho kinase inhibitor, ripasudil, affects metabolic function and cell viability in donor human corneal endothelial cells (HCECs).Endothelial cell-Descemet membrane (EDM) tissues were treated with 10 μM ripasudil and assayed for mitochondrial and glycolytic activity using extracellular flux analysis and then compared to untreated controls. In addition, EDM tissues with a 24-h ripasudil treatment and control tissues were exposed to 1 μM staurosporine to induce apoptosis and then analyzed for cell viability using apoptosis and necrosis assays.Mitochondrial respiration metrics, specifically maximal respiration (P = 0.758) and spare respiratory capacity (P = 0.777), did not differ among the 1-h ripasudil treatment, 24-h treatment, and untreated tissues. Glycolytic activity assays showed an increase in glycolytic capacity at 1 h compared to the 24-h exposure group (P = 0.049) and controls (P = 0.009). Following exposure to staurosporine, the percentage of apoptotic HCECs was lower (P = 0.009) in ripasudil-treated tissues (2.473%, standard error of the mean [SEM] 0.477%) compared to untreated controls (3.349%, SEM 0.566%). In contrast, the percentage of necrotic HCECs decreased but did not differ statistically (P = 0.158) between ripasudil-treated (3.789%, SEM 0.487%) and untreated (4.567%, SEM 0.571%) tissues.Exposures to ripasudil did not result in any detectable reduction in metabolic function for HCECs in an ex vivo donor tissue model, and an increase in glycolytic activity at the 1-h time point was detected. In addition, HCECs treated with ripasudil gained a protective effect against induced apoptosis, suggesting that ripasudil may help improve the integrity of the corneal endothelium.

Mitochondrial function is essential for the viability of aerobic eukaryotic cells, as mitochondria provide energy through the generation of adenosine triphosphate (ATP), regulate cellular metabolism, provide redox balancing, participate in immune signaling, and can initiate apoptosis. Mitochondria are dynamic organelles that participate in a cyclical and ongoing process of regeneration and autophagy (clearance), termed mitophagy specifically for mitochondrial (macro)autophagy. An imbalance in mitochondrial function toward mitochondrial dysfunction can be catastrophic for cells and has been characterized in several common ophthalmic diseases. In this article, we review mitochondrial homeostasis in detail, focusing on the balance of mitochondrial dynamics including the processes of fission and fusion, and provide a description of the mechanisms involved in mitophagy. Furthermore, this article reviews investigations of ocular diseases with impaired mitophagy, including Fuchs endothelial corneal dystrophy, primary open-angle glaucoma, diabetic retinopathy, and age-related macular degeneration, as well as several primary mitochondrial diseases with ocular phenotypes that display impaired mitophagy, including mitochondrial encephalopathy lactic acidosis stroke, Leber hereditary optic neuropathy, and chronic progressive external ophthalmoplegia. The results of various studies using cell culture, animal, and human tissue models are presented and reflect a growing awareness of mitophagy impairment as an important feature of ophthalmic disease pathology. As this review indicates, it is imperative that mitophagy be investigated as a targetable mechanism in developing therapies for ocular diseases characterized by oxidative stress and mitochondrial dysfunction.

The extracellular matrix (ECM) plays a crucial role in all parts of the eye, from maintaining clarity and hydration of the cornea and vitreous to regulating angiogenesis, intraocular pressure maintenance, and vascular signaling. This review focuses on the interactions of the ECM for homeostasis of normal physiologic functions of the cornea, vitreous, retina, retinal pigment epithelium, Bruch’s membrane, and choroid as well as trabecular meshwork, optic nerve, conjunctiva and tenon’s layer as it relates to glaucoma. A variety of pathways and key factors related to ECM in the eye are discussed, including but not limited to those related to transforming growth factor-β, vascular endothelial growth factor, basic-fibroblastic growth factor, connective tissue growth factor, matrix metalloproteinases (including MMP-2 and MMP-9, and MMP-14), collagen IV, fibronectin, elastin, canonical signaling, integrins, and endothelial morphogenesis consistent of cellular activation-tubulogenesis and cellular differentiation-stabilization. Alterations contributing to disease states such as wound healing, diabetes-related complications, Fuchs endothelial corneal dystrophy, angiogenesis, fibrosis, age-related macular degeneration, retinal detachment, and posteriorly inserted vitreous base are also reviewed.

Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. These problems are thought to arise from accumulated reactive oxygen species (ROS) inside cells. Oxidative stress acting on the cells of organs and tissues preserved in hypothermic conditions before surgery, as is the case for cornea transplantation, is thought to be a major reason behind cell death prior to surgery and decreased graft survival after transplantation. We have recently discovered that ubiquinol – the reduced and active form of coenzyme Q10 and a powerful antioxidant – significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Docking studies showed that γ-CD has the strongest binding affinity with ubiquinol compared to α- or β-CD. Complexed ubiquinol showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products including Optisol-GS® corneal storage medium, balanced salt solution for intraocular irrigation, and topical Refresh® artificial tear eye drops. Greater ROS scavenging activity was noted in a cell model with high basal metabolism and ROS generation (A549) and in HCEC-B4G12 human corneal endothelial cells after treatment with ubiquinol/γ-CD compared to free ubiquinol. Furthermore, complexed ubiquinol was more effective at lowering ROS, and at far lower concentrations, compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected HCEC-B4G12 cells against erastin-induced ferroptosis . No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Using a vertical diffusion system, a topically applied inclusion complex of γ-CD and a lipophilic dye (coumarin-6) demonstrated transcorneal penetrance in porcine corneas and the capacity for the γ-CD vehicle to deliver drug to the corneal endothelium. Using the same model, topically applied ubiquinol/γ-CD complex penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol. Lastly, the penetrance of ubiquinol/γ-CD complex was assayed using human donor corneas preserved for 7 days in Optisol-GS® per standard industry practices, and demonstrated higher amounts of ubiquinol retained in the corneal endothelium compared to free ubiquinol. In summary, ubiquinol complexed with γ-CD is a highly stable composition that can be incorporated into a variety of aqueous-phase products for ophthalmic use including donor corneal storage media and topical eye drops to scavenge ROS and protect corneal endothelial cells against oxidative damage .

