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Sun D, Moore S, Jakobs TC. Optic nerve astrocyte reactivity protects function in experimental glaucoma and other nerve injuries. J Exp Med 2017;214(5):1411-1430.Abstract
Reactive remodeling of optic nerve head astrocytes is consistently observed in glaucoma and other optic nerve injuries. However, it is unknown whether this reactivity is beneficial or harmful for visual function. In this study, we used the Cre recombinase (Cre)-loxP system under regulation of the mouse glial fibrillary acidic protein promoter to knock out the transcription factor signal transducer and activator of transcription 3 (STAT3) from astrocytes and test the effect this has on reactive remodeling, ganglion cell survival, and visual function after experimental glaucoma and nerve crush. After injury, STAT3 knockout mice displayed attenuated astrocyte hypertrophy and reactive remodeling; astrocytes largely maintained their honeycomb organization and glial tubes. These changes were associated with increased loss of ganglion cells and visual function over a 30-day period. Thus, reactive astrocytes play a protective role, preserving visual function. STAT3 signaling is an important mediator of various aspects of the reactive phenotype within optic nerve astrocytes.
Ramke M, Lee JY, Dyer DW, Seto D, Rajaiya J, Chodosh J. The 5'UTR in human adenoviruses: leader diversity in late gene expression. Sci Rep 2017;7(1):618.Abstract

Human adenoviruses (HAdVs) shut down host cellular cap-dependent mRNA translation while initiating the translation of viral late mRNAs in a cap-independent manner. HAdV 5' untranslated regions (5'UTRs) are crucial for cap-independent initiation, and influence mRNA localization and stability. However, HAdV translational regulation remains relatively uncharacterized. The HAdV tripartite leader (TPL), composed of three introns (TPL 1-3), is critical to the translation of HAdV late mRNA. Herein, we annotated and analyzed 72 HAdV genotypes for the HAdV TPL and another previously described leader, the i-leader. Using HAdV species D, type 37 (HAdV-D37), we show by reverse transcription PCR and Sanger sequencing that mRNAs of the HAdV-D37 E3 transcription unit are spliced to the TPL. We also identified a polycistronic mRNA for RID-α and RID-β. Analysis of the i-leader revealed a potential open reading frame within the leader sequence and the termination of this potential protein in TPL3. A potential new leader embedded within the E3 region was also detected and tentatively named the j-leader. These results suggest an underappreciated complexity of post-transcriptional regulation, and the importance of HAdV 5'UTRs for precisely coordinated viral protein expression along the path from genotype to phenotype.

Tahvildari M, Emami-Naeini P, Omoto M, Mashaghi A, Chauhan SK, Dana R. Treatment of donor corneal tissue with immunomodulatory cytokines: a novel strategy to promote graft survival in high-risk corneal transplantation. Sci Rep 2017;7(1):971.Abstract

Antigen-presenting cells (APCs) play an important role in transplant rejection and tolerance. In high-risk corneal transplantation, where the graft bed is inflamed and vascularized, immature APCs in the donor corneal stroma quickly mature and migrate to lymphoid tissues to sensitize host T cells. In this study, using a mouse model of corneal transplantation, we investigated whether enrichment of tolerogenic APCs (tolAPCs) in donor corneas can enhance graft survival in corneal allograft recipients with inflamed graft beds. Treatment of donor corneas with interleukin-10 (IL-10) and transforming growth factor-β1 (TGFβ1) altered the phenotype and function of tissue-residing APCs. Transplantation of these tolAPC-enriched corneas decreased frequencies of interferon gamma (IFNγ)(+) effector T cells (Teffs), as well as allosensitization in the hosts, diminished graft infiltration of CD45(+) and CD4(+) cells, and significantly improved corneal allograft survival compared to saline-injected controls. These data provide a novel approach for tolAPC-based immunotherapy in transplantation by direct cytokine conditioning of the donor tissue.

