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2.  Surgical Approaches to Gene and Stem Cell Therapy for Retinal Disease 
Human Gene Therapy  2011;22(5):531-535.
Gene and cell therapy holds tremendous promise for treating a variety of acquired and inherited disorders of the retina that impact on vision. Much has been written about the impact of vectors in delivering genes to cells of the retina in vivo. A critically important component of these kinds of therapies is the procedure used to introduce the cells or vectors. Drs. Stout and Francis provide a broad overview of gene and cell therapies for diseases of the retina, focusing on the procedures that could be used for delivery.
doi:10.1089/hum.2011.060
PMCID: PMC3132008  PMID: 21480778
3.  Simultaneous visualization and cell-specific confirmation of RNA and protein in the mouse retina 
Molecular Vision  2014;20:1366-1373.
Purpose
Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffin embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue.
Methods
Whole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fibrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fluorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy.
Results
A fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue.
Conclusions
The protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffin-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.
PMCID: PMC4169891  PMID: 25352743
4.  Occult nonmetallic intraocular foreign bodies presenting as fulminant uveitis: a case series and review of the literature 
Intraocular foreign bodies (IOFBs) can complicate globe trauma and are associated with a high incidence of severe vision loss. Occult IOFBs present a particular challenge as they are not diagnosed promptly and tend to present with advanced complications, including endophthalmitis and retinal detachment. In this report, we present three cases of occult nonmetallic IOFBs presenting as fulminant uveitis, and we also review the literature.
doi:10.2147/OPTH.S47339
PMCID: PMC3770888  PMID: 24039395
intraocular foreign body; endophthalmitis; vitrectomy; trauma
5.  The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells 
PLoS ONE  2012;7(6):e31260.
Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases.
doi:10.1371/journal.pone.0031260
PMCID: PMC3382240  PMID: 22761647
6.  Subretinal Transplantation of Forebrain Progenitor Cells in Non-human Primates: Survival and Intact Retinal Function 
Purpose
Cell-based therapy rescues retinal structure and function in rodent models of retinal disease, but translation to clinic will require more information about consequences of transplantation in an eye closely resembling the human eye. Therefore we explored donor cell behavior using human cortical neural progenitor cells (hNPCctx) introduced into the subretinal space of normal rhesus macaques.
Methods
hNPCctx transduced with Green Fluorescent Protein (hNPCctx-GFP) were delivered bilaterally into the subretinal space of six normal adult rhesus macaques under conditions paralleling those of the human operating room. Outcome measures included clinical parameters of surgical success, multifocal electroretinogram (mfERG) and histopathological analyses performed between 3 and 39 days post-engraftment. To test the effects of GFP transduction on cell bioactivity, hNPCctx –GFP from the same batch were also injected into RCS rats and compared with non-labeled hNPCctx.
Results
Studies using RCS rats indicated that GFP transduction did not alter the ability of the cells to rescue vision. After cells were introduced into the monkey subretinal space by a pars plana transvitreal approach, the resulting detachment was rapidly resolved and retinal function showed little or no disturbance in mfERG recordings. Retinal structure was unaffected and no signs of inflammation or rejection were seen. Donor cells survived as a single layer in the subretinal space and no cells migrated into the inner retina.
Conclusions
Human neural progenitor cells can be introduced into a primate eye without complication, using an approach that would be suitable for extrapolation to human patients.
doi:10.1167/iovs.08-2908
PMCID: PMC2826708  PMID: 19234356
7.  Rhesus monkeys and humans share common susceptibility genes for age-related macular disease 
Human Molecular Genetics  2008;17(17):2673-2680.
Age-related macular degeneration (AMD), a complex multigenic disorder and the most common cause of vision loss in the elderly, is associated with polymorphisms in the LOC387715/ARMS2 and HTRA1 genes on 10q26. Like humans, macaque monkeys possess a macula and develop age-related macular pathologies including drusen, the phenotypic hallmark of AMD. We genotyped a cohort of 137 unrelated rhesus macaques with and without macular drusen. As in humans, one variant within LOC387715/ARMS2 and one in HTRA1 were significantly associated with affected status. HTRA1 and the predicted LOC387715/ARMS2 gene were both transcribed in rhesus and human retina and retinal pigment epithelium. Among several primate species, orthologous exons for the human LOC387715/ARMS2 gene were present only in Old World monkeys and apes. In functional analyses, the disease-associated HTRA1 polymorphism resulted in a 2-fold increase in gene expression, supporting a role in pathogenesis. These results demonstrate that two genes associated with AMD in humans are also associated with macular disease in rhesus macaques and that one of these genes is specific to higher primates. This is the first evidence that humans and macaques share the same genetic susceptibility factors for a common complex disease.
doi:10.1093/hmg/ddn167
PMCID: PMC2733804  PMID: 18535016
8.  Human CHN1 mutations hyperactivate α2-chimaerin and cause Duane’s retraction syndrome 
Science (New York, N.Y.)  2008;321(5890):839-843.