Purpose To assess how trypan blue staining affects Descemet membrane endothelial keratoplasty (DMEK) graft visibility and corneal endothelial cell (CEC) mitochondrial respiration. Methods DMEK grafts (n = 20) were stained with trypan blue 0.06% for 1, 3, 5, or 10 minutes. Each graft was injected into an artificial anterior chamber. Surgery was simulated with tapping and sweeping motions on the corneal surface and injections of balanced salt solution (BSS). Graft visibility was assessed at 5, 10, 20, and 30 minutes. Effects of trypan blue on mitochondrial respiration were assessed using primary CECs cultured from donor corneas (n = 43). Treatment wells exposed to trypan blue 0.06% (1, 5, or 30 minutes) and donor-matched control wells to methylene blue 1% (1 minute) or BSS (1, 5, or 30 minutes) were assayed for key respiration parameters. Results After 5 minutes of surgical manipulation, grafts stained for 5 minutes were significantly more visible than grafts stained for 1 or 3 minutes; there was no added benefit of staining for 10 minutes. After 10 minutes of surgical manipulation, grafts stained for 3 minutes were more visible than grafts stained for 1 minute, without additional benefits of staining ≥5 minutes. No visibility differences were observed after ≥20 minutes of surgical manipulation. CEC mitochondrial respiration did not change significantly following trypan blue exposure for all intervals tested compared to BSS. Conclusions Staining DMEK grafts with trypan blue for 3 to 5 minutes optimizes visibility during surgical manipulation without mitochondrial impairment. Corneal surgeons learning DMEK will benefit from optimizing this critical step.

PURPOSE To quantify changes in endothelial cell density (ECD) of donor corneal tissue in relation to the presence or absence of a medical history of diabetes mellitus diagnosis, treatment, and complications. METHODS A retrospective review was performed for all corneas collected at Iowa Lions Eye Bank between January 2012 and December 2015. For purposes of analysis, donor corneas were divided into 4 groups: nondiabetic, non-insulin-dependent diabetic, insulin-dependent diabetic without medical complications due to diabetes, and insulin-dependent diabetic with medical complications due to diabetes. ECD values (obtained through specular microscopy) and transplant suitability for endothelial transplantation (determined by the standard protocol of the eye bank) were compared among groups using linear mixed model analysis. RESULTS In total, 4185 corneas from 2112 donors were included for analysis. Insulin-dependent diabetic samples with medical complications due to diabetes (N = 231 from 119 donors) showed lower ECD values compared with nondiabetic samples (-102 cells/mm, P = 0.049) and non-insulin-dependent diabetic samples (-117 cells/mm, P = 0.031). ECD values did not differ significantly among the remaining groups. The likelihood of suitability for endothelial transplantation did not differ among all 4 groups. CONCLUSIONS Corneas from donors with insulin-dependent diabetes mellitus and medical complications resulting from the disease have lower mean ECD values compared with other donors. However, our analysis suggests that these corneas are equally likely to be included in the donor pool for corneal transplantation. Additional studies are needed to determine the mechanism(s) contributing to cell loss in donors with advanced diabetes and to assess associated endothelial cell functional impairment.

Purpose To determine the incidence of positive corneoscleral donor rim fungal cultures after keratoplasty and to report clinical outcomes of grafts with culture-positive donor rims. Design Retrospective cohort study. Participants Consecutive donor corneas and keratoplasty recipients at a single tertiary referral center over 20 years. Methods Patient charts were reviewed to determine the incidence of positive donor rim fungal cultures and clinical outcomes of all grafts using contaminated tissue. Main Outcome Measures The primary outcome measures were positive donor rim fungal culture results and the development of postkeratoplasty fungal infection using corresponding corneal tissue. The secondary outcome measure was the impact of postoperative prophylaxis on donor tissue-associated infections. Results A total of 3414 keratoplasty cases were included in the statistical analysis. Seventy-one cases (2.1%) were associated with a fungal culture-positive donor rim. Candida species were cultured in 40 cases (56.3%). There was a higher incidence of positive rim cultures over the last 5 years of the analytic period compared with the first 15 years ( P = 0.018). Fungal keratitis developed in 4 cases (5.6%), and all patients required further surgical intervention to achieve cure. There were no cases of fungal endophthalmitis. Empiric antimycotic prophylaxis initiated at the time of positive culture result reduced the incidence of keratitis from 15.8% in untreated cases to 1.9% in treated cases ( P = 0.056). Conclusions Positive donor rim fungal cultures are uncommon, but carry an unacceptably high risk of postoperative fungal infection. This risk may be reduced with prophylactic antimycotic therapy when culture-positive donor rims are identified.

Purpose: To compare Descemet membrane endothelial keratoplasty (DMEK) outcomes using nondiabetic grafts in diabetic and nondiabetic recipients. Methods: All eyes that underwent DMEK between February 2013 and October 2016 (follow-up ≥3 months, without prior keratoplasty) were included. Recipients were divided into diabetic (insulin dependent [IDDM] or noninsulin dependent [NIDDM]) and nondiabetic groups. Main outcome measures included postoperative visual acuity, rebubble procedure rates, and graft failure rates. Results: Of 334 eyes (243 subjects) included for analysis, 63 eyes (18.8%) were from diabetic recipients. At each timepoint, best-corrected visual acuity trended lower for IDDM recipients compared to NIDDM and nondiabetic recipients. There were no statistically significant differences in rebubble rates of diabetic compared to nondiabetic recipients (20.6% vs. 12.9%, p = 0.17), or IDDM compared to nondiabetic recipients (27.3% vs. 12.9%, p = 0.08; hazard ratio 2.26). Overall, 13 grafts (3.9%) failed (mean follow-up, 565 days; range, 90–1293 days). Graft failures did not differ between diabetic and nondiabetic recipients (4.0% vs. 4.9%, p = 0.15) regardless of subgroup (p = 0.36). Conclusions: DMEK provides excellent outcomes for patients with and without diabetes. DMEK outcomes were excellent with improvements in visual acuity and low rates of graft failure. Our findings were unable to determine differences between rebubble procedure rates but do emphasize the need for further research using stratified groups based on diabetes severity. Keywords: Corneal endothelium, DMEK, Diabetes mellitus, Descemet membrane, Endothelial keratoplasty, Corneal transplantation

