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1.
Int J Mol Sci ; 22(4)2021 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-33672445

RESUMEN

Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders.


Asunto(s)
Edición Génica , Células Madre Pluripotentes Inducidas/patología , Fagocitosis , Epitelio Pigmentado de la Retina/patología , Retinitis Pigmentosa/patología , Diferenciación Celular/genética , Línea Celular , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/ultraestructura , Mutación/genética , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/patología , Segmento Externo de las Células Fotorreceptoras Retinianas/ultraestructura , Epitelio Pigmentado de la Retina/ultraestructura , Retinitis Pigmentosa/genética , Tirosina Quinasa c-Mer/genética , Tirosina Quinasa c-Mer/metabolismo
2.
Int J Mol Sci ; 22(4)2021 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-33567500

RESUMEN

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness worldwide in the elderly population. In our previous studies, we found that deficiencies in the nuclear factor, erythroid 2 like 2 (NFE2L2) and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) genes caused AMD-like pathological phenotypes in mice. In the present work, we show hijacked epithelial-mesenchymal transition (EMT) due to the common loss of PGC-1α and NFE2L2 (double knock-out, dKO) genes in aged animals. The implanted area was assessed by histology, immunohistochemistry and transmission electron microscopy. Confocal microscopy revealed altered regions in the filamentous actin ring. This contrasted with hexagonal RPE morphology in wild-type mice. The ultrastructural RPE features here illustrated loss of apical microvilli, alteration of cell-cell contact, loss of basal in-folding with deposits on Bruch's membrane, and excessive lipofuscin deposition in dKO samples. We also found the expression of epithelial-mesenchymal transition transcription factors, such as Snail, Slug, collagen 1, vimentin and OB-cadherin, to be significantly different in dKO RPEs. An increased immunoreactivity of senescence markers p16, DEC1 and HMGB1 was also noted. These findings suggest that EMT and senescence pathways may intersect in the retinas of dKO mice. Both processes can be activated by damage to the RPE, which may be caused by increased oxidative stress resulting from the absence of NFE2L2 and PGC-1α genes, important for antioxidant defense. This dKO model may provide useful tools for studying AMD pathogenesis and evaluating novel therapies for this disease.


Asunto(s)
Senescencia Celular , Transición Epitelial-Mesenquimal , Mitocondrias/patología , Factor 2 Relacionado con NF-E2/fisiología , Estrés Oxidativo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/fisiología , Epitelio Pigmentado de la Retina/patología , Animales , Degeneración Macular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal
3.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33572787

RESUMEN

Age-related macular degeneration (AMD) is the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris among elderly individuals and is the leading cause of blindness worldwide. Thus, a better understanding of the underlying mechanisms in retinal tissue activated by blue light exposure is important for developing novel treatment and intervention strategies. In this study, blue-light-emitting diodes with a wavelength of 440 nm were applied to RPE cells at a dose of 3.7 ± 0.75 mW/cm2 for 24 h. ARPE-19 cells were used to investigate the underlying mechanism induced by blue light exposure. A trypan blue exclusion assay was used for the cell viability determination. Flow cytometry was used for apoptosis rate detection and autophagy analysis. An immunofluorescence microscopy analysis was used to investigate cellular oxidative stress and DNA damage using DCFDA fluorescence staining and an anti-γH2AX antibody. Blue light exposure of zebrafish larvae was established to investigate the effect on retinal tissue development in vivo. To further demonstrate the comprehensive effect of blue light on ARPE-19 cells, next-generation sequencing (NGS) was performed for an ingenuity pathway analysis (IPA) to reveal additional related mechanisms. The results showed that blue light exposure caused a decrease in cell proliferation and an increase in apoptosis in ARPE-19 cells in a time-dependent manner. Oxidative stress increased during the early stage of 2 h of exposure and activated DNA damage in ARPE-19 cells after 8 h. Furthermore, autophagy was activated in response to blue light exposure at 24-48 h. The zebrafish larvae model showed the unfavorable effect of blue light in prohibiting retinal tissue development. The RNA-Seq results confirmed that blue light induced cell death and participated in tissue growth inhibition and maturation. The current study reveals the mechanisms by which blue light induces cell death in a time-dependent manner. Moreover, both the in vivo and NGS data uncovered blue light's effect on retinal tissue development, suggesting that exposing children to blue light could be relatively dangerous. These results could benefit the development of preventive strategies utilizing herbal medicine-based treatments for eye diseases or degeneration in the future.


