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Bestrophinopathy: An RPE-photoreceptor interface disease.

Mon, 2018-02-05 09:46
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Bestrophinopathy: An RPE-photoreceptor interface disease.

Prog Retin Eye Res. 2017 May;58:70-88

Authors: Guziewicz KE, Sinha D, Gómez NM, Zorych K, Dutrow EV, Dhingra A, Mullins RF, Stone EM, Gamm DM, Boesze-Battaglia K, Aguirre GD

Abstract
Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies.

PMID: 28111324 [PubMed - indexed for MEDLINE]

MMP19 expression in the human optic nerve.

Mon, 2018-01-22 04:35
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MMP19 expression in the human optic nerve.

Mol Vis. 2016;22:1429-1436

Authors: Chirco KR, Hazlewood RJ, Miller K, Workalemahu G, Jampol LM, Lesser GR, Mullins RF, Kuehn MH, Fingert JH

Abstract
PURPOSE: The defining feature of glaucoma is excavation of the optic nerve head; however, the mechanism of this loss of tissue is not well understood. We recently discovered a copy number variation upstream of matrix metalloproteinase 19 (MMP19) in a large, autosomal dominant pedigree with a congenital malformation of the optic disc called cavitary optic disc anomaly (CODA). Patients with CODA have abnormal optic discs that exhibit an excavated shape similar to cupping seen in glaucoma. The goal of this study is to characterize the localization of MMP19 within the human optic nerve.
METHODS: The MMP19 protein in the optic nerve was evaluated with western blot analysis and with immunohistochemistry in sagittal and en face/cross sections of optic nerves obtained from healthy human donor eyes.
RESULTS: The MMP19 protein was detected in the human optic nerve, retina, and RPE/choroid with western blot analysis, with highest expression in the retina and the optic nerve. Using immunohistochemistry, MMP19 was localized within the optic nerve to the extracellular space within the septa that separate bundles of optic nerve axons into fascicles. The presence of MMP19 within the optic nerve septa was further confirmed by the colocalization of MMP19 to this structure with type IV collagen. Strong labeling of MMP19 was also detected in the arachnoid layer of the optic nerve sheath. Finally, immunohistochemistry of the optic nerve cross sections demonstrated that MMP19 shows a peripheral to central gradient, with more abundant labeling along the edges of the optic nerve and in the arachnoid layer than in the center of the nerve.
CONCLUSIONS: Abundant MMP19 was detected in the optic nerve head, the primary site of pathology in patients with CODA. The localization of MMP19 to the optic nerve septa is consistent with its predicted secretion and accumulation within the extracellular spaces of this tissue. Moreover, the lateral localization of MMP19 observed in the optic nerve cross sections suggests that it might have a role in regulating adhesion to the optic nerve to the scleral canal and remodeling the extracellular matrix that provides the structural integrity of the optic disc. Dysregulation of MMP19 production might, therefore, undermine the connections between the optic nerve and the scleral canal and cause a collapse of the optic disc and the development of CODA. Similar processes might also be at work in the formation of optic disc cupping in glaucoma.

PMID: 28003733 [PubMed - indexed for MEDLINE]

Using Patient-Specific Induced Pluripotent Stem Cells and Wild-Type Mice to Develop a Gene Augmentation-Based Strategy to Treat CLN3-Associated Retinal Degeneration.

Mon, 2018-01-22 04:35
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Using Patient-Specific Induced Pluripotent Stem Cells and Wild-Type Mice to Develop a Gene Augmentation-Based Strategy to Treat CLN3-Associated Retinal Degeneration.

Hum Gene Ther. 2016 Oct;27(10):835-846

Authors: Wiley LA, Burnight ER, Drack AV, Banach BB, Ochoa D, Cranston CM, Madumba RA, East JS, Mullins RF, Stone EM, Tucker BA

Abstract
Juvenile neuronal ceroid lipofuscinosis (JNCL) is a childhood neurodegenerative disease with early-onset, severe central vision loss. Affected children develop seizures and CNS degeneration accompanied by severe motor and cognitive deficits. There is no cure for JNCL, and patients usually die during the second or third decade of life. In this study, independent lines of induced pluripotent stem cells (iPSCs) were generated from two patients with molecularly confirmed mutations in CLN3, the gene mutated in JNCL. Clinical-grade adeno-associated adenovirus serotype 2 (AAV2) carrying the full-length coding sequence of human CLN3 was generated in a U.S. Food and Drug Administration-registered cGMP facility. AAV2-CLN3 was efficacious in restoring full-length CLN3 transcript and protein in patient-specific fibroblasts and iPSC-derived retinal neurons. When injected into the subretinal space of wild-type mice, purified AAV2-CLN3 did not show any evidence of retinal toxicity. This study provides proof-of-principle for initiation of a clinical trial using AAV-mediated gene augmentation for the treatment of children with CLN3-associated retinal degeneration.