The objective of this study was to characterize the proteome of the corneal endothelial cell layer and its basement membrane (Descemet membrane) in humans with various severities of type II diabetes mellitus compared to controls, and identify differentially expressed proteins across a range of diabetic disease severities that may influence corneal endothelial cell health. Endothelium-Descemet membrane complex tissues were peeled from transplant suitable donor corneas. Protein fractions were isolated from each sample and subjected to multidimensional liquid chromatography and tandem mass spectrometry. Peptide spectra were matched to the human proteome, assigned gene ontology, and grouped into protein signaling pathways unique to each of the disease states. We identified an average of 12,472 unique proteins in each of the endothelium-Descemet membrane complex tissue samples. There were 2,409 differentially expressed protein isoforms that included previously known risk factors for type II diabetes mellitus related to metabolic processes, oxidative stress, and inflammation. Gene ontology analysis demonstrated that diabetes progression has many protein footprints related to metabolic processes, binding, and catalysis. The most represented pathways involved in diabetes progression included mitochondrial dysfunction, cell-cell junction structure, and protein synthesis regulation. This proteomic dataset identifies novel corneal endothelial cell and Descemet membrane protein expression in various stages of diabetic disease. These findings give insight into the mechanisms involved in diabetes progression relevant to the corneal endothelium and its basement membrane, prioritize new pathways for therapeutic targeting, and provide insight into potential biomarkers for determining the health of this tissue.

The events that lead to choroidal neovascularization in eyes with age-related macular degeneration are poorly understood. One possibility that has been explored in a number of studies is that macrophages can promote neovascular changes. In this manuscript we summarize the evidence for inflammation in general and macrophages in particular in pathologic neovascularization, and discuss how the diverse functions of these cells may promote or inhibit macular disease. We also discuss some of the conflicting findings regarding the role of macrophages in experimental choroidal neovascularization in mouse models, and suggest areas for future research.

As tissue engineering and drug delivery applications increase in both number and complexity, the demand for new synthetic biocompatible polymers with precisely tailored properties grows accordingly. Block copolymers are a particularly promising biomaterial as the physical and physiological properties of these polymers can be closely controlled through manipulation of the type and organization of the blocks in the polymer's backbone. In this work, poly(ethylene glycol) (PEG) and poly(lactic acid) (PLA) were incorporated into PEG–PLA–PEG block macromonomers with (meth)acrylate functionality to form photopolymerizable, highly cross-linked polymers for potential use in a variety of biomedical applications. Simply by directing the PLA:PEG ratio in these macromonomers, the hydrophobicity, physical behavior, degradation, and biocompatibility of the resulting polymer were controlled. Specifically, it was found that by increasing the PLA:PEG ratio, the degree of water uptake and the mechanical strength of the material is significantly decreased, while the glass transition temperature and degradation of the PEG–PLA polymers are delayed. Additionally, the biocompatibility of the PEG–PLA polymers is significantly influenced by the chemical composition of the material as increased PLA generally yields greater cell compatibility. By demonstrating the versatility of the photopolymerizable PEG–PLA polymers, the results of this study indicate that these materials have the potential to serve as a synthetic biomaterial platform, in which the properties of the polymer can be tailored to a variety of tissue engineering or drug delivery applications.

Mutations that disrupt the coding sequence of Nav1.5, the cardiac Na+ channel encoded by SCN5A, cause inherited arrhythmias and cardiomyopathy. Changes in Nav1.5 splice isoforms, localization and function have also been associated with prognosis in acquired heart disease. However, the role of genetic variants in SCN5A gene regulatory sequences (e.g. those modulating expression) remains poorly understood. MicroRNAs (miRs) have emerged as vital gene regulators in heart, having roles in cardiogenesis, contractile function, and disease. These small non-coding RNAs are loaded into Argonaute proteins to direct post-transcriptional gene suppression by base-pairing with target transcripts, and genetic variants altering miR binding sequences may influence disease. Here, we used high-throughput biochemical means (HITS-CLIP) to generate the first transcriptome-wide map of miR binding events in human heart (4000 sites across >2200 genes). Our query of the data uncovered >500 common SNPs residing in or nearby miR target sites. Among these, we identified a synonymous SNP (rs1805126, T/C) which alters a conserved miR-24 binding site in the terminal coding exon of SCN5A. We hypothesized that this SNP affects Nav1.5 expression and contributes to clinical outcomes in heart failure. In vitro experiments showed that miR-24 suppresses Na+ current on patch clamp analysis (~5-fold; p1800 subjects with EFs≤30% and implantable cardioverter defibrillators (ICDs) from GRADE, a prospective multicenter study of genetic modulators of heart failure outcomes. We found that CC homozygotes had worse survival rates than T-allele carriers (HR=1.5, p=0.001), but no change in appropriate ICD shocks, a surrogate for sudden death. Together, these data reveal a novel miR-24:SCN5A:SNP regulatory axis that points to a surprising link between cardiac Na+ channel levels and non-arrhythmic death in heart failure. Overall, this work represents a key advance in exploring the interface of cardiac miRs with genetics and disease, perhaps signifying a broader functional relevance for “silent” SNPs.

