Lam JD, Oh DJ, Wong LL, Amarnani D, Park-Windhol C, Sanchez AV, Cardona-Velez J, McGuone D, Stemmer-Rachamimov AO, Eliott D, Bielenberg DR, van Zyl T, Shen L, Gai X, D'Amore PA, Kim LA, Arboleda-Velasquez JF. Identification of RUNX1 as a Mediator of Aberrant Retinal Angiogenesis. Diabetes 2017;66(7):1950-1956.Abstract
Proliferative diabetic retinopathy (PDR) is a common cause of blindness in the developed world's working adult population and affects those with type 1 and type 2 diabetes. We identified Runt-related transcription factor 1 (RUNX1) as a gene upregulated in CD31(+) vascular endothelial cells obtained from human PDR fibrovascular membranes (FVMs) via transcriptomic analysis. In vitro studies using human retinal microvascular endothelial cells (HRMECs) showed increased RUNX1 RNA and protein expression in response to high glucose, whereas RUNX1 inhibition reduced HRMEC migration, proliferation, and tube formation. Immunohistochemical staining for RUNX1 showed reactivity in vessels of patient-derived FVMs and angiogenic tufts in the retina of mice with oxygen-induced retinopathy, suggesting that RUNX1 upregulation is a hallmark of aberrant retinal angiogenesis. Inhibition of RUNX1 activity with the Ro5-3335 small molecule resulted in a significant reduction of neovascular tufts in oxygen-induced retinopathy, supporting the feasibility of targeting RUNX1 in aberrant retinal angiogenesis.
Olivares AM, Jelcick AS, Reinecke J, Leehy B, Haider A, Morrison MA, Cheng L, Chen DF, DeAngelis MM, Haider NB. Multimodal Regulation Orchestrates Normal and Complex Disease States in the Retina. Sci Rep 2017;7(1):690.Abstract

Regulation of biological processes occurs through complex, synergistic mechanisms. In this study, we discovered the synergistic orchestration of multiple mechanisms regulating the normal and diseased state (age related macular degeneration, AMD) in the retina. We uncovered gene networks with overlapping feedback loops that are modulated by nuclear hormone receptors (NHR), miRNAs, and epigenetic factors. We utilized a comprehensive filtering and pathway analysis strategy comparing miRNA and microarray data between three mouse models and human donor eyes (normal and AMD). The mouse models lack key NHRS (Nr2e3, RORA) or epigenetic (Ezh2) factors. Fifty-four total miRNAs were differentially expressed, potentially targeting over 150 genes in 18 major representative networks including angiogenesis, metabolism, and immunity. We identified sixty-eight genes and 5 miRNAS directly regulated by NR2E3 and/or RORA. After a comprehensive analysis, we discovered multimodal regulation by miRNA, NHRs, and epigenetic factors of three miRNAs (miR-466, miR1187, and miR-710) and two genes (Ell2 and Entpd1) that are also associated with AMD. These studies provide insight into the complex, dynamic modulation of gene networks as well as their impact on human disease, and provide novel data for the development of innovative and more effective therapeutics.

Islam R, Eidet JR, Badian RA, Lippestad M, Messelt E, Griffith M, Dartt DA, Utheim TP. Tissue Harvesting Site and Culture Medium Affect Attachment, Growth, and Phenotype of Ex Vivo Expanded Oral Mucosal Epithelial Cells. Sci Rep 2017;7(1):674.Abstract

Transplantation of cultured oral mucosal epithelial cells (OMECs) is a promising treatment strategy for limbal stem cell deficiency. In order to improve the culture method, we investigated the effects of four culture media and tissue harvesting sites on explant attachment, growth, and phenotype of OMECs cultured from Sprague-Dawley rats. Neither choice of media or harvesting site impacted the ability of the explants to attach to the culture well. Dulbecco's modified Eagle's medium/Ham's F12 (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI) supported the largest cellular outgrowth. Fold outgrowth was superior from LL explants compared to explants from the buccal mucosa (BM), HP, and transition zone of the lower lip (TZ) after six-day culture. Putative stem cell markers were detected in cultures grown in DMEM and RPMI. In DMEM, cells from TZ showed higher colony-forming efficiency than LL, BM, and HP. In contrast to RPMI, DMEM both expressed the putative stem cell marker Bmi-1 and yielded cell colonies. Our data suggest that OMECs from LL and TZ cultured in DMEM give rise to undifferentiated cells with high growth capacity, and hence are the most promising for treatment of limbal stem cell deficiency.