PMID: 27400765 [PubMed - indexed for MEDLINE]

Drusen on Demand? Authors Describe a Novel Culture System for Generating subRPE Deposits.

Mon, 2018-01-08 00:57
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Drusen on Demand? Authors Describe a Novel Culture System for Generating subRPE Deposits.

Invest Ophthalmol Vis Sci. 2017 02 01;58(2):720

Authors: Mullins RF

PMID: 28152142 [PubMed - indexed for MEDLINE]

CLINICOPATHOLOGICAL CORRELATION IN A PATIENT WITH PREVIOUSLY TREATED BIRDSHOT CHORIORETINOPATHY.

Mon, 2018-01-08 00:57
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CLINICOPATHOLOGICAL CORRELATION IN A PATIENT WITH PREVIOUSLY TREATED BIRDSHOT CHORIORETINOPATHY.

Retin Cases Brief Rep. 2017 Fall;11(4):344-347

Authors: Sohn EH, Chirco KR, Folk JC, Mullins RF

Abstract
PURPOSE: Birdshot chorioretinopathy (BCR) is a bilateral, chronic uveitis primarily involving the posterior segment that often results in progressive vision loss. Histopathology on eyes with BCR has been limited, but we had the rare opportunity to study the eyes of a donor with BCR. We sought to compare immunolabeling in the eyes of this donor who was treated with immunosuppression for over 30 years to age-matched controls.
METHODS: From each eye, a macular punch and superotemporal regions were used for cryostat sectioning, and immunohistochemistry was performed on the sections using antibodies directed against CD45, intercellular adhesion molecule-1, IBA1, and GFAP. The vasculature-binding lectin, Ulex europaeus agglutinin-I (UEA-I), was also used to perform lectin histochemistry.
RESULTS: At death, her visual acuity was 20/25 right eye, 20/250 left eye with extensive chorioretinal atrophy, vascular attenuation, and disk pallor. Compared with controls, the BCR donor had extensive degeneration of the outer nuclear layer and retinal pigment epithelium as well as choroidal thinning with inner retinal preservation. Loss of UEA-I+ choroidal endothelial cells was extensive, and atypical intercellular adhesion molecule-1 labeling and IBA+ microglia/macrophages were present along with widespread GFAP labeling throughout the retina.
CONCLUSION: The BCR may cause progressive chorioretinal and optic atrophy with long-standing increased leukocyte abundance throughout the retina and microglial activation especially at the retina-choroid interface.

PMID: 27465484 [PubMed - indexed for MEDLINE]

APOPTOSIS AND ANGIOFIBROSIS IN DIABETIC TRACTIONAL MEMBRANES AFTER VASCULAR ENDOTHELIAL GROWTH FACTOR INHIBITION: Results of a Prospective Trial. Report No. 2.

Sun, 2017-12-03 16:29

APOPTOSIS AND ANGIOFIBROSIS IN DIABETIC TRACTIONAL MEMBRANES AFTER VASCULAR ENDOTHELIAL GROWTH FACTOR INHIBITION: Results of a Prospective Trial. Report No. 2.

Retina. 2017 Nov 22;:

Authors: Jiao C, Eliott D, Spee C, He S, Wang K, Mullins RF, Hinton DR, Sohn EH

Abstract
PURPOSE: We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy.
METHODS: Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed.
RESULTS: High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF.
CONCLUSION: Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.

PMID: 29190236 [PubMed - as supplied by publisher]

Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury.

Sun, 2017-12-03 16:29
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Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury.

FASEB J. 2017 Nov;31(11):4903-4916

Authors: Zhao Z, Liang Y, Liu Y, Xu P, Flamme-Wiese MJ, Sun D, Sun J, Mullins RF, Chen Y, Cai J

Abstract
γδ T cells located near the epithelial barrier are integral components of local inflammatory and innate immune responses. We have previously reported the presence of choroidal γδ T cells in a model of chronic degeneration of the retinal pigment epithelium (RPE). The goals of the current study were to further define the functions of choroidal γδ T cells and to explore the underlying mechanisms of their action. Our data demonstrate that choroidal γδ T cells are activated by RPE injury in response to NaIO3 treatment, and that they express genes that encode immunosuppressive cytokines, such as IL-4 and IL-10. γδ-T-cell-deficient mice developed profound RPE and retinal damage at doses that caused minimal effects in wild-type mice, and adoptive transfer of γδ T cells prevented sensitization. Intravitreal injection of IL-4 and IL-10 ameliorated RPE toxicity that was induced by NaIO3Ex vivo coculture of γδ T cells with RPE explants activated the production of anti-inflammatory cytokines via an aryl hydrocarbon receptor (AhR)-dependent mechanism. AhR deficiency abolished the protective effects of γδ T cells after adoptive transfer. Collectively, these findings define important roles for choroid γδ T cells in maintaining tissue homeostasis in the outer retina.-Zhao, Z., Liang, Y., Liu, Y., Xu, P., Flamme-Wiese, M. J., Sun, D., Sun, J., Mullins, R. F., Chen, Y., Cai, J. Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury.