Background The purpose of this project was to identify short hairpin RNA (shRNA) sequences that can suppress expression of human CAPN5 in which gain-of-function mutants cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). We created HEK293T cells that stably express an ADNIV disease allele, CAPN5-p.R243L. Transfection protocols were optimized for neuroblastoma SHSY5Y cells. The gene silencing effect of four different shRNA plasmids that target CAPN5 was tested. RNA and protein expression was determined using quantitative RT-PCR and immunoblot analysis. Findings Two of four shRNA plasmids reduced mutant CAPN5 RNA in a stable cell line. Similar knockdown was observed in SH-SY5Y cells that natively express CAPN5. Lactose dehydrogenase assays showed that down-regulation of CAPN5 was not cytotoxic. Conclusions CAPN5 expression can be suppressed by shRNA-based RNA interference. Further testing in ADNIV models will determine the potential of gene silencing as a strategy to treat, delay, or prevent blindness in ADNIV patients.

Importance Differences in geographical protein expression in the human choroid–retinal pigment epithelial (RPE) complex may explain molecular predisposition of regions to ophthalmic diseases such as age-related macular degeneration. Objective To characterize the proteome of the human choroid-RPE complex and to identify differentially expressed proteins in specific anatomic regions. Design, Setting, and Participants Experimental study of choroid-RPE tissue from 3 nondiseased eyes. The choroid-RPE complex underwent biopsy from beneath the foveal, macular, and peripheral retina. Protein fractions were isolated and subjected to multidimensional liquid chromatography and tandem mass spectrometry. A bioinformatic pipeline matched peptide spectra to the human proteome, assigned gene ontology classification, and identified protein signaling pathways unique to each of the choroid-RPE regions. Main Outcomes and Measures Mean number of mass spectra, statistically significant differentially expressed proteins, gene ontology classification, and pathway representation. Results We identified a mean of 4403 unique proteins in each of the foveal, macular, and peripheral choroid-RPE tissues. Six hundred seventy-one differentially expressed proteins included previously known risk factors for retinal diseases related to oxidative stress, inflammation, and the complement cascade. Gene ontology analysis showed that unique categories in the foveal and macular regions included immune process proteins as well as protein complexes and plasma membrane proteins. The peripheral region contained unique antioxidant activity proteins. Many proteins had the highest expression in the foveal or macular regions, including inflammation-related proteins HLA-A, HLA-B, and HLA-C antigens; intercellular adhesion molecule 1 (ICAM-1); S100; transcription factor ERG; antioxidant superoxide dismutase 1 (SOD1); chloride intracellular channel 6 ion (CLIC6); activators of the complement cascade C1q, C6, and C8; and complement factor H. Proteins with higher expression in the periphery included bestrophin 1 (BEST1), transcription factor RNA binding motif protein 39 (RBM39), inflammatory mediator macrophage migration inhibitory factor, antioxidant SOD3, ion channel voltage-dependent anion-selective channel protein 3 (VDAC3), and complement inhibitor CD55. The complement activation was among the highest represented pathways ( P Conclusions and Relevance This proteomic data set identifies novel molecular signatures in anatomically sensitive regions of the choroid-RPE complex. The findings give mechanistic insight into choroid-RPE function, reveal important choroid-RPE processes, and prioritize new pathways for therapeutic targeting.

Exome sequencing indicated that the gene encoding the calpain-5 protease, CAPN5, is the likely cause of retinal degeneration and autoimmune uveitis in human patients with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). To explore the mechanism of ADNIV, a human CAPN5 disease allele was expressed in mouse retinas with a lentiviral vector created to express either the wild-type human (h) CAPN5 or the ADNIV mutant hCAPN5-R243L allele under a rhodopsin promoter with tandem green fluorescent protein (GFP) expression. Vectors were injected into the subretinal space of perinatal mice. Mouse phenotypes were analyzed using electroretinography, histology and inflammatory gene expression profiling. Mouse calpain-5 showed high homology to its human ortholog with >98% sequence identity that includes the ADNIV mutant residue. Calpain-5 protein was expressed in the inner and outer segments of the photoreceptors and in the outer plexiform layer. Expression of the hCAPN5-R243L allele caused loss of the electroretinogram b-wave, photoreceptor degeneration and induction of immune cell infiltration and inflammatory genes in the retina, recapitulating major features of the ADNIV phenotype. Intraocular neovascularization and fibrosis were not observed during the study period. Our study shows that expression of the hCAPN5-R243L disease allele elicits an ADNIV-like disease in mice. It further suggests that ADNIV is due to CAPN5 gain-of-function rather than haploinsufficiency, and retinal expression may be sufficient to generate an autoimmune response. Genetic models of ADNIV in the mouse can be used to explore protease mechanisms in retinal degeneration and inflammation as well as preclinical therapeutic testing.

Proteomic analysis is important in the examination of complex extracellular matrices such as the vitreous where several tissues both inside the eye and remote to the eye contribute to the diseased state. In these cases, genomic analysis of local tissue gene expression may be insufficient or misleading. By switching the emphasis from diagnostic biomarkers to biomarkers with therapeutic potential, we can create rationale therapeutic strategies for blinding vitreoretinal diseases. The same strategy can extend to other ocular tissues. We are just beginning to understand the molecular constituents of the vitreous in health and disease, and translational proteomics may more effectively direct efforts to cure blindness.

Biorepositories are collections of surgically obtained human tissues for current and future investigations of disease mechanisms, therapeutics, and diagnostics. In ophthalmology, a critical challenge is how to interface the operating room with the laboratory. To attain standards required for basic research, clinical and research teams must cooperate to collect, annotate, and store specimens that yield consistent results required for advanced molecular techniques. We developed an efficient platform for obtaining vitreous and other eye tissues from the operating room and transferring them to the lab. The platform includes a mobile lab cart for on-site tissue processing, a multi-user, web-based database for point-of-care phenotypic capture, and an integrated data tracking system for long-term storage. These biorepository instruments have proven essential for our studies in ophthalmic disease proteomics. This system can be implemented in other operating rooms and laboratories for a variety of biological tissues.