Asunto(s)
Autofagia/efectos de la radiación , Daño del ADN/efectos de la radiación , Luz/efectos adversos , Degeneración Macular/etiología , Estrés Oxidativo/efectos de la radiación , Epitelio Pigmentado de la Retina/efectos de la radiación , Animales , Línea Celular , Modelos Animales de Enfermedad , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Pez Cebra
4.
Biomed Pharmacother ; 133: 111041, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33378949

RESUMEN

Poly (ADP-ribose) polymerase 1 (PARP1)-dependent cell death in the retinal pigment epithelium (RPE) is implicated in dry age-related macular degeneration (AMD). Although PARP1 inhibitors are available for treating dry AMD, their delivery route is not ideal for patients. The aim of this study was to test the efficacy of a novel PARP1-inhibitory compound (PIC) in vitro and in vivo. This study presents PIC, a novel small molecule, with superior efficacy to PARP1 inhibitors in the market. PIC demonstrated a distinctive inhibitory profile against PARP isotypes than the FDA-approved PARP1 inhibitors. PIC inhibited PARP1 activation at an IC50 of 0.41 ± 0.15 nM in an enzyme-based assay in vitro and at IC50 and EC50 in ARPE-19 cells of 0.11 ± 0.02 nM and 0.22 ± 0.02 nM, respectively, upon H2O2 insult. PIC also moderated mitochondrial fission and depolarization and maintained cellular energy levels under oxidative stress in ARPE-19 cells. Furthermore, PIC demonstrated good corneal penetration in a rat model, presenting PIC as a promising candidate for eye drop therapeutics for dry AMD. When PIC was administered as an eye drop formulation, RPE morphology was preserved, maintaining the thickness of the outer nuclear layers under sodium iodate (SI) treatment in rats. In SI-treated rabbits, eye drop administration of PIC also retained the structural and functional integrity when analyzed using funduscopy and electroretinogram. Collectively, our data portray PIC as an attractive treatment measure for dry AMD.


Asunto(s)
Degeneración Macular/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Administración Oftálmica , Animales , Antioxidantes/farmacología , Línea Celular , Modelos Animales de Enfermedad , Humanos , Yodatos , Degeneración Macular/inducido químicamente , Degeneración Macular/enzimología , Degeneración Macular/patología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Absorción Ocular , Soluciones Oftálmicas , Estrés Oxidativo/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Conejos , Ratas Sprague-Dawley , Epitelio Pigmentado de la Retina/enzimología , Epitelio Pigmentado de la Retina/patología
5.
Am J Ophthalmol ; 223: 129-139, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33342759

RESUMEN

PURPOSE: To assess the relationship between demographics, clinical characteristics, and structural optical coherence tomography (OCT) findings and the development of sight-threatening macular complications (choroidal neovascularization [CNV], large areas of retinal pigment epithelium [RPE] atrophy, and cystoid macular degeneration [CMD]) in a cohort of eyes with "resolved" chronic central serous chorioretinopathy (CSC) at study baseline. DESIGN: Retrospective cohort study. METHODS: In this study, a total of 71 eyes with "resolved" (absence of subretinal fluid) chronic CSC at baseline and 36 months of regular follow-up examinations were retrospectively enrolled. Structural OCT scans were reviewed. Baseline OCT qualitative features reflecting distress of the neuroretina, RPE, or choroid were assessed and included ellipsoid zone discontinuity, outer nuclear layer (ONL) thinning; presence of hyper-reflective intraretinal foci; dome-shaped pigment epithelium detachment (PED); hyper-reflective flat, irregular PED; hyporeflective flat, irregular PED; and inner choroidal attenuation. OCT images obtained at follow-up visits were also reviewed for development of macular complications (CNV, large areas of RPE atrophy [at least 250 µm in diameter], and CMD). Main outcome measurements included incidence of macular complications and hazard ratio (HR) for demographics, clinical characteristics, and OCT risk factors. RESULTS: At month 36, 20 eyes (28.2%) developed macular complications. Nine eyes (12.7%) displayed CNV, 9 eyes (12.7%) had large areas of RPE atrophy, and 2 eyes (2.8%) developed cystoid macular degeneration. The following factors were associated with an increased risk of development of CNV: intraretinal hyper-reflective foci had an HR of 11.58 (95% confidence interval [CI]: 1.10-37.24; P = .040); inner choroidal attenuation had an HR of 9.66 (95% CI: 1.07-22.34; P = .043); and the presence of macular complications in the fellow eye had an HR of 20.17 (95% CI: 1.34-39.41; P = .030). Factors associated with the development of RPE atrophy were also identified: ONL thinning had an HR of 13.47 (95% CI: 1.10-39.86; P = .042); dome-shaped PED had an HR of 21.40 (95% CI: 1.50-41.10; P = .031); and inner choroidal attenuation had an HR of 13.20 (95% CI: 1.07-39.32; P = .044). CONCLUSIONS: OCT risk factors were identified for the development of macular complications in eyes with chronic CSC. Findings may help in the identification of high-risk patients.