Paschalis EI, Zhou C, Lei F, Scott N, Kapoulea V, Robert M-C, Vavvas D, Dana R, Chodosh J, Dohlman CH. Mechanisms of Retinal Damage after Ocular Alkali Burns. Am J Pathol 2017;187(6):1327-1342.Abstract
Alkali burns to the eye constitute a leading cause of worldwide blindness. In recent case series, corneal transplantation revealed unexpected damage to the retina and optic nerve in chemically burned eyes. We investigated the physical, biochemical, and immunological components of retinal injury after alkali burn and explored a novel neuroprotective regimen suitable for prompt administration in emergency departments. Thus, in vivo pH, oxygen, and oxidation reduction measurements were performed in the anterior and posterior segment of mouse and rabbit eyes using implantable microsensors. Tissue inflammation was assessed by immunohistochemistry and flow cytometry. The experiments confirmed that the retinal damage is not mediated by direct effect of the alkali, which is effectively buffered by the anterior segment. Rather, pH, oxygen, and oxidation reduction changes were restricted to the cornea and the anterior chamber, where they caused profound uveal inflammation and release of proinflammatory cytokines. The latter rapidly diffuse to the posterior segment, triggering retinal damage. Tumor necrosis factor-α was identified as a key proinflammatory mediator of retinal ganglion cell death. Blockade, by either monoclonal antibody or tumor necrosis factor receptor gene knockout, reduced inflammation and retinal ganglion cell loss. Intraocular pressure elevation was not observed in experimental alkali burns. These findings illuminate the mechanism by which alkali burns cause retinal damage and may have importance in designing therapies for retinal protection.
Konstantinou EK, Notomi S, Kosmidou C, Brodowska K, Al-Moujahed A, Nicolaou F, Tsoka P, Gragoudas E, Miller JW, Young LH, Vavvas DG. Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity. Sci Rep 2017;7:46581.Abstract

Verteporfin (VP) was first used in Photodynamic therapy, where a non-thermal laser light (689 nm) in the presence of oxygen activates the drug to produce highly reactive oxygen radicals, resulting in local cell and tissue damage. However, it has also been shown that Verteporfin can have non-photoactivated effects such as interference with the YAP-TEAD complex of the HIPPO pathway, resulting in growth inhibition of several neoplasias. More recently, it was proposed that, another non-light mediated effect of VP is the formation of cross-linked oligomers and high molecular weight protein complexes (HMWC) that are hypothesized to interfere with autophagy and cell growth. Here, in a series of experiments, using human uveal melanoma cells (MEL 270), human embryonic kidney cells (HEK) and breast cancer cells (MCF7) we showed that Verteporfin-induced HMWC require the presence of light. Furthermore, we showed that the mechanism of this cross-linking, which involves both singlet oxygen and radical generation, can occur very efficiently even after lysis of the cells, if the lysate is not protected from ambient light. This work offers a better understanding regarding VP's mechanisms of action and suggests caution when one studies the non-light mediated actions of this drug.

Michalak SM, Whitman MC, Park JG, Tischfield MA, Nguyen EH, Engle EC. Ocular Motor Nerve Development in the Presence and Absence of Extraocular Muscle. Invest Ophthalmol Vis Sci 2017;58(4):2388-2396.Abstract

Purpose: To spatially and temporally define ocular motor nerve development in the presence and absence of extraocular muscles (EOMs). Methods: Myf5cre mice, which in the homozygous state lack EOMs, were crossed to an IslMN:GFP reporter line to fluorescently label motor neuron cell bodies and axons. Embryonic day (E) 11.5 to E15.5 wild-type and Myf5cre/cre:IslMN:GFP whole mount embryos and dissected orbits were imaged by confocal microscopy to visualize the developing oculomotor, trochlear, and abducens nerves in the presence and absence of EOMs. E11.5 and E18.5 brainstems were serially sectioned and stained for Islet1 to determine the fate of ocular motor neurons. Results: At E11.5, all three ocular motor nerves in mutant embryos approached the orbit with a trajectory similar to that of wild-type. Subsequently, while wild-type nerves send terminal branches that contact target EOMs in a stereotypical pattern, the Myf5cre/cre ocular motor nerves failed to form terminal branches, regressed, and by E18.5 two-thirds of their corresponding motor neurons died. Comparisons between mutant and wild-type embryos revealed novel aspects of trochlear and oculomotor nerve development. Conclusions: We delineated mouse ocular motor nerve spatial and temporal development in unprecedented detail. Moreover, we found that EOMs are not necessary for initial outgrowth and guidance of ocular motor axons from the brainstem to the orbit but are required for their terminal branching and survival. These data suggest that intermediate targets in the mesenchyme provide cues necessary for appropriate targeting of ocular motor axons to the orbit, while EOM cues are responsible for terminal branching and motor neuron survival.