We performed whole-exome sequencing of a family with autosomal dominant Dandy-Walker malformation and occipital cephaloceles (ADDWOC) and detected a mutation in the extracellular matrix protein encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1 binding partner. Structural modeling the NID1-LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the extracellular matrix in the pathogenesis of Dandy-Walker spectrum disorders.

MicroRNAs (miRs) have emerged as key biological effectors in human health and disease. These small noncoding RNAs are incorporated into Argonaute (Ago) proteins, where they direct post-transcriptional gene silencing via base-pairing with target transcripts. Although miRs have become intriguing biological entities and attractive therapeutic targets, the translational impacts of miR research remain limited by a paucity of empirical miR targeting data, particularly in human primary tissues. Here, to improve our understanding of the diverse roles miRs play in cardiovascular function and disease, we applied high-throughput methods to globally profile miR:target interactions in human heart tissues. We deciphered Ago2:RNA interactions using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts. Our initial exploration of this interactome revealed an abundance of miR target sites in gene coding regions, including several sites pointing to new miR-29 functions in regulating cardiomyocyte calcium, growth and metabolism. Also, we uncovered several clinically-relevant interactions involving common genetic variants that alter miR targeting events in cardiomyopathy-associated genes. Overall, these data provide a critical resource for bolstering translational miR research in heart, and likely beyond.

Purpose: To compare vitreous biopsy methods using analysis platforms used in proteomics biomarker discovery. Methods: Vitreous biopsies from 10 eyes were collected sequentially using a 23-gauge needle and a 23-gauge vitreous cutter instrument. Paired specimens were evaluated by UV absorbance spectroscopy, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Results: The total protein concentration obtained with a needle and vitrectomy instrument biopsy averaged 1.10 mg/mL (standard error of the mean = 0.35) and 1.13 mg/mL (standard error of the mean = 0.25), respectively. In eight eyes with low or medium viscidity, there was a very high correlation (R2 = 0.934) between the biopsy methods. When data from 2 eyes with high viscidity vitreous were included, the correlation was reduced (R2 = 0.704). The molecular weight protein sodium dodecyl sulfate–polyacrylamide gel electrophoresis profiles of paired needle and vitreous cutter samples were similar, except for a minority of pairs with single band intensity variance. Using LC-MS/MS, equivalent peptides were identified with similar frequencies (R2 ≥ 0.90) in paired samples. Conclusion: Proteins and peptides collected from vitreous needle biopsies are nearly equivalent to those obtained from a vitreous cutter instrument. This study suggests both techniques may be used for most proteomic and biomarker discovery studies of vitreoretinal diseases, although a minority of proteins and peptides may differ in concentration.

Neovascular AMD involves the activation of choroidal endothelial cells to increase their inflammatory and angiogenic behaviors. Elastin derived peptides (EDPs) can elicit some of these phenotypic changes in endothelial cells. This investigation was performed to follow up on those findings by determining a receptor for these peptides in the human eye as well as evaluating the effects of elevated EDPs on choroidal cells in vitro and in vivo.The expression of elastin receptor genes including GLB1 was analyzed using reverse transcription PCR. Migration of choroidal endothelial cells was quantified in the presence of inhibitors to different EDP binding proteins. C57BL6 mice were injected with EDPs and studied by electroretinography, transmission electron microscopy, and microarray analysis.An alternatively spliced form of beta-galactosidase (GLB1) is present on human choroidal endothelial cells and acts as a receptor for EDPs. Elevated levels of circulating EDPs do not affect retinal function in the mouse, but do increase the expression and deposition of collagen IV in the RPE/choroid complex.EDPs may play a role in neovascular AMD by binding to and inducing neovascular phenotypes in choroidal endothelial cells through their receptor, GLB1. These peptides also cause an increased mRNA expression and deposition of collagen IV in the RPE/choroid, which may alter diffusion properties between the retina and choriocapillaris.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.(1-5) Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. (6-9) An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

Age-related macular degeneration (AMD) is a common, potentially blinding disorder affecting the aged. In advanced stages of the disease, vision in the macular region can be severely compromised by either atrophic changes in the retinal pigment epithelium (RPE), overlying retina, and underlying choriocapillaris or compromise of Bruch's membrane and subsequent invasion of choroidal endothelial cells into the subretinal space, with severe consequences for vision.1,2 Although outcomes are more favorable in patients with neovascular disease than was previously possible,3 a better understanding of cellular events that occur in the earliest stages of disease will lead to improved future therapies. Clinically, the earliest stages of AMD are characterized by changes in the evenness of the RPE pigmentation and the observation of deposits in Bruch's membrane, termed drusen.4–6 Because drusen and other sub-RPE deposits (basal laminar deposit and basal linear deposit, also referred to as diffuse drusen) are diagnostic and predictive of AMD,7 there has been considerable interest in defining the composition of sub-RPE deposits as well as understanding their origin. Immunohistochemical8–11 and biochemical12,13 studies performed on human donor eyes with drusen have identified an array of molecular species associated with these deposits, including inflammatory mediators and lipids of retinal and choroidal origin. Interestingly, the formation of drusen does not appear to be spatially random but is influenced by the anatomy of the underlying choroid. Friedman noted in whole mounts that drusen were likely to develop over what were interpreted as collecting venules of the choroid.14 It was further noted by Sarks et al.15 that the smallest, earliest drusen were likely to accumulate over choriocapillary pillars at what were called “entrapment sites.” The relationship of drusen to vessel walls, rather than lumens, was confirmed in a series of whole mounted choroids by Lengyel et al.,16 which is further supported by findings of RPE protein deposition in Bruch's membrane at pillar sites.17 The mechanism(s) driving sub-RPE deposit formation, and the basis for the predilection of these deposits to form in areas over intercapillary pillars or devoid of capillary lumens, is unknown. One might speculate that, if drusen preferentially develop over regions of capillary lumen-depleted choroid, reduced vascular perfusion may contribute to drusen formation. To further understand the relationship between drusen and other sub-RPE deposits with the choriocapillaris, we performed a series of morphometric experiments in which we assessed the relationship of the vasculature with sub-RPE deposits in the human macula. The density of pathologic deposits was strongly linked to the density of the choroidal vasculature, with eyes having the most drusen showing the lowest vascular density. We also found that choroidal “ghost” vessels, remnants of previously healthy capillaries, increased in abundance with increasing drusen density. We discuss these findings in the context of drusen biogenesis and the pathophysiology of AMD.