Asunto(s)
Coriorretinopatía Serosa Central/complicaciones , Neovascularización Coroidal/epidemiología , Edema Macular/epidemiología , Epitelio Pigmentado de la Retina/patología , Tomografía de Coherencia Óptica , Adulto , Atrofia , Neovascularización Coroidal/diagnóstico por imagen , Enfermedad Crónica , Colorantes/administración & dosificación , Femenino , Angiografía con Fluoresceína , Estudios de Seguimiento , Humanos , Verde de Indocianina/administración & dosificación , Edema Macular/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Estudios Retrospectivos , Factores de Riesgo , Líquido Subretiniano , Agudeza Visual/fisiología
6.
Am J Ophthalmol ; 223: 160-168, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33342761

RESUMEN

PURPOSE: To reveal the characteristics of ocular changes in patients with biallelic CRB1 mutations. DESIGN: Comparative exome sequencing and retrospective case series on clinical data. METHODS: Seventy-four patients from 63 families with biallelic potential pathogenic variants in CRB1 were selected from our in-house exome sequencing. The clinical data were reviewed and evaluated in detail, including best-corrected visual acuity, fundus photography, optical coherence tomography (OCT), and electroretinogram (ERG). RESULTS: Biallelic CRB1 variants, involving 45 variants including 23 novel, were identified in 40 novel families based on exome sequencing. Analyzing clinical data of the 74 individuals from 63 families revealed the following CRB1-associated phenotypes: (1) early-onset reduced visual acuity with congenital nystagmus; (2) 2 types of characteristic retinal changes including yellowish geographic macular degeneration (YMD) or nummular pigment deposits (NPD) at posterior retina with bone-spicule pigmentation at midperipheral retina; (3) undetectable rod and cone responses on ERG; (4) cystoid macular edema or macular atrophy on OCT. YMD and NPD are unique and CRB1-associated. Long-term follow-up examination as well as age- and variant-dependent phenotypic analysis suggested YMD is the early fundus change that would gradually progress to NPD. CONCLUSIONS: YMD and NPD are 2 major characteristic CRB1-associated fundus changes and the former one will advance to the latter with age. Recognizing such characteristic signs associated with biallelic CRB1 variants may be of value in areas without widespread access to genetic testing where a more targeted approach is needed and might be biomarkers for evaluation of effects for future intervention.


Asunto(s)
Proteínas del Ojo/genética , Atrofia Geográfica/genética , Proteínas de la Membrana/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Degeneración Retiniana/genética , Epitelio Pigmentado de la Retina/patología , Adolescente , Adulto , Niño , Preescolar , Análisis Mutacional de ADN , Electrorretinografía , Salud de la Familia , Femenino , Angiografía con Fluoresceína , Fondo de Ojo , Estudios de Asociación Genética , Pruebas Genéticas , Atrofia Geográfica/diagnóstico , Humanos , Lactante , Recién Nacido , Masculino , Linaje , Degeneración Retiniana/diagnóstico , Estudios Retrospectivos , Tomografía de Coherencia Óptica , Agudeza Visual , Secuenciación del Exoma Completo
7.
J Vis Exp ; (166)2020 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-33369607

RESUMEN

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.


Asunto(s)
Elementos Transponibles de ADN/genética , Estrés Oxidativo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Transfección , Antioxidantes/farmacología , Biomarcadores/metabolismo , Recuento de Células , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/química , Células Epiteliales/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/aislamiento & purificación , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/aislamiento & purificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Peróxido de Hidrógeno/toxicidad , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/aislamiento & purificación , Factores de Crecimiento Nervioso/metabolismo , Neuroprotección/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serpinas/genética , Serpinas/aislamiento & purificación , Serpinas/metabolismo , Donantes de Tejidos , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo
8.
PLoS One ; 15(12): e0243626, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33347461

