Retinal Degenerations

Benaglio P, San Jose PF, Avila-Fernandez A, Ascari G, Harper S, Manes G, Ayuso C, Hamel C, Berson EL, Rivolta C. Mutational screening of splicing factor genes in cases with autosomal dominant retinitis pigmentosa. Mol Vis 2014;20:843-51.Abstract

PURPOSE: Mutations in genes encoding proteins from the tri-snRNP complex of the spliceosome account for more than 12% of cases of autosomal dominant retinitis pigmentosa (adRP). Although the exact mechanism by which splicing factor defects trigger photoreceptor death is not completely clear, their role in retinitis pigmentosa has been demonstrated by several genetic and functional studies. To test for possible novel associations between splicing factors and adRP, we screened four tri-snRNP splicing factor genes (EFTUD2, PRPF4, NHP2L1, and AAR2) as candidate disease genes. METHODS: We screened up to 303 patients with adRP from Europe and North America who did not carry known RP mutations. Exon-PCR and Sanger methods were used to sequence the NHP2L1 and AAR2 genes, while the sequences of EFTUD2 and PRPF4 were obtained by using long-range PCRs spanning coding and non-coding regions followed by next-generation sequencing. RESULTS: We detected novel missense changes in individual patients in the sequence of the genes PRPF4 and EFTUD2, but the role of these changes in relationship to disease could not be verified. In one other patient we identified a novel nucleotide substitution in the 5' untranslated region (UTR) of NHP2L1, which did not segregate with the disease in the family. CONCLUSIONS: The absence of clearly pathogenic mutations in the candidate genes screened in our cohort suggests that EFTUD2, PRPF4, NHP2L1, and AAR2 are either not involved in adRP or are associated with the disease in rare instances, at least as observed in this study in patients of European and North American origin.

Bujakowska KM, Consugar M, Place E, Harper S, Lena J, Taub DG, White J, Navarro-Gomez D, Weigel DiFranco C, Farkas MH, Gai X, Berson EL, Pierce EA. Targeted exon sequencing in Usher syndrome type I. Invest Ophthalmol Vis Sci 2014;55(12):8488-96.Abstract

PURPOSE: Patients with Usher syndrome type I (USH1) have retinitis pigmentosa, profound congenital hearing loss, and vestibular ataxia. This syndrome is currently thought to be associated with at least six genes, which are encoded by over 180 exons. Here, we present the use of state-of-the-art techniques in the molecular diagnosis of a cohort of 47 USH1 probands. METHODS: The cohort was studied with selective exon capture and next-generation sequencing of currently known inherited retinal degeneration genes, comparative genomic hybridization, and Sanger sequencing of new USH1 exons identified by human retinal transcriptome analysis. RESULTS: With this approach, we were able to genetically solve 14 of the 47 probands by confirming the biallelic inheritance of mutations. We detected two likely pathogenic variants in an additional 19 patients, for whom family members were not available for cosegregation analysis to confirm biallelic inheritance. Ten patients, in addition to primary disease-causing mutations, carried rare likely pathogenic USH1 alleles or variants in other genes associated with deaf-blindness, which may influence disease phenotype. Twenty-one of the identified mutations were novel among the 33 definite or likely solved patients. Here, we also present a clinical description of the studied cohort at their initial visits. CONCLUSIONS: We found a remarkable genetic heterogeneity in the studied USH1 cohort with multiplicity of mutations, of which many were novel. No obvious influence of genotype on phenotype was found, possibly due to small sample sizes of the genotypes under study.