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. (1,2) Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. (1,2) The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

The complement system plays a crucial role in the progression of age-related macular degeneration (AMD). In this study, the authors sought to evaluate the pathophysiologic roles of complement components C3a and C5a in the human choroid in AMD.Human RPE/choroid was assayed for the presence of C3a and C5a receptors (C3aR and C5aR) using RT-PCR and immunohistochemistry. Choroidal endothelial cell migration and proliferation were evaluated in the presence of C5a. Organ cultures of human choroid were incubated in C5a or bovine serum albumin (BSA) followed by quantitative immunohistochemistry and quantitative PCR for ICAM-1. AMD patients and controls were genotyped at SNPs in the C5R1 and C3AR1 genes.C5aR, but not C3aR, was detected in human choroid. C5a did not promote endothelial cell migration or proliferation. However, choriocapillaris endothelial cells in organ culture responded to C5a by increasing ICAM-1 mRNA and protein. No significant association of SNP genotypes was detected in AMD patients at the C3AR1 and C5R1 genes.The generation of C5a peptides may lead to activation of choriocapillaris endothelial cells in AMD. Activation of the choroidal endothelium may affect the progression of AMD by recruitment of monocytes, leading to additional sequelae of AMD pathogenesis.

Background Age-related macular degeneration (AMD) is a common disease of the elderly that leads to loss of the central visual field due to atrophic or neovascular events. Evidence from human eyes and animal models suggests an important role for macrophages and endothelial cell activation in the pathogenesis of AMD. We sought to determine whether common ancestral variants in genes encoding the selectin family of proteins are associated with AMD. Methods Expression of E-selectin, L-selectin and P-selectin was examined in choroid and retina by quantitative PCR and immunofluorescence. Samples from patients with AMD (n = 341) and controls (n = 400) were genotyped at a total of 34 SNPs in the SELE, SELL and SELP genes. Allele and genotype frequencies at these SNPs were compared between AMD patients and controls as well as between subtypes of AMD (dry, geographic atrophy, and wet) and controls. Results High expression of all three selectin genes was observed in the choroid as compared to the retina. Some selectin labeling of retinal microglia, drusen cores and the choroidal vasculature was observed. In the genetic screen of AMD versus controls, no positive associations were observed for SELE or SELL. One SNP in SELP (rs3917751) produced p-values < 0.05 (uncorrected for multiple measures). In the subtype analyses, 6 SNPs (one in SELE, two in SELL, and three in SELP) produced p-values < 0.05. However, when adjusted for multiple measures with a Bonferroni correction, only one SNP in SELP (rs3917751) produced a statistically significant p-value (p = 0.0029). Conclusions This genetic screen did not detect any SNPs that were highly associated with AMD affection status overall. However, subtype analysis showed that a single SNP located within an intron of SELP (rs3917751) is statistically associated with dry AMD in our cohort. Future studies with additional cohorts and functional assays will clarify the biological significance of this discovery. Based on our findings, it is unlikely that common ancestral variants in the other selectin genes (SELE and SELL) are risk factors for AMD. Finally, it remains possible that sporadic or rare mutations in SELE, SELL, or SELP have a role in the pathogenesis of AMD.

To determine the safety and efficacy of a femtosecond laser (IntraLase) and manual microkeratome (Moria ALTK) in creating precut endothelial keratoplasty donor tissue.Sixteen corneoscleral buttons from 8 donors were evaluated within 2 days of the death of the donor. The mean donor age was 72 years, and mean death-to-preservation time was 11 hours. Eight eyes underwent deep lamellar keratectomy by using the femtosecond laser (IntraLase: firing rate, 30 kHz; lamellar cut energy, 7.4 microJ; side cut energy, 5.5 microJ; spot size, 10 microm; diameter, 9.0 mm; depth, 400 microm; spiral pattern), whereas the other 8 eyes were cut by using the Moria ALTK microkeratome (350-microm head). Ultrasonic pachymetry and endothelial cell density (ECD) were performed before and after keratectomy. The residual stromal bed was examined with electron microscopy to determine the smoothness of the surface. Cell viability was assessed by using a transferase dUTP nick end labeling (TUNEL) assay.The mean preoperative pachymetry was similar in the microkeratome group and femtosecond laser group (P = 0.239). The microkeratome group obtained a consistently deeper keratectomy of 446 +/- 25 versus 400 +/- 41 microm in the laser group (P = 0.023). Similarly, the residual stromal bed was thinner in the microkeratome group (115 +/- 28.5 vs. 177 +/- 42 microm; P = 0.005). There was no statistically significant difference in the ECD between the 2 groups preoperatively or at 48 hours after keratectomy. Compared with the preoperative state, there was a 1% and 4% reduction of ECD in the microkeratome and femtosecond laser groups, respectively. Scanning electron microscopy of the stromal surface consistently showed a smoother contour in the manual microkeratome group. TUNEL assays indicate no significant endothelial cell loss in either the microkeratome group or the femtosecond laser group.The femtosecond laser (30 kHz) and the manual microkeratome are equally effective in creating precut endothelial keratoplasty donor tissue, with no detrimental effect on endothelial cell density. The microkeratome creates a smoother stromal surface and thinner endothelial discs. The femtosecond laser lamellar dissection depth is less deep, and the stromal surface is less smooth. This particular feature of femtosecond laser keratectomy may improve disc adherence, which continues to be a problem in endothelial keratoplasty. A prospective, randomized study is needed to evaluate postoperative vision and disc adherence by using both technologies in endothelial keratoplasty.