RESUMEN

Proliferative vitreoretinopathy (PVR) is a progressive disease that develops in a subset of patients who undergo surgery for retinal detachment repair, and results in significant vision loss. PVR is characterized by the migration of retinal pigment epithelial (RPE) cells into the vitreous cavity, where they undergo epithelial-to-mesenchymal transition and form contractile membranes within the vitreous and along the retina, resulting in recurrent retinal detachments. Currently, surgical intervention is the only treatment for PVR and there are no pharmacological agents that effectively inhibit or prevent PVR formation. Here, we show that a single intravitreal injection of the polyether ionophore salinomycin (SNC) effectively inhibits the formation of PVR in a mouse model with no evidence of retinal toxicity. After 4 weeks, fundus photography and optical coherence tomography (OCT) demonstrated development of mean PVR grade of 3.5 (SD: 1.3) in mouse eyes injected with RPE cells/DMSO (vehicle), compared to mean PVR grade of 1.6 (SD: 1.3) in eyes injected with RPE cells/SNC (p = 0.001). Additionally, immunohistochemistry analysis showed RPE cells/SNC treatment reduced both fibrotic (αSMA, FN1, Vim) and inflammatory (GFAP, CD3, CD20) markers compared to control RPE cells/DMSO treatment. Finally, qPCR analysis confirmed that Tgfß, Tnfα, Mcp1 (inflammatory/cytokine markers), and Fn1, Col1a1 and Acta2 (fibrotic markers) were significantly attenuated in the RPE cells/SNC group compared to RPE/DMSO control. These results suggest that SNC is a potential pharmacologic agent for the prevention of PVR in humans and warrants further investigation.


Asunto(s)
Ionóforos/uso terapéutico , Piranos/uso terapéutico , Vitreorretinopatía Proliferativa/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Femenino , Inyecciones Intravítreas , Ionóforos/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Piranos/administración & dosificación , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología , Vitreorretinopatía Proliferativa/patología
9.
PLoS One ; 15(9): e0237078, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32881874

RESUMEN

PURPOSE: To investigate the association between outer retinal layer metrics, including photoreceptor outer segment volume, on spectral-domain optical coherence tomography (OCT) and brain volume on MRI in normal aging, Alzheimer's disease and Parkinson's disease. METHODS: This was an exploratory analysis of a cross-sectional cohort study that was approved by the Cleveland Clinic Institutional Review Board to evaluate neurodegenerative disorders. Subjects aged ≥ 50 were recruited. A comprehensive neurological exam, brain MRI with volumetric evaluation, and OCT were performed for each subject. Outer retinal layer parameters, including ellipsoid zone (EZ) to retinal pigment epithelium (RPE) volume (i.e., surrogate for panmacular photoreceptor outer segment volume), were evaluated with a novel OCT analysis platform. RESULTS: Of 85 subjects, 64 eyes of 64 subjects met MRI and OCT quality control criteria. Total brain volume (%ICV) significantly correlated with EZ-RPE volume in the normal cognition control group (n = 31, Pearson correlation coefficient 0.514, P < .01), the Parkinson's disease group (n = 19, Pearson correlation coefficient 0.482, P = .04), and the Alzheimer's dementia group (n = 14, Pearson correlation coefficient 0.526, P = .05). Multiple linear regression analysis revealed that photoreceptor outer segment (i.e., EZ-RPE) volume was an independent, influential factor on total brain volume in all study subjects (Coefficient 15.2, 95% confidence interval 7.8-22.6, P < .001). CONCLUSION: Outer retinal parameters on OCT may serve as a novel biomarker related to brain volume. This correlation was noted in control subjects suggesting a possible developmental link between retina and brain volume. This relationship was also maintained with atrophic neurodegenerative disorders. Further research is needed to explore possible threshold differences for underlying neurodegenerative disorders.


Asunto(s)
Envejecimiento/patología , Encéfalo/patología , Enfermedades Neurodegenerativas/patología , Segmento Externo de las Células Fotorreceptoras Retinianas/patología , Anciano , Encéfalo/diagnóstico por imagen , Femenino , Humanos , Modelos Lineales , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Análisis Multivariante , Enfermedades Neurodegenerativas/diagnóstico por imagen , Tamaño de los Órganos , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Epitelio Pigmentado de la Retina/patología , Tomografía de Coherencia Óptica
10.
Middle East Afr J Ophthalmol ; 27(2): 128-130, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32874047

RESUMEN

We report two cases with foveal congenital simple hamartoma of the retinal pigment epithelium (CSHRPE), as both patients presented to our retina services complaining of a unilateral decreased vision. Full ophthalmic examination and multimodal imaging were performed including fundus photography, fundus autofluorescence, optical coherence tomography, fluorescein angiography, and electrophysiological testing. Both patients presented with 20/80 vision in the affected eyes. Foveal CSHRPE was found in both eyes, along with parapapillary hyperpigmented rim, multiple pinpoint macular lesions, and few posterior pole hyperpigmented lesions. Multifocal electroretinogram showed diminished central amplitude in both eyes, with three-dimensional topography map showing blunted foveal peaks in one eye and the absence of a central peak in the other patient. Both patients had a stable vision and clinical examination of the CSHRPE during 5 and 6 years follow up, respectively. Foveal CSHRPE is usually symptomatic and results in a decline in visual acuity. Follow-up of these patients showed stable vision and clinical examination.