Lundgren P, Wilde Å, Löfqvist C, Smith LEH, Hård A-L, Hellström A. Weight at first detection of retinopathy of prematurity predicts disease severity. Br J Ophthalmol 2014;98(11):1565-9.Abstract
OBJECTIVE: To investigate whether postnatal weight at first detection of retinopathy of prematurity (ROP) can predict preterm infants who will develop severe ROP warranting treatment. DESIGN: This modern, population-based cohort included 147 infants born at gestational age (GA) <32 weeks in the Gothenburg region during 2011-2012 and screened for ROP at Sahlgrenska University hospital. GA, birth weight (BW), and weekly postnatal weight from birth until postmenstrual age (PMA) 40 weeks data were retrospectively retrieved. Birth weight SD scores (BWSDS) were calculated. ROP data, including first detected ROP stage, maximal ROP stage, ROP treatment, and PMA at first detected sign of ROP were also retrieved. Weight SDS (WSDS) at first ROP detection was calculated. RESULTS: Stepwise multivariate logistic regression analysis revealed that the best fit-model of risk factors for developing severe ROP warranting treatment included; GA (OR=0.28, CI 95% 0.12 to 0.66, p<0.01) and WSDS at first ROP detection (OR=0.22, CI 95% 0.05 to 0.89, p<0.05). CONCLUSIONS: Low weight and low WSDS at first ROP detection can be useful predictors for ROP warranting treatment.
Yao J, Ko CW, Baranov PY, Regatieri CV, Redenti S, Tucker BA, Mighty J, Tao SL, Young MJ. Enhanced differentiation and delivery of mouse retinal progenitor cells using a micropatterned biodegradable thin-film polycaprolactone scaffold. Tissue Eng Part A 2014;Abstract

The deterioration of retinal tissue in advanced stages of retinitis pigmentosa and age-related macular degeneration and the lack of signaling cues for laminar regeneration are significant challenges highlighting the need for a tissue-engineering approach to retinal repair. In this study, we fabricated a biodegradable thin-film polycaprolactone (PCL) scaffold with varying surface topographies using microfabrication techniques. Mouse retinal progenitor cells (mRPC) cultured on PCL scaffolds exhibited enhanced potential to differentiate towards a photoreceptor fate in comparison to mRPCs cultured on control substrates, suggesting that PCL scaffolds are promising as substrates to guide differentiation of mRPCs towards a photoreceptor fate in vitro prior to transplantation. When co-cultured with the retinal explants of rhodopsin null mice, mRPC/PCL constructs showed increased mRPC integration rates compared to directly applied dissociated mRPCs. Moreover, these mRPC/PCL constructs could be delivered into the sub-retinal space of rhodopsin null mice with minimal disturbance of the host retina. Whether co-cultured with retinal explants or transplanted into the sub-retinal space, newly integrated mRPCs localized to the outer nuclear layer and expressed appropriate markers of photoreceptor fate. Thus, the PCL scaffold provides a platform to guide differentiation and organized deliver of mRPCs as a practical strategy to repair damaged retina.

Birsner AE, Benny O, D'Amato RJ. The corneal micropocket assay: a model of angiogenesis in the mouse eye. J Vis Exp 2014;(90)Abstract
The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.
Hu Y, Lin H, Dib B, Atik A, Bouzika P, Lin C, Yan Y, Tang S, Miller JW, Vavvas DG. Cholesterol crystals induce inflammatory cytokines expression in a human retinal pigment epithelium cell line by activating the NF-κB pathway. Discov Med 2014;18(97):7-14.Abstract

PURPOSE: To investigate the expression of inflammatory cytokines in ARPE-19 cells after stimulation with cholesterol crystals. METHODS: APRE-19 cells were cultured, primed with IL-1α, and treated with cholesterol crystals under different concentrations. Inflammatory cytokines (mature-IL-1β, IL-6, and IL-8) in supernatant and inflammatory cytokines (pro-IL-1β, IL-18) in cell lysate were detected by western blot. The NF-κB pathway inhibitor BAY 11-7082 was used to determine the pathway of cytokine expression. RESULTS: Cholesterol crystals did not induce the nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome, but did increase pro-IL-1β expression in ARPE-19 cells. Cholesterol crystals increased pro-IL-1β expression by activating the NF-κB pathway. Cholesterol crystal activation of the NF-κB pathway also leads to increased IL-6 and IL-8 expression. CONCLUSION: Cholesterol crystals can induce inflammatory cytokine expression in ARPE-19 cells by activating the NF-κB pathway.