Idiopathic macular holes are full-thickness neurosensory retinal defects in the fovea that typically result in moderate to severe vision loss.1–3 Central to the pathogenesis of macular holes is anomalous vitreomacular traction and incomplete posterior vitreous detachment, which often leaves remnants of the vitreous cortex on the internal limiting membrane (ILM) surface.4–6 Vitrectomy is a highly successful procedure for achieving macular hole closure and improved visual acuity.7–11 As surgical methods have evolved to include gas tamponade, epiretinal membrane (ERM) peeling, and ILM peeling, rates of macular hole closure now exceed 90%, with low recurrence rates in most reported series.12–16 Various surgical techniques involve peeling the ILM from the superficial retina. Most use creation of a defect in the ILM, grasping an exposed edge with intraocular forceps, and extending a circumferential tear around the hole. Alternatively, the ILM can be directly grasped with forceps and peeled from the retinal surface. Dissection of the transparent 10-μm-thick ILM is a surgically challenging step and may be facilitated by the use of adjuvant dyes such as indocyanine green to improve visualization and surgical success.17 Adjuvant stains may also alter the ILM structure and assist in creating a cleavage plane between the ILM and underlying retinal layers.18 Application of dyes, however, may be toxic to the retinal cells and/or retinal pigment epithelium, resulting in reduced postoperative visual acuity despite successful hole closure.19 In this report, we describe a technique of ILM abrasion performed to address elements of tangential traction on the retinal surface to achieve successful macular hole closure without complete removal of the ILM. Such a technique may limit the loss of adjacent tissue of the inner retina and eliminate the risk of adjuvant dye toxicity. The technique uses a Tano diamond-dusted membrane scraper (DDMS; Synergetics, Inc., O'Fallon, MO, USA) to abrade the ILM.20 Recently, we showed a 94% success rate in 100 eyes that underwent ILM abrasion with a DDMS as an alternative to traditional ILM peeling at the time of macular hole repair.21 Abrasion of the ILM with a DDMS at the time of pars plana vitrectomy is sufficient to achieve high rates of macular hole closure, and this technique avoids complete removal of the retinal ILM basement membrane and subjacent tissues and provides hole closure rates that are comparable to those in traditional ILM peeling.21 In this study, we used two methods to ascertain the effect of ILM abrasion on the retina, and we describe the immunohistochemical characteristics of retinal tissue removed by the DDMS from surgical specimens with macular holes. We also used electron microscopy to examine the retinal tissue remaining in human donor eyes with macular holes following the same ILM abrasion technique.

In order to understand the extent of choriocapillary endothelial cell activation in different topographic regions of the eye, we sought to compare the localization of intercellular adhesion molecule-1 (ICAM-1) in macular and peripheral regions of human eyes.Sections of sucrose-embedded human donor eyes that included the macula and ora serrata were evaluated for ICAM-1 and ICAM-2 immunoreactivity with monoclonal and polyclonal antibodies. Patterns of ICAM-1 labeling in peripheral and macular regions were examined in 20 eyes. Morphometric analyses of anti-ICAM-1 labeling intensity in the choriocapillaris were performed using ImageJ software on a series of macular and extramacular punches from nine eyes. Quantitative PCR analysis for ICAM-1 mRNA was performed on the RPE-choroid from the same regions from six of the same eyes, and Western blots of samples treated or untreated with N-glycosidase were performed to compare retinal and choroidal ICAM-1.ICAM-1 labeling of the choriocapillaris was typically more intense in the macula than in the peripheral choroid in human donor eyes (14/20). ICAM-2 was also detected in the choriocapillaris and retinal vessels. Morphometric measurements confirmed a significant macular-extramacular difference in ICAM-1 in six of nine eyes (p0.05), with 1 of 9 eyes showing the opposite pattern. This pattern was not noted for endogenous alkaline phosphatase or ICAM-2. The opposite pattern was noted in the external limiting membrane (ELM), which exhibited more intense ICAM-1 labeling in the far periphery than in the macula. On Western blots, choroidal ICAM-1 exhibited a greater molecular weight than the retinal form, with most of the apparent weight difference due to N-linked carbohydrate chains.The regional differences in ICAM-1 distribution in the choriocapillaris may indicate that this region is subject to increased leukocyte trafficking. In view of the role of inflammatory processes in age-related macular degeneration (AMD), we propose that the higher level of ICAM-1 protein in the macular choriocapillaris may impart greater susceptibility of the macula to immune cell-mediated damage in AMD.

Choroidal neovascularization (CNV) is a complication of multiple eye diseases, including age-related macular degeneration, that usually results in irreversible vision loss. It is characterized by proliferation and growth of choroidal blood vessels through Bruch's membrane into the subpigment epithelial and/or subretinal space. The purpose of this study was to characterize the carbohydrate groups associated with CNV by lectin histochemistry.Frozen sections from three human eyes with CNV (two fixed eyes and one unfixed eye) were prepared. Sections containing choroidal neovascular membranes were incubated with a battery of biotinylated lectins directed against a number of distinct oligosaccharide moieties. Lectin labeling of the vessels in CNV was visualized with avidin-Texas red.Several carbohydrate groups were preferentially associated with the vascular elements in CNV. Glycoconjugates that react with lectins derived from wheat germ, soybean, and hairy vetch seed (sWGA, SBA, and VVA, respectively) all showed reactivity with CNV vessels that was higher than the labeling of the surrounding matrix. SBA and sWGA also reacted with CNV vessels at low concentrations at which normal retinal and choroidal vessels were largely unlabeled.Choroidal neovascular membranes possess a distinct set of carbohydrate moieties. These data may be valuable in understanding endothelial cell biology in CNV.