Pacouret S, Bouzelha M, Shelke R, Andres-Mateos E, Xiao R, Maurer A, Mevel M, Turunen H, Barungi T, Penaud-Budloo M, Broucque F, Blouin V, Moullier P, Ayuso E, Vandenberghe LH. AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations. Mol Ther 2017;25(6):1375-1386.Abstract
Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.
Jamuar SS, Schmitz-Abe K, D'Gama AM, Drottar M, Chan W-M, Peeva M, Servattalab S, Lam A-TN, Delgado MR, Clegg NJ, Zayed ZA, Dogar MA, Alorainy IA, Jamea AA, Abu-Amero K, Griebel M, Ward W, Lein ES, Markianos K, Barkovich JA, Robson CD, Grant EP, Bosley TM, Engle EC, Walsh CA, Yu TW. Biallelic mutations in human DCC cause developmental split-brain syndrome. Nat Genet 2017;49(4):606-612.Abstract

Motor, sensory, and integrative activities of the brain are coordinated by a series of midline-bridging neuronal commissures whose development is tightly regulated. Here we report a new human syndrome in which these commissures are widely disrupted, thus causing clinical manifestations of horizontal gaze palsy, scoliosis, and intellectual disability. Affected individuals were found to possess biallelic loss-of-function mutations in the gene encoding the axon-guidance receptor 'deleted in colorectal carcinoma' (DCC), which has been implicated in congenital mirror movements when it is mutated in the heterozygous state but whose biallelic loss-of-function human phenotype has not been reported. Structural MRI and diffusion tractography demonstrated broad disorganization of white-matter tracts throughout the human central nervous system (CNS), including loss of all commissural tracts at multiple levels of the neuraxis. Combined with data from animal models, these findings show that DCC is a master regulator of midline crossing and development of white-matter projections throughout the human CNS.