Sahin O, Ziaei A. The role of methotrexate in resolving ocular inflammation after specific therapy for presumed latent syphilitic uveitis and presumed tuberculosis-related uveitis. Retina 2014;34(7):1451-9.Abstract
PURPOSE: This study was designed to investigate whether the antiinflammatory and antiproliferative activity of oral and intravitreal methotrexate (MTX) suppresses intraocular inflammation in patients with presumed latent syphilitic uveitis and presumed tuberculosis-related uveitis. METHODS: Interventional prospective study including three cases with presumed latent syphilitic uveitis treated with intravenous penicillin and oral MTX, and two cases with presumed tuberculosis-related uveitis treated with standard antituberculosis therapy and intravitreal MTX injections. Treatment efficacy of all cases was assessed by best-corrected visual acuity, fundus fluorescein angiography, and optical coherence tomography. RESULTS: Four eyes of 3 patients with presumed latent syphilitic uveitis had improved best-corrected visual acuity, suppression of intraocular inflammation, and resolution of cystoid macular edema in 6 months with oral MTX therapy. No recurrence of intraocular inflammation was observed in 6 months to 18 months of follow-up period after cessation of MTX. Two eyes of two patients with presumed tuberculosis-related uveitis showed improved best-corrected visual acuity, suppression of intraocular inflammation, and resolution of cystoid macular edema after intravitreal injections of MTX. No recurrence of intraocular inflammation was observed in 6 months to 8 months of follow-up period after cessation of antituberculous therapy. CONCLUSION: For the first time in the treatment of presumed latent syphilitic uveitis and presumed tuberculosis-related uveitis, we believe that MTX might have an adjunctive role to suppress intraocular inflammation, reduce uveitic macular edema, and prevent the recurrences of the diseases.
Patel M, Vavvas DG. Spontaneous resolution of a postvitrectomy macular hole retinal detachment. Retin Cases Brief Rep 2014;8(3):161-3.Abstract

PURPOSE: The purpose of this report was to describe a case of spontaneous resolution of a large postvitrectomy macular hole retinal detachment. METHODS: Case report and optical coherence tomography imaging. RESULTS: A 64-year-old man with history of macula-off retinal detachment and 4 previous vitrectomies in the left eye developed a macular hole and associated retinal detachment 3 months after his last vitreoretinal surgery. Two months later, examination revealed that the macular hole had spontaneously closed, and the retinal detachment had resolved. CONCLUSION: Spontaneous resolution of macular hole-associated retinal detachment in a previously vitrectomized eye has not been reported previously. Changes in tangential traction by the associated epiretinal membrane, improvement of the cystoid changes noted at the edge of the macular hole, and/or proliferation of glial tissue to bridge the hole, along with the absorption of the subretinal fluid by the retinal pigment epithelium pump contributed to this rare event have been hyphothesized.

Sweigard HJ, Yanai R, Gaissert P, Saint-Geniez M, Kataoka K, Thanos A, Stahl GL, Lambris JD, Connor KM. The alternative complement pathway regulates pathological angiogenesis in the retina. FASEB J 2014;28(7):3171-82.Abstract
A defining feature in proliferative retinopathies is the formation of pathological neovessels. In these diseases, the balance between neovessel formation and regression determines blindness, making the modulation of neovessel growth highly desirable. The role of the immune system in these retinopathies is of increasing interest, but it is not completely understood. We investigated the role of the alternative complement pathway during the formation and resolution of aberrant neovascularization. We used alternative complement pathway-deficient (Fb(-/-)) mice and age- and strain-matched control mice to assess neovessel development and regression in an oxygen-induced retinopathy (OIR) mouse model. In the control mice, we found increased transcription of Fb after OIR treatment. In the Fb(-/-) mice, we prepared retinal flatmounts and identified an increased number of neovessels, peaking at postnatal day 17 (P17; P=0.001). Subjecting human umbilical vein endothelial cells (HUVECs) to low oxygen, mimicking a characteristic of neovessels, decreased the expression of the complement inhibitor Cd55. Finally, using laser capture microdissection (LCM) to isolate the neovessels after OIR, we found decreased expression of Cd55 (P=0.005). Together, our data implicate the alternative complement pathway in facilitating neovessel clearance by down-regulating the complement inhibitor Cd55 specifically on neovessels, allowing for their targeted removal while leaving the established vasculature intact.-Sweigard, J. H., Yanai, R., Gaissert, P., Saint-Geniez, M., Kataoka, K., Thanos, A., Stahl, G. L., Lambris, J. D., Connor, K. M. The alternative complement pathway regulates pathological angiogenesis in the retina.
Farkas MH, Lew DS, Sousa ME, Bujakowska K, Chatagnon J, Bhattacharya SS, Pierce EA, Nandrot EF. Mutations in pre-mRNA processing factors 3, 8, and 31 cause dysfunction of the retinal pigment epithelium. Am J Pathol 2014;184(10):2641-52.Abstract