Roohipoor R, Yaseri M, Teymourpour A, Kloek C, Miller JB, Loewenstein JI. Early Performance on an Eye Surgery Simulator Predicts Subsequent Resident Surgical Performance. J Surg Educ 2017;Abstract

OBJECTIVE: To examine early performance on an eye surgery simulator and its relationship to subsequent live surgical performance in a single large residency program. DESIGN: Retrospective study. SETTING: Massachusetts Eye and Ear, Harvard Medical School, Department of Ophthalmology. METHODS: In a retrospective study, we compared performance of 30 first-year ophthalmology residents on an eye surgery simulator to their surgical skills as third-year residents. Variables collected from the eye surgery simulator included scores on the following modules of the simulator (Eyesi, VRmagic, Mannheim, Germany): antitremor training level 1, bimanual training level 1, capsulorhexis level 1 (configured), forceps training level 1, and navigation training level 1. Subsequent surgical performance was assessed using the total number of phacoemulsification cataract surgery cases for each resident, as well as the number performed as surgeon during residency and scores on global rating assessment of skills in intraocular surgery (GRASIS) scales during the third year of residency. Spearman correlation coefficients were calculated between each of the simulator performance and subsequent surgical performance variables. We also compared variables in a small group of residents who needed extra help in learning cataract surgery to the other residents in the study. MAIN OUTCOME MEASURES: Relationships between Eyesi scores early in residency and surgical performance measures in the final year of residency. RESULTS: A total of 30 residents had Eyesi data from their first year of residency and had already graduated so that all subsequent surgical performance data were available. There was a significant correlation between capsulorhexis task score on the simulator and total surgeries (r = 0.745, p = 0.008). There was a significant correlation between antitremor training level 1 (r = 0.554, p = 0.040), and forceps training level 1 (r = 0.622, p = 0.023) with primary surgery numbers. There was a significant correlation between forceps training level 1 (r = 0.811, p = 0.002), and navigation training level 1 (r = 0.692, p =0.013) with total GRASIS score. There was a significant inverse correlation between total GRASIS score and residents in need of extra help (r = -0.358, p =0.003). CONCLUSION: Module scores on an eye surgery simulator early in residency may predict a resident׳s future performance in the operating room. These scores may allow early identification of residents in need of supplemental training in cataract surgery.

Spencer C, Abend S, McHugh KJ, Saint-Geniez M. Identification of a synergistic interaction between endothelial cells and retinal pigment epithelium. J Cell Mol Med 2017;21(10):2542-2552.Abstract
The retinal pigment epithelium located between the neurosensory retina and the choroidal vasculature is critical for the function and maintenance of both the photoreceptors and underlying capillary endothelium. While the trophic role of retinal pigment epithelium on choroidal endothelial cells is well recognized, the existence of a reciprocal regulatory function of endothelial cells on retinal pigment epithelium cells remained to be fully characterized. Using a physiological long-term co-culture system, we determined the effect of retinal pigment epithelium-endothelial cell heterotypic interactions on cell survival, behaviour and matrix deposition. Human retinal pigment epithelium and endothelial cells were cultured on opposite sides of polyester transwells for up to 4 weeks in low serum conditions. Cell viability was quantified using a trypan blue assay. Cellular morphology was evaluated by H&E staining, S.E.M. and immunohistochemistry. Retinal pigment epithelium phagocytic function was examined using a fluorescent bead assay. Gene expression analysis was performed on both retinal pigment epithelium and endothelial cells by quantitative PCR. Quantification of extracellular matrix deposition was performed on decellularized transwells stained for collagen IV, fibronectin and fibrillin. Our results showed that presence of endothelial cells significantly improves retinal pigment epithelium maturation and function as indicated by the induction of visual cycle-associated genes, accumulation of a Bruch's membrane-like matrix and increase in retinal pigment epithelium phagocytic activity. Co-culture conditions led to increased expression of anti-angiogenic growth factors and receptors in both retinal pigment epithelium and endothelial cells compared to monoculture. Tube-formation assays confirmed that co-culture with retinal pigment epithelium significantly decreased the angiogenic phenotype of endothelial cells. These findings provide evidence of critical interdependent interactions between retinal pigment epithelium and endothelial cell involved in the maintenance of retinal homeostasis.
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.

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.

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.

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