Daniel E, Pistilli M, Kothari S, Khachatryan N, Kaçmaz OR, Gangaputra SS, Sen NH, Suhler EB, Thorne JE, Foster SC, Jabs DA, Nussenblatt RB, Rosenbaum JT, Levy-Clarke GA, Bhatt NP, Kempen JH, for Group SITEDR. Risk of Ocular Hypertension in Adults with Noninfectious Uveitis. Ophthalmology 2017;124(8):1196-1208.Abstract
PURPOSE: To describe the risk and risk factors for ocular hypertension (OHT) in adults with noninfectious uveitis. DESIGN: Retrospective, multicenter, cohort study. PARTICIPANTS: Patients aged ≥18 years with noninfectious uveitis seen between 1979 and 2007 at 5 tertiary uveitis clinics. METHODS: Demographic, ocular, and treatment data were extracted from medical records of uveitis cases. MAIN OUTCOME MEASURES: Prevalent and incident OHT with intraocular pressures (IOPs) of ≥21 mmHg, ≥30 mmHg, and increase of ≥10 mmHg from documented IOP recordings (or use of treatment for OHT). RESULTS: Among 5270 uveitic eyes of 3308 patients followed for OHT, the mean annual incidence rates for OHT ≥21 mmHg and OHT ≥30 mmHg are 14.4% (95% confidence interval [CI], 13.4-15.5) and 5.1% (95% CI, 4.7-5.6) per year, respectively. Statistically significant risk factors for incident OHT ≥30 mmHg included systemic hypertension (adjusted hazard ratio [aHR], 1.29); worse presenting visual acuity (≤20/200 vs. ≥20/40, aHR, 1.47); pars plana vitrectomy (aHR, 1.87); history of OHT in the other eye: IOP ≥21 mmHg (aHR, 2.68), ≥30 mmHg (aHR, 4.86) and prior/current use of IOP-lowering drops or surgery in the other eye (aHR, 4.17); anterior chamber cells: 1+ (aHR, 1.43) and ≥2+ (aHR, 1.59) vs. none; epiretinal membrane (aHR, 1.25); peripheral anterior synechiae (aHR, 1.81); current use of prednisone >7.5 mg/day (aHR, 1.86); periocular corticosteroids in the last 3 months (aHR, 2.23); current topical corticosteroid use [≥8×/day vs. none] (aHR, 2.58); and prior use of fluocinolone acetonide implants (aHR, 9.75). Bilateral uveitis (aHR, 0.69) and previous hypotony (aHR, 0.43) were associated with statistically significantly lower risk of OHT. CONCLUSIONS: Ocular hypertension is sufficiently common in eyes treated for uveitis that surveillance for OHT is essential at all visits for all cases. Patients with 1 or more of the several risk factors identified are at particularly high risk and must be carefully managed. Modifiable risk factors, such as use of corticosteroids, suggest opportunities to reduce OHT risk within the constraints of the overriding need to control the primary ocular inflammatory disease.
Adini A, Adini I, Chi Z-L, Derda R, Birsner AE, Matthews BD, D'Amato RJ. A novel strategy to enhance angiogenesis in vivo using the small VEGF-binding peptide PR1P. Angiogenesis 2017;20(3):399-408.Abstract
Therapeutic angiogenesis is an experimental frontier in vascular biology that seeks to deliver angiogenic growth factors to ischemic or injured tissues to promote targeted formation of new blood vessels as an alternative approach to surgical revascularization procedures. Vascular endothelial growth factor (VEGF) is a potent angiogenic signal protein that is locally upregulated at sites of tissue injury. However, therapies aimed at increasing VEGF levels experimentally by injecting VEGF gene or protein failed to improve outcomes in human trials in part due to its short half-life and systemic toxicity. We recently designed a novel 12-amino acid peptide (PR1P) whose sequence was derived from an extracellular VEGF-binding domain of the pro-angiogenic glycoprotein prominin-1. In this study, we characterized the molecular binding properties of this novel potential therapeutic for targeted angiogenesis and provided the foundation for its use as an angiogenic molecule that can potentiate endogenous VEGF. We showed that PR1P bound VEGF directly and enhanced VEGF binding to endothelial cells and to VEGF receptors VEGFR2 and neuropilin-1. PR1P increased angiogenesis in the murine corneal micropocket assay when combined with VEGF, but had no activity without added VEGF. In addition, PR1P also enhanced angiogenesis in murine choroidal neovascularization and wound-healing models and augmented reperfusion in a murine hind-limb ischemia model. Together our data suggest that PR1P enhanced angiogenesis by potentiating the activity of endogenous VEGF. In so doing, this novel therapy takes advantage of endogenous VEGF gradients generated in injured tissues and may improve the efficacy of and avoid systemic toxicity seen with previous VEGF therapies.
Wu S, Fan Y, Wu D, Hong J, Xu J. The association of maternal factors with epibulbar dermoid of newborn: a retrospective, matched case-control study. Eye (Lond) 2017;31(7):1099-1105.Abstract
PurposeTo determine the association of maternal factors and exposure during pregnancy with the incidence in newborns of epibulbar dermoid (ED), a congenital ocular surface benign tumor.Patients and methodsThis is a retrospective, paired case-control study in which 121 children with ED (case group) and 121 children without ED (control group) were recruited. Questionnaire-based interviews with mothers of participants were performed and maternal medical records during pregnancy were reviewed. The questionnaire investigated basic information, personal history, environmental exposure, exposure to maternal diseases, symptoms and corresponding medical treatments during pregnancy, and parental socioeconomic status. The case and control participants were matched for sex, birth weight, gestational age, and parental socioeconomic status level. Univariate and multivariate logistic regression analyses were conducted with ED as the main outcome variable.ResultsFactors significantly associated with ED were: history of maternal inevitable miscarriage (odds ratio (OR), 2.59; 95% confidence intervals (CI), 1.13-5.90), common cold in the first trimester (OR, 3.94; CI, 1.74-8.93), and paternal smoke exposure >half a pack per day during pregnancy (OR, 4.81; CI, 1.74-13.28).ConclusionHistory of maternal miscarriage, common cold exposure in the first trimester, and paternal smoking (>half a pack per day) during pregnancy could result in significant risk factors for ED of newborns. These data also imply that paternal smoking delivers nicotine to maternal respiratory system and uterine microenvironment that may both affect microvascular development and predispose the fetus to future ED.
Tsikata E, Laíns I, Gil J, Marques M, Brown K, Mesquita T, Melo P, da Luz Cachulo M, Kim IK, Vavvas D, Murta JN, Miller JB, Silva R, Miller JW, Chen TC, Husain D. Automated Brightness and Contrast Adjustment of Color Fundus Photographs for the Grading of Age-Related Macular Degeneration. Transl Vis Sci Technol 2017;6(2):3.Abstract