Asunto(s)
Hamartoma/congénito , Enfermedades de la Retina/congénito , Epitelio Pigmentado de la Retina/anomalías , Adulto , Técnicas de Diagnóstico Oftalmológico , Electrorretinografía , Angiografía con Fluoresceína , Fóvea Central/patología , Fondo de Ojo , Hamartoma/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Imagen Multimodal , Enfermedades de la Retina/diagnóstico , Epitelio Pigmentado de la Retina/patología , Tomografía de Coherencia Óptica/métodos , Agudeza Visual/fisiología
11.
PLoS One ; 15(8): e0236283, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32764794

RESUMEN

Geographic atrophy (GA) is an advanced form of dry age-related macular degeneration (AMD), in which local inflammation and hyperactivity of the complement pathway have been implicated in its pathophysiology. This study explores whether any surrogate biomarkers are specifically associated with GA. Plasma from subjects with GA, intermediate dry AMD and non-AMD control were evaluated in 2 cohorts. Cohort 1 was assayed in a 320-analyte Luminex library. Statistical analysis was performed using non-parametric and parametric methods (Kruskal-Wallis, principal component analysis, partial least squares and multivariate analysis of variance (MANOVA) and univariate ANCOVAs). Bioinformatic analysis was conducted and identified connections to the amyloid pathway. Statistically significant biomarkers identified in Cohort 1 were then re-evaluated in Cohort 2 using individual ELISA and multiplexing. Of 320 analytes in Cohort 1, 273 were rendered measurable, of which 56 were identified as changing. Among these markers, 40 were identified in univariate ANCOVAs. Serum amyloid precursor protein (sAPP) was analyzed by a separate ELISA and included in further analyses. The 40 biomarkers, sAPP and amyloid-ß (Aß) (1-42) (included for comparison) were evaluated in Cohort 2. This resulted in 11 statistically significant biomarkers, including sAPP and Aß(1-40), but not Aß(1-42). Other biomarkers identified included serum proteases- tissue plasminogen activator, tumor-associated trypsinogen inhibitor, matrix metalloproteinases 7 and 9, and non-proteases- insulin-like growth factor binding protein 6, AXL receptor tyrosine kinase, omentin, pentraxin-3 and osteopontin. Findings suggest that there is a preferential processing of APP to Aß(1-40) over Aß(1-42), and a potential role for the carboxylase activity of the γ-secretase protein, which preferentially splices sAPPß to Aß(1-40). Other markers are associated with the breakdown and remodeling of the extracellular matrix, and loss of homeostasis, possibly within the photoreceptor-retinal pigment epithelium-choriocapillaris complex. These data suggest novel disease pathways associated with GA pathogenesis and could provide potential novel targets for treatment of GA.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Atrofia Geográfica/sangre , Degeneración Macular/complicaciones , Epitelio Pigmentado de la Retina/patología , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Biomarcadores/metabolismo , Estudios de Cohortes , Biología Computacional , Femenino , Fondo de Ojo , Atrofia Geográfica/diagnóstico , Atrofia Geográfica/etiología , Atrofia Geográfica/patología , Humanos , Degeneración Macular/sangre , Degeneración Macular/patología , Masculino , Persona de Mediana Edad , Imagen Óptica , Transducción de Señal , Activador de Tejido Plasminógeno
12.
PLoS One ; 15(7): e0236298, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32701996

RESUMEN

Degeneration of the retinal pigment epithelium (RPE) plays a central role in age-related macular degeneration (AMD). Throughout life, RPE cells are challenged by a variety of cytotoxic stressors, some of which are cumulative with age and may ultimately contribute to drusen and lipofuscin accumulation. Stressors such as these continually damage RPE cells resulting in a state of chronic wounding. Current cell-based platforms that model a state of chronic RPE cell wounding are limited, and the RPE cellular response is not entirely understood. Here, we used the electric cell-substrate impedance sensing (ECIS) system to induce a state of acute or chronic wounding on differentiated human fetal RPE cells to analyze changes in the wound repair response. RPE cells surrounding the lesioned area employ both cell migration and proliferation to repair wounds but fail to reestablish their original cell morphology or density after repetitive wounding. Chronically wounded RPE cells develop phenotypic AMD characteristics such as loss of cuboidal morphology, enlarged size, and multinucleation. Transcriptomic analysis suggests a systemic misregulation of RPE cell functions in bystander cells, which are not directly adjacent to the wound. Genes associated with the major RPE cell functions (LRAT, MITF, RDH11) significantly downregulate after wounding, in addition to differential expression of genes associated with the cell cycle (CDK1, CDC6, CDC20), inflammation (IL-18, CCL2), and apoptosis (FAS). Interestingly, repetitive wounding resulted in prolonged misregulation of genes, including FAS, LRAT, and PEDF. The use of ECIS to induce wounding resulted in an over-representation of AMD-associated genes among those dysregulated genes, particularly genes associated with advanced AMD. This simple system provides a new model for further investigation of RPE cell wound response in AMD pathogenesis.