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is an autoimmune condition of the eye that sequentially mimics uveitis, retinitis pigmentosa, and proliferative diabetic retinopathy as it progresses to complete blindness. We identified two different missense mutations in the CAPN5 gene in three ADNIV kindreds. CAPN5 encodes calpain-5, a calcium-activated cysteine protease that is expressed in retinal photoreceptor cells. Both mutations cause mislocalization from the cell membrane to the cytosol, and structural modeling reveals that both mutations lie within a calcium-sensitive domain near the active site. CAPN5 is only the second member of the large calpain gene family to cause a human Mendelian disorder, and this is the first report of a specific molecular cause for autoimmune eye disease. Further investigation of these mutations is likely to provide insight into the pathophysiologic mechanisms of common diseases ranging from autoimmune disorders to diabetic retinopathy., Author Summary We care for several families with an inherited form of autoimmune inflammation inside the eye. The patients also develop bleeding, scar tissue, and eventually blindness. Using advanced gene analysis methods, we discovered the cause of this disease is gene mutations in the CAPN5 gene. This gene makes a protein, calpain-5, which belongs to a family of calcium-activated enzymes that slice other proteins inside cells. Calpain-5 is expressed in the retina, and the disease mutations alter its location inside the cell. Future studies to understand how this protein causes inflammation and bleeding inside the eye will help us develop treatments for this condition and more common eye diseases with inflammation and bleeding.

Age-related macular degeneration (AMD) is a major cause of blindness. The neovascular (wet) form of AMD is characterized by the abnormal growth of choroidal blood vessels into the sub-retinal space of the macula. This multistep process is likely to be initiated by the breakdown of Bruch's membrane which, when intact, prevents pathologic angiogenesis. In this process, choroidal ECs may migrate from the choroid into the sub-RPE and/or sub-retinal space. These ECs proliferate and form tubes (tubulogenesis), ultimately reorganizing their junctions to increase permeability across the newly formed vascular wall. The neovascular process in AMD can result in serous detachment of the retinal pigmented epithelium (RPE) and/or neurosensory retinal detachment, as well as fibrous disciform scarring beneath the retina, causing a catastrophic decrease in visual acuity.1-5 Current treatments for neovascular AMD are focused primarily on vascular endothelial growth factor (VEGF)-mediated processes.6 While VEGF is a potent inducer of angiogenesis, understanding the role of additional angiogenic stimuli would be invaluable for the development of improved treatments.7 Elastin is a glycoprotein consisting of cross-linked 72 kDa tropoelastin subunits and is an abundant component of the extracellular matrix (ECM) of arteries, lung, and skin.8 Breakdown of elastin results in the formation of elastin-derived peptides (EDPs), cross-linked fragments of tropoelastin of varying sizes.9 These subunits have been shown to initiate EC migration and tubulogenesis in chicken chorio-allantoic membranes, human microvascular ECs (cell line HMEC-1), and human umbilical vein ECs.10 Currently, it is unknown how these peptides are able to activate ECs and whether they are capable of activating ECs from other tissues, such as the choroid. It is plausible that EDPs bind to elastin binding proteins on the cell surface, inducing angiogenic behaviors such as cell migration and/or proliferation. Several lines of evidence suggest abnormal elastin metabolism occurs in Bruch's membrane in AMD. First, early onset choroidal neovascularization has been shown in patients with pseudoxanthoma elasticum. These patients may be at risk for developing choroidal neovascular membranes because of abnormalities in the elastic layer of Bruch's membrane, including breaks, clinically defined as angioid streaks.11,12 Second, fibulin-5 missense mutations have been identified in association with AMD.13 This mutation may contribute to AMD development by affecting the elastic layer of Bruch's membrane, since fibulin-5 participates in elastogenesis14,15 and is localized to Bruch's membrane in human eyes.16 Third, correlations between the integrity of the elastic layer of Bruch's membrane and AMD have also been made. Light and electron microscopic studies of human eyes have found that Bruch's membrane becomes calcified and increasingly fragmented with increasing severity of AMD.17,18 It is likely that fragmentation of Bruch's membrane in the macula renders it more susceptible to the formation of neovascular membranes. There is also evidence for abnormal systemic elastin metabolism in AMD. Blumenkranz et al.11 found a correlation between choroidal neovascularization (CNV) and elastotic degeneration. Patients with exudative AMD demonstrated a greater than twofold increase in their susceptibility to elastotic degeneration of relatively sun-protected areas of the skin in dermal biopsies, suggesting that AMD is associated with systemic elastin abnormalities.11 Serum levels of EDPs in patients with exudative AMD have also been found to be significantly higher than levels in non-exudative AMD patients and control patients,19 which may possibly be due to elevated levels of MMP-9.20 Thus, numerous lines of evidence suggest a relationship between altered elastin metabolism and AMD progression. It is feasible that EDPs, derived from serum and/or fragmentation of Bruch's membrane, could promote the pathogenesis observed in AMD. One interpretation of these findings is that systemic and local defects in elastin physiology lead to a compromised barrier for CNV in Bruch's membrane. A second, non-exclusive, possibility is that fragments of elastin itself, generated by systemic or local elastolysis, promote angiogenesis in choroidal ECs. To clarify the functional impact of elastin on the severity of exudative AMD, we sought to determine whether EDPs promote an angiogenic phenotype in choroidal ECs. During our investigation, we found that EDPs are capable of promoting choroidal EC migration. These results support the idea that abnormal elastin metabolism in AMD patients promotes disease progression toward neovascular membrane formation.