PURPOSE: The purpose of this study was to develop an algorithm to automatically standardize the brightness, contrast, and color balance of digital color fundus photographs used to grade AMD and to validate this algorithm by determining the effects of the standardization on image quality and disease grading. METHODS: Seven-field color photographs of patients (>50 years) with any stage of AMD and a control group were acquired at two study sites, with either the Topcon TRC-50DX or Zeiss FF-450 Plus cameras. Field 2 photographs were analyzed. Pixel brightness values in the red, green, and blue (RGB) color channels were adjusted in custom-built software to make the mean brightness and contrast of the images equal to optimal values determined by the Age-Related Eye Disease Study (AREDS) 2 group. RESULTS: Color photographs of 370 eyes were analyzed. We found a wide range of brightness and contrast values in the images at baseline, even for those taken with the same camera. After processing, image brightness variability (brightest image-dimmest image in a color channel) was reduced 69-fold, 62-fold, and 96-fold for the RGB channels. Contrast variability was reduced 6-fold, 8-fold, and 13-fold, respectively, after adjustment. Of the 23% images considered nongradable before adjustment, only 5.7% remained nongradable. CONCLUSIONS: This automated software enables rapid and accurate standardization of color photographs for AMD grading. TRANSLATIONAL RELEVANCE: This work offers the potential to be the future of assessing and grading AMD from photos for clinical research and teleimaging.

Stagner AM, Jakobiec FA, Fay A. Primary orbital synovial sarcoma: A clinicopathologic review with a differential diagnosis and discussion of molecular genetics. Surv Ophthalmol 2017;62(2):227-236.Abstract

Synovial sarcoma is a soft-tissue sarcoma of the extremities developing in young adults that has rarely been reported in the orbit. Synovial sarcoma is associated with a unique translocation, resulting in an SYT-SSX fusion gene. We analyze 7 published periocular cases, together with the current one, to gain a better appreciation of the features of the tumor in this location and to compare the findings with those derived from nonophthalmic studies. An inferior orbital mass developed in a 31-year-old woman after experiencing periorbital and hemifacial pain for more than a decade. Radiographically, the mass was circumscribed and displayed coarse internal calcifications. A large but subtotal excision with histopathologic examination disclosed a primitive tumor composed of spindled and ovoid cells. Immunohistochemistry demonstrated positivity for nuclear transducin-like enhancer of split 1 and membranous CD99, typical for synovial sarcoma. Fluorescence in situ hybridization identified a (X,18) translocation in the tumor cells. The patient underwent postoperative adjuvant proton beam radiotherapy with a good response that has been maintained during 1 year of follow-up. Orbital soft-tissue tumors of all types are increasingly identified by their distinctive genetic signatures that offer more specificity than standard immunohistochemical tests.

VanderVeen DK, Melia M, Yang MB, Hutchinson AK, Wilson LB, Lambert SR. Anti-Vascular Endothelial Growth Factor Therapy for Primary Treatment of Type 1 Retinopathy of Prematurity: A Report by the American Academy of Ophthalmology. Ophthalmology 2017;124(5):619-633.Abstract

PURPOSE: To review the available evidence on the ocular safety and efficacy of anti-vascular endothelial growth factor (VEGF) agents for the treatment of retinopathy of prematurity (ROP) compared with laser photocoagulation therapy. METHODS: A literature search of the PubMed and Cochrane Library databases was conducted last on September 6, 2016, with no date restrictions and limited to articles published in English. This search yielded 311 citations, of which 37 were deemed clinically relevant for full-text review. Thirteen of these were selected for inclusion in this assessment. The panel methodologist assigned ratings to the selected articles according to the level of evidence. RESULTS: Of the 13 citations, 6 articles on 5 randomized clinical trials provided level II evidence supporting the use of anti-VEGF agents, either as monotherapy or in combination with laser therapy. The primary outcome for these articles included recurrence of ROP and the need for retreatment (3 articles), retinal structure (2 articles), and refractive outcome (1 article). Seven articles were comparative case series that provided level III evidence. The primary outcomes included the effects of anti-VEGF treatment on development of peripheral retinal vessels (1 article), refractive outcomes (1 article), or both structural and refractive or visual outcomes (5 articles). CONCLUSIONS: Current level II and III evidence indicates that intravitreal anti-VEGF therapy is as effective as laser photocoagulation for achieving regression of acute ROP. Although there are distinct ocular advantages to anti-VEGF pharmacotherapy for some cases (such as eyes with zone I disease or aggressive posterior ROP), the disadvantages are that the ROP recurrence rate is higher, and vigilant and extended follow-up is needed because retinal vascularization is usually incomplete. After intravitreal injection, bevacizumab can be detected in serum within 1 day, and serum VEGF levels are suppressed for at least 8 to 12 weeks. The effects of lowering systemic VEGF levels on the developing organ systems of premature infants are unknown, and there are limited long-term data on potential systemic and neurodevelopmental effects after anti-VEGF use for ROP treatment. Anti-VEGF agents should be used judiciously and with awareness of the known and unknown or potential side effects.