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2017

Ung C, Sanchez A, Shen L, Davoudi S, Ahmadi T, Navarro-Gomez D, Chen C, Hancock H, Penman A, Hoadley S, Consugar M, Restrepo C, Shah V, Arboleda-Velasquez J, Sobrin L, Gai X, Kim L. Whole exome sequencing identification of novel candidate genes in patients with proliferative diabetic retinopathy. Vision Res. 2017;
Rare or novel gene variants in patients with proliferative diabetic retinopathy may contribute to disease development. We performed whole exome sequencing (WES) on patients at the phenotypic extremes of diabetic retinal complications: 57 patients diagnosed with proliferative diabetic retinopathy (PDR) as cases and 13 patients with no diabetic retinopathy despite at least 10years of type 2 diabetes as controls. Thirty-one out of the 57 cases and all 13 controls were from the African American Proliferative Diabetic Retinopathy Study (AA). The rest of the cases were of mixed ethnicities (ME). WES identified 721 candidate genes with rare or novel non-synonymous variants found in at least one case with PDR and not present in any controls. After filtering for genes with null alleles in greater than two cases, 28 candidate genes were identified in our ME cases and 16 genes were identified in our AA cases. Our analysis showed rare and novel variants within these genes that could contribute to the development of PDR, including rare non-synonymous variants in FAM132A, SLC5A9, ZNF600, and TMEM217. We also found previously unidentified variants in VEGFB and APOB. We found that VEGFB, VPS13B, PHF21A, NAT1, ZNF600, PKHD1L1 expression was reduced in human retinal endothelial cells (HRECs) cultured under high glucose conditions. In an exome sequence analysis of patients with PDR, we identified variants in genes that could contribute to pathogenesis. Six of these genes were further validated and found to have reduced expression in HRECs under high glucose conditions, suggestive of an important role in the development of PDR.
Islam R, Eidet JR, Badian R, 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.

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.

Jamuar S, Schmitz-Abe K, D’Gama A, Drottar M, Chan WM, Peeva M, Servattalab S, Lam AT, Delgado M, Clegg N, Zayed ZA, Dogar MA, Alorainy I, Jamea AA, Abu-Amero K, Griebel M, Ward W, Lein E, Markianos K, Barkovich J, Robson C, Grant E, Bosley T, Engle E, Walsh C, Yu T. Biallelic mutations in human DCC cause developmental split-brain syndrome. Nat Genet. 2017;49(4):606–612.

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.

Konstantinou E, Notomi S, Kosmidou C, Brodowska K, Al-Moujahed A, Nicolaou F, Tsoka P, Gragoudas E, Miller J, Young L, Vavvas D. 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.

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.

Lam J, Oh D, Wong L, Amarnani D, Park-Windhol C, Sanchez A, Cardona-Velez J, McGuone D, Stemmer-Rachamimov A, Eliott D, Bielenberg D, Zyl T, Shen L, Gai X, D’Amore P, Kim L, Arboleda-Velasquez J. Identification of RUNX1 as a Mediator of Aberrant Retinal Angiogenesis. Diabetes. 2017;66(7):1950–1956.
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.
Michalak S, Whitman M, Park J, Tischfield M, Nguyen E, Engle E. Ocular Motor Nerve Development in the Presence and Absence of Extraocular Muscle. Invest Ophthalmol Vis Sci. 2017;58(4):2388–2396.

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.

Olivares, Jelcick, Reinecke, Leehy, Haider, Morrison, Cheng, Chen, DeAngelis, Haider. Multimodal Regulation Orchestrates Normal and Complex Disease States in the Retina. Sci Rep. 2017;7(1):690.

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.

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 L. AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations. Mol Ther. 2017;25(6):1375–1386.
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.
Paschalis E, Zhou C, Lei F, Scott N, Kapoulea V, Robert MC, Vavvas D, Dana R, Chodosh J, Dohlman C. Mechanisms of Retinal Damage after Ocular Alkali Burns. Am J Pathol. 2017;187(6):1327–1342.
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.
Adini A, Adini I, Chi ZL, Derda R, Birsner A, Matthews B, D’Amato R. A novel strategy to enhance angiogenesis in vivo using the small VEGF-binding peptide PR1P. Angiogenesis. 2017;20(3):399–408.
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.