Mutations in the ubiquitously expressed pre-mRNA processing factors 3, 8, and 31 (PRPF3, PRPF8, and PRPF31) cause nonsyndromic dominant retinitis pigmentosa in humans, an inherited retinal degeneration. It is unclear what mechanisms, or which cell types of the retina, are affected. Transgenic mice with the human mutations in these genes display late-onset morphological changes in the retinal pigment epithelium (RPE). To determine whether the observed morphological changes are preceded by abnormal RPE function, we investigated its phagocytic function in Prpf3(T494M/T494M), Prpf8(H2309P/H2309P), and Prpf31(+/-) mice. We observe decreased phagocytosis in primary RPE cultures from mutant mice, and this is replicated by shRNA-mediated knockdown of PRPF31 in human ARPE-19 cells. The diurnal rhythmicity of phagocytosis is almost lost, indicated by the marked attenuation of the phagocytic burst 2 hours after light onset. The strength of adhesion between RPE apical microvilli and photoreceptor outer segments also declined during peak adhesion in all mutants. In all models, at least one of the receptors involved in binding and internalization of shed photoreceptor outer segments was subjected to changes in localization. Although the mechanism underlying these changes in RPE function is yet to be elucidated, these data are consistent with the mouse RPE being the primary cell affected by mutations in the RNA splicing factors, and these changes occur at an early age.

Song D, Grieco S, Li Y, Hunter A, Chu S, Zhao L, Song Y, DeAngelis RA, Shi L-Y, Liu Q, Pierce EA, Nishina PM, Lambris JD, Dunaief JL. A murine RP1 missense mutation causes protein mislocalization and slowly progressive photoreceptor degeneration. Am J Pathol 2014;184(10):2721-9.Abstract
Mutations in the RP1 gene can cause retinitis pigmentosa. We identified a spontaneous L66P mutation caused by two adjacent point mutations in the Rp1 gene in a colony of C57BL/6J mice. Mice homozygous for the L66P mutation exhibited slow, progressive photoreceptor degeneration throughout their lifespan. Optical coherence tomography imaging found abnormal photoreceptor reflectivity at 1 month of age. Histology found shortening and disorganization of the photoreceptor inner and outer segments and progressive thinning of the outer nuclear layer. Electroretinogram a- and b-wave amplitudes were decreased with age. Western blot analysis found that the quantity and size of the mutated retinitis pigmentosa 1 (RP1) protein were normal. However, immunohistochemistry found that the mutant Rp1 protein partially mislocalized to the transition zone of the shortened axonemes. This mutation disrupted colocalization with cytoplasmic microtubules in vitro. In conclusion, the L66P mutation in the first doublecortin domain of the Rp1 gene impairs Rp1 protein localization and function, leading to abnormalities in photoreceptor outer segment structure and progressive photoreceptor degeneration. This is the first missense mutation in Rp1 shown to cause retinal degeneration. It provides a unique, slowly progressive photoreceptor degeneration model that mirrors the slow degeneration kinetics in most patients with retinitis pigmentosa.
Kazlauskas A. Plakophilin-2 promotes activation of epidermal growth factor receptor. Mol Cell Biol 2014;34(20):3778-9.Abstract

While growth factor-driven dimerization of receptor tyrosine kinases (RTKs) is a simple and intuitive mechanism of activating RTKs, K.-I. Arimoto et al. (Mol. Cell. Biol. 34:3843-3854, 2014, doi:10.1128/MCB.00758-14) describe a novel means of promoting the activity of RTKs. Namely, plakophilin-2 (PKP2) associates with the epidermal growth factor receptor (EGFR) and enhances its ligand-dependent and ligand-independent activity. This discovery suggests that antagonizing PKP2 may be a new therapeutic opportunity to combat tumors in which activation of EGFR contributes to pathogenesis.