Asunto(s)
Degeneración Macular/patología , Modelos Biológicos , Enfermedad Aguda , Efecto Espectador , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Enfermedad Crónica , Feto/patología , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Cinética , Degeneración Macular/genética , Epitelio Pigmentado de la Retina/embriología , Epitelio Pigmentado de la Retina/patología , Transcriptoma/genética , Cicatrización de Heridas
14.
Am J Pathol ; 190(9): 1813-1822, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32473920

RESUMEN

Diabetic retinopathy (DR) is the leading cause of visual impairment in developed nations. Though plasma microRNA-93 (miR-93) is associated with the risk of DR, the function and regulatory mechanism of miR-93 during DR remains unclear. Blood samples were collected from 12 DR patients and 12 healthy controls. Primary human retinal pigment epithelium (RPE) cells and ARPE-19 cells were cultured in 5 mmol/L or 33 mmol/L d-glucose medium. Long noncoding (lnc) RNA MEG3 and miR-93 expression was detected by real-time quantitative PCR. The effect of MEG3 and miR-93 on high glucose (HG)-induced apoptosis was detected by MTT and flow cytometry. IL-6 and tumor necrosis factor-α levels were detected by enzyme-linked immunosorbent assay. The relationships among MEG3, miR-93, and Nrf2 (also known as NFE2L2) were explored via dual-luciferase reporter assay. lncRNA MEG3 and Nrf2 were decreased and miR-93 was increased in blood samples of DR patients and HG-treated human RPE and ARPE-19 cells. Overexpression of miR-93 inhibited cell proliferation and promoted apoptosis, whereas overexpression of Nrf2 or MEG3 promoted proliferation and suppressed apoptosis and inflammation. In addition, MEG3 targeted miR-93 and down-regulated miR-93. Moreover, miR-93 directly targeted Nrf2 and negatively regulated Nrf2. This study suggests that lncRNA MEG3 depresses HG-induced apoptosis and inflammation of RPE via miR-93/Nrf2 axis, providing a novel perspective on the genesis and development of DR.


Asunto(s)
Retinopatía Diabética/metabolismo , MicroARNs/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , ARN Largo no Codificante/metabolismo , Epitelio Pigmentado de la Retina/patología , Apoptosis/fisiología , Línea Celular , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Retinopatía Diabética/patología , Regulación de la Expresión Génica , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal/fisiología
15.
Sci Rep ; 10(1): 7273, 2020 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-32350384

RESUMEN

We have developed a new technique to study the integrity, morphology and functionality of the retinal neurons and the retinal pigment epithelium (RPE). Young and old control albino (Sprague-Dawley) and pigmented (Piebald Virol Glaxo) rats, and dystrophic albino (P23H-1) and pigmented (Royal College of Surgeons) rats received a single intravitreal injection of 3% Fluorogold (FG) and their retinas were analyzed from 5 minutes to 30 days later. Retinas were imaged in vivo with SD-OCT and ex vivo in flat-mounts and in cross-sections. Fifteen minutes and 24 hours after intravitreal administration of FG retinal neurons and the RPE, but no glial cells, were labeled with FG-filled vesicles. The tracer reached the RPE 15 minutes after FG administration, and this labeling remained up to 30 days. Tracing for 15 minutes or 24 hours did not cause oxidative stress. Intraretinal tracing delineated the pathological retinal remodelling occurring in the dystrophic strains. The RPE of the P23H-1 strain was highly altered in aged animals, while the RPE of the RCS strain, which is unable to phagocytose, did not accumulate the tracer even at young ages when the retinal neural circuit is still preserved. In both dystrophic strains, the RPE cells were pleomorphic and polymegathic.