The RacGAP molecule α2-chimaerin is implicated in neuronal signaling pathways required for precise guidance of developing corticospinal axons. We now demonstrate that a variant of Duane’s retraction syndrome, a congenital eye movement disorder in which affected individuals show aberrant development of axon projections to the extraocular muscles, can result from gain-of-function heterozygous missense mutations in CHN1 that increase α2-chimaerin RacGAP activity in vitro. A subset of mutations enhances α2-chimaerin membrane translocation and/or α2-chimaerin’s previously unrecognized ability to form a complex with itself. In ovo expression of mutant CHN1 alters the development of ocular motor axons. These data demonstrate that human CHN1 mutations can hyperactivate α2-chimaerin and result in aberrant cranial motor neuron development.
doi:10.1126/science.1156121
PMCID: PMC2593867  PMID: 18653847
9.  Prediction of Cis-Regulatory Elements Controlling Genes Differentially Expressed by Retinal and Choroidal Vascular Endothelial Cells 
Cultured endothelial cells of the human retina and choroid demonstrate distinct patterns of gene expression. We hypothesized that differential gene expression reflected differences in the interactions of transcription factors and respective cis-regulatory motifs(s) in these two emdothelial cell subpopulations, recognizing that motifs often exist as modules. We tested this hypothesis in silico by using TRANSFAC Professional and CisModule to identify cis-regulatory motifs and modules in genes that were differentially expressed by human retinal versus choroidal endothelial cells, as identified by analysis of a microarray data set. Motifs corresponding to eight transcription factors were significantly (p < 0.05) differentially abundant in genes that were relatively highly expressed in retinal (i.e., GCCR, HMGIY, HSF1, p53, VDR) or choroidal (i.e., E2F, YY1, ZF5) endothelial cells. Predicted cis-regulatory modules were quite different for these two groups of genes. Our findings raise the possibility of exploiting specific cis-regulatory motifs to target therapy at the ocular endothelial cells subtypes responsible for neovascular age-related macular degeneration or proliferative diabetic retinopathy.
doi:10.1007/s12177-008-9007-1
PMCID: PMC2573398  PMID: 19122891
endothelial cell; retina; choroid; cis-regulatory motif; cis-regulatory module
11.  Prediction of cis-regulatory elements controlling genes differentially expressed by retinal and choroidal vascular endothelial cells 
Cultured endothelial cells of the human retina and choroid demonstrate distinct patterns of gene expression. We hypothesized that differential gene expression reflected differences in the interactions of transcription factors and respective cis-regulatory motifs(s) in these two endothelial cell subpopulations, recognizing that motifs often exist as modules. We tested this hypothesis in silico by using TRANSFAC Professional and CisModule to identify cis-regulatory motifs and modules in genes that were differentially expressed by human retinal versus choroidal endothelial cells, as identified by analysis of a microarray data set. Motifs corresponding to eight transcription factors were significantly (p < 0.05) differentially abundant in genes that were relatively highly expressed in retinal (i.e., glucocorticoid receptor, high mobility group AT-hook 1, heat shock transcription factor 1, p53, vitamin D receptor) or choroidal (i.e., transcription factor E2F, Yin Yang 1, zinc finger 5) endothelial cells. Predicted cis-regulatory modules were quite different for these two groups of genes. Our findings raise the possibility of exploiting specific cis-regulatory motifs to target therapy at the ocular endothelial cells subtypes responsible for neovascular age-related macular degeneration or proliferative diabetic retinopathy.
Electronic supplementary material
The online version of this article (doi:10.1007/s12177-008-9007-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s12177-008-9007-1
PMCID: PMC2573398  PMID: 19122891
Endothelial cell; Retina; Choroid; cis-Regulatory motif; cis-Regulatory module
12.  Internal En Bloc Resection and Genetic Analysis of Retinal Capillary Hemangioblastoma 
Archives of ophthalmology  2007;125(9):1189-1193.
Objective
To report the clinical outcomes and molecular genetics of nongermline retinal hemangioblastoma managed by surgical resection.
Methods
Retrospective case series of 3 patients aged 16 to 46 years treated at a tertiary care referral center (Casey Eye Institute, Portland, Oregon). Tumors 7 to 9 mm in diameter were removed from 3 consecutive eyes (in 3 patients) via internal en bloc surgical resection using a bimanual technique. Samples of DNA from 2 of 3 tumors were tested for von Hippel-Lindau gene (VHL) mutations as a clue to the molecular basis for spontaneously occurring hemangioblastoma. Main outcome measures were morbidity, visual acuity, resolution of macular exudates, and presence of VHL markers.
Results
Visual acuity improved or remained stable in all patients. All 3 developed cataracts, extracted in 2 instances. Histopathological findings were typical of retinal hemangioblastoma in all cases. The cells from one patient’s DNA sample showed loss of heterozygosity for the VHL gene, while no genetic abnormalities were detected in the other patient’s DNA sample.
Conclusions
Our patients’ favorable outcomes suggest that surgical resection is an option for patients with large retinal hemangioblastomas. In addition, ours may be the first report of retinal hemangioblastoma unassociated with a VHL mutation.
doi:10.1001/archopht.125.9.1189
PMCID: PMC2140243  PMID: 17846357

Results 1-12 (12)