PMID: 28729290 [PubMed - indexed for MEDLINE]

From compliment to insult: genetics of the complement system in physiology and disease in the human retina.

Sun, 2017-12-03 16:29
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From compliment to insult: genetics of the complement system in physiology and disease in the human retina.

Hum Mol Genet. 2017 Aug 01;26(R1):R51-R57

Authors: Mullins RF, Warwick AN, Sohn EH, Lotery AJ

Abstract
Age-related macular degeneration (AMD) is a major cause of visual impairment that affects the central retina. Genome wide association studies and candidate gene screens have identified members of the complement pathway as contributing to the risk of AMD. In this review, we discuss the complement system, its importance in retinal development and normal physiology, how its dysregulation may contribute to disease, and how it might be targeted to prevent damage to the aging choriocapillaris in AMD.

PMID: 28482029 [PubMed - indexed for MEDLINE]

Prevascularized silicon membranes for the enhancement of transport to implanted medical devices.

Sun, 2017-12-03 16:29
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Prevascularized silicon membranes for the enhancement of transport to implanted medical devices.

J Biomed Mater Res B Appl Biomater. 2016 Nov;104(8):1602-1609

Authors: Worthington KS, Wiley LA, Mullins RF, Tucker BA, Nuxoll E

Abstract
Recent advances in drug delivery and sensing devices for in situ applications are limited by the diffusion-limiting foreign body response of fibrous encapsulation. In this study, we fabricated prevascularized synthetic device ports to help mitigate this limitation. Membranes with rectilinear arrays of square pores with widths ranging from 40 to 200 μm were created using materials (50 μm thick double-sided polished silicon) and processes (photolithography and directed reactive ion etching) common in the manufacturing of microfabricated sensors. Vascular endothelial cells responded to membrane geometry by either forming vascular tubes that extended through the pore or completely filling membrane pores after 4 days in culture. Although tube formation began to predominate overgrowth around 75 μm and continued to increase at even larger pore sizes, tubes formed at these large pore sizes were not completely round and had relatively thin walls. Thus, the optimum range of pore size for prevascularization of these membranes was estimated to be 75-100 μm. This study lays the foundation for creating a prevascularized port that can be used to reduce fibrous encapsulation and thus enhance diffusion to implanted medical devices and sensors. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1602-1609, 2016.

PMID: 26316050 [PubMed - indexed for MEDLINE]

ASSESSMENT OF AAV SEROTYPE TROPISM IN HUMAN RETINAL EXPLANTS.

Sun, 2017-11-26 14:12
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ASSESSMENT OF AAV SEROTYPE TROPISM IN HUMAN RETINAL EXPLANTS.

Hum Gene Ther. 2017 Nov 21;:

Authors: Wiley LA, Burnight E, Kaalberg EE, Jiao C, Riker MJ, Halder JA, Luse MA, Han IC, Russell SR, Sohn EH, Stone E, Tucker BA, Mullins RF

Abstract
Advances in the discovery of the causes of monogenic retinal disorders, combined with technologies for the delivery of DNA to the retina, offer enormous opportunities for the treatment of previously untreatable blinding diseases. However, for gene augmentation to be most effective, vectors that have the correct cell-type specificity are needed. While animal models are very useful, they often exhibit differences in retinal cell surface receptors compared to human retina. In this study, we evaluated the use of an ex vivo organotypic explant system to test the transduction efficiency and tropism of 7 different adeno-associated viral type 2 (AAV2) serotypes in human retina and retinal pigment epithelium-choroid: AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, and AAV2/9, all driving expression of GFP under control of the cytomegalovirus promoter. After 7 days in culture, we found that AAV2/4 and AAV2/5 are particularly efficient at transducing photoreceptor cells and that AAV2/5 is highly specific to the outer nuclear layer. To validate the authenticity of the organotypic culture system, we also compared the transduction of the same set of AAVs in a pig model, in which subretinal injections in vivo were compared to cultured and transduced organotypic cultures ex vivo. This study shows how different AAV serotypes behave in the human retina and will provide insight for further investigation of each of these serotypes for gene augmentation-based treatment of inherited retinal degeneration.

PMID: 29160116 [PubMed - as supplied by publisher]

Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies.

Sun, 2017-11-05 06:18
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Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies.

Elife. 2016 Nov 15;5:

Authors: Lee WH, Higuchi H, Ikeda S, Macke EL, Takimoto T, Pattnaik BR, Liu C, Chu LF, Siepka SM, Krentz KJ, Rubinstein CD, Kalejta RF, Thomson JA, Mullins RF, Takahashi JS, Pinto LH, Ikeda A

Abstract
While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases.

PMID: 27863209 [PubMed - indexed for MEDLINE]