Asunto(s)
Rastreo Celular , Fagocitosis , Degeneración Retiniana , Neuronas Retinianas , Epitelio Pigmentado de la Retina , Estilbamidinas/farmacología , Animales , Femenino , Ratas , Ratas Sprague-Dawley , Degeneración Retiniana/diagnóstico por imagen , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Neuronas Retinianas/metabolismo , Neuronas Retinianas/patología , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología
16.
Nat Commun ; 11(1): 2549, 2020 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-32439975

RESUMEN

Mitochondria undergo dynamic fusion/fission, biogenesis and mitophagy in response to stimuli or stresses. Disruption of mitochondrial homeostasis could lead to cell senescence, although the underlying mechanism remains unclear. We show that deletion of mitochondrial phosphatase PGAM5 leads to accelerated retinal pigment epithelial (RPE) senescence in vitro and in vivo. Mechanistically, PGAM5 is required for mitochondrial fission through dephosphorylating DRP1. PGAM5 deletion leads to increased mitochondrial fusion and decreased mitochondrial turnover. As results, cellular ATP and reactive oxygen species (ROS) levels are elevated, mTOR and IRF/IFN-ß signaling pathways are enhanced, leading to cellular senescence. Overexpression of Drp1 K38A or S637A mutant phenocopies or rescues mTOR activation and senescence in PGAM5-/- cells, respectively. Young but not aging Pgam5-/- mice are resistant to sodium iodate-induced RPE cell death. Our studies establish a link between defective mitochondrial fission, cellular senescence and age-dependent oxidative stress response, which have implications in age-related diseases.


Asunto(s)
Senescencia Celular , Dinámicas Mitocondriales , Fosfoproteínas Fosfatasas/metabolismo , Factores de Edad , Animales , Línea Celular , Dinaminas/genética , Dinaminas/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Estrés Oxidativo/genética , Fosfoproteínas Fosfatasas/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Transducción de Señal
17.
Invest Ophthalmol Vis Sci ; 61(5): 32, 2020 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-32428233

RESUMEN

Purpose: Because air pollution has been linked to glaucoma and AMD, we characterized the relationship between pollution and retinal structure. Methods: We examined data from 51,710 UK Biobank participants aged 40 to 69 years old. Ambient air pollution measures included particulates and nitrogen oxides. SD-OCT imaging measured seven retinal layers: retinal nerve fiber layer, ganglion cell-inner plexiform layer, inner nuclear layer, outer plexiform layer + outer nuclear layer, photoreceptor inner segments, photoreceptor outer segments, and RPE. Multivariable regression was used to evaluate associations between pollutants (per interquartile range increase) and retinal thickness, adjusting for age, sex, race, Townsend deprivation index, body mass index, smoking status, and refractive error. Results: Participants exposed to greater particulate matter with an aerodynamic diameter of <2.5 µm (PM2.5) and higher nitrogen oxides were more likely to have thicker retinal nerve fiber layer (ß = 0.28 µm; 95% CI, 0.22-0.34; P = 3.3 × 10-20 and ß = 0.09 µm; 95% CI, 0.04-0.14; P = 2.4 × 10-4, respectively), and thinner ganglion cell-inner plexiform layer, inner nuclear layer, and outer plexiform layer + outer nuclear layer thicknesses (P < 0.001). Participants resident in areas of higher levels of PM2.5 absorbance were more likely to have thinner retinal nerve fiber layer, inner nuclear layer, and outer plexiform layer + outer nuclear layers (ß = -0.16 [95% CI, -0.22 to -0.10; P = 5.7 × 10-8]; ß = -0.09 [95% CI, -0.12 to -0.06; P = 2.2 × 10-12]; and ß = -0.12 [95% CI, -0.19 to -0.05; P = 8.3 × 10-4], respectively). Conclusions: Greater exposure to PM2.5, PM2.5 absorbance, and nitrogen oxides were all associated with apparently adverse retinal structural features.


Asunto(s)
Contaminación del Aire/efectos adversos , Fibras Nerviosas/efectos de los fármacos , Óxidos de Nitrógeno/efectos adversos , Material Particulado/efectos adversos , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Ganglionares de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Adulto , Anciano , Bancos de Muestras Biológicas , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fibras Nerviosas/patología , Células Fotorreceptoras de Vertebrados/patología , Enfermedades de la Retina/inducido químicamente , Células Ganglionares de la Retina/patología , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Epitelio Pigmentado de la Retina/patología , Encuestas y Cuestionarios , Tomografía de Coherencia Óptica , Reino Unido
18.
Am J Ophthalmol ; 218: 136-147, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32446735