Valdez CN, Arboleda-Velasquez JF, Amarnani DS, Kim LA, D'Amore PA. Retinal microangiopathy in a mouse model of inducible mural cell loss. Am J Pathol 2014;184(10):2618-26.Abstract
Diabetes can lead to vision loss because of progressive degeneration of the neurovascular unit in the retina, a condition known as diabetic retinopathy. In its early stages, the pathology is characterized by microangiopathies, including microaneurysms, microhemorrhages, and nerve layer infarcts known as cotton-wool spots. Analyses of postmortem human retinal tissue and retinas from animal models indicate that degeneration of the pericytes, which constitute the outer layer of capillaries, is an early event in diabetic retinopathy; however, the relative contribution of specific cellular components to the pathobiology of diabetic retinopathy remains to be defined. We investigated the phenotypic consequences of pericyte death on retinal microvascular integrity by using nondiabetic mice conditionally expressing a diphtheria toxin receptor in mural cells. Five days after administering diphtheria toxin in these adult mice, changes were observed in the retinal vasculature that were similar to those observed in diabetes, including microaneurysms and increased vascular permeability, suggesting that pericyte cell loss is sufficient to trigger retinal microvascular degeneration. Therapies aimed at preventing or delaying pericyte dropout may avoid or attenuate the retinal microangiopathy associated with diabetes.
Sandberg MA, Pearce EN, Harper S, Weigel-DiFranco C, Hart L, Rosner B, Berson EL. The relationship of central foveal thickness to urinary iodine concentration in retinitis pigmentosa with or without cystoid macular edema. JAMA Ophthalmol 2014;132(10):1209-14.Abstract

IMPORTANCE: Current treatments for cystoid macular edema (CME) in retinitis pigmentosa (RP) are not always effective, may lead to adverse effects, and may not restore visual acuity. The present research lays the rationale for evaluating whether an iodine supplement could reduce CME in RP. OBJECTIVE: To determine whether central foveal thickness (CFT) in the presence of CME is related to dietary iodine intake inferred from urinary iodine concentration (UIC) in nonsmoking adults with RP. DESIGN, SETTING, AND PARTICIPANTS: We performed a cross-sectional observational study of 212 nonsmoking patients aged 18 to 69 years referred to our institution for RP with visual acuity of no worse than 20/200 in at least 1 eye. EXPOSURE: Retinitis pigmentosa with or without CME. MAIN OUTCOMES AND MEASURES: With the eye as the unit of analysis, the relationship of log CFT measured by optical coherence tomography to UIC measured from multiple spot samples and represented as a 3-level classification variable (<100, 100-199, and ≥200 µg/L), assigning greater weight to patients with more reliable UIC estimates. RESULTS: Analyses were limited to 199 patients after excluding 11 who failed to return urine samples for measuring UIC and 2 outliers for UIC. Of the 199 patients, 36.2% had CME in 1 or both eyes. Although log CFT was inversely related to UIC based on findings from all eyes (P = .02), regression of log CFT on UIC separately for eyes with and without CME showed a strong inverse significant relationship for the former group (P < .001) and no significant relationship for the latter group (P = .66) as tested. For the eyes with CME, CFT ranged from a geometric mean of 267 µm for a median UIC of less than 100 µg/L to a geometric mean of 172 µm for a median UIC of 200 µg/L or greater. In contrast, we found no significant association between CME prevalence and UIC based on the entire sample as tested (odds ratio, 1.01 [95% CI, 0.38-2.67]; P = .99). CONCLUSIONS AND RELEVANCE: A higher UIC in nonsmoking adults with RP was significantly associated with less central foveal swelling in eyes with CME. Additional study is required to determine whether an iodine supplement can limit or reduce the extent of CME in patients with RP.

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