RESUMEN

PURPOSE: To investigate the development and progression of retinal pigment epithelial and outer retinal atrophy (RORA) secondary to maternally inherited diabetes and deafness (MIDD). DESIGN: Retrospective observational case series. METHODS: Thirty-six eyes of 18 patients (age range, 22.4-71.6 years) with genetically proven MIDD and serial optical coherence tomography (OCT) images were included. As proposed reference standard to diagnose and stage atrophy, OCT images were longitudinally evaluated and analyzed for presence and precursors of RORA. RORA was defined as an area of (1) hypertransmission, (2) disruption of the retinal pigment epithelium, (3) photoreceptor degeneration, and (4) absence of other signs of a retinal pigment epithelial tear. RESULTS: The majority of patients revealed areas of RORA in a circular area around the fovea of between 5° and 15° eccentricity. Over the observation time (range, 0.5-8.5 years), evidence for a consistent sequence of OCT features from earlier disease stages to the end stage of RORA could be found, starting with loss of ellipsoid zone and subretinal deposits, followed by loss of external limiting membrane and loss of retinal pigment epithelium with hypertransmission of OCT signal into the choroid, and leading to loss of the outer nuclear layer bordered by hyporeflective wedges. Outer retinal tabulations seemed to develop in regions of coalescent areas of RORA. CONCLUSIONS: The development and progression of RORA could be tracked in MIDD patients using OCT images, allowing potential definition of novel surrogate markers. Similarities to OCT features in age-related macular degeneration, where mitochondrial dysfunction has been implicated in the pathogenesis, support wide-ranging benefits from proof-of-concept studies in MIDD.


Asunto(s)
Sordera/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Enfermedades Mitocondriales/complicaciones , Distrofias Retinianas/diagnóstico , Epitelio Pigmentado de la Retina/patología , Adulto , Anciano , Atrofia , Sordera/diagnóstico , Diabetes Mellitus Tipo 2/diagnóstico , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Mitocondriales/diagnóstico , Distrofias Retinianas/etiología , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Estudios Retrospectivos , Tomografía de Coherencia Óptica , Agudeza Visual/fisiología , Adulto Joven
19.
Am J Ophthalmol ; 217: 252-260, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32442431

RESUMEN

PURPOSE: To determine whether there are differences in the prevalence of intraretinal pigment migration (IPM) across ages and genetic causes of inherited retinal dystrophies (IRDs). DESIGN: Retrospective cohort study. METHODS: Patients were evaluated at a single tertiary referral center. All patients with a clinical diagnosis of IRD and confirmatory genetic testing were included in these analyses. A total of 392 patients fit inclusion criteria, and 151 patients were excluded based on inconclusive genetic testing. Patients were placed into 3 groups, ciliary and ciliary-related photoreceptor, nonciliary photoreceptor, and retinal pigment epithelium (RPE), based on the cellular expression of the gene and the primary affected cell type. The presence of IPM was evaluated by using slit lamp biomicroscopy, indirect ophthalmoscopy, and wide-field color fundus photography. RESULTS: IPM was seen in 257 of 339 patients (75.8%) with mutations in photoreceptor-specific genes and in 18 of 53 patients (34.0%) with mutations in RPE-specific genes (P < .0001). Pairwise analysis following stratification by age and gene category suggested significant differences at all age groups between patients with mutations in photoreceptor-specific genes and patients with mutations in RPE-specific genes (P < .05). A fitted multivariate logistic regression model was produced and demonstrated that the incidence of IPM increases as a function of both age and gene category. CONCLUSIONS: IPM is a finding more commonly observed in IRDs caused by mutations in photoreceptor-specific genes than RPE-specific genes. The absence of IPM does not always rule out IRD and should raise suspicion for disease mutations in RPE-specific genes.


Asunto(s)
Envejecimiento , Distrofias Retinianas/diagnóstico , Epitelio Pigmentado de la Retina/patología , Tomografía de Coherencia Óptica/métodos , Adulto , Movimiento Celular , Femenino , Estudios de Seguimiento , Humanos , Masculino , Oftalmoscopía , Distrofias Retinianas/congénito , Estudios Retrospectivos , Microscopía con Lámpara de Hendidura
20.
Invest Ophthalmol Vis Sci ; 61(5): 10, 2020 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-32396631

RESUMEN

Purpose: A burst in phagocytosis of spent photoreceptor outer fragments by RPE is a rhythmic process occurring 1 to 2 hours after the onset of light. This phenomenon is considered crucial for the health of the photoreceptors and RPE. We have recently reported that dopamine, via dopamine 2 receptor (D2R), shifts the circadian rhythm in the RPE. Methods: Here, we first investigated the impact of the removal of D2R on the daily peak of phagocytosis by RPE and then we analyzed the function and morphology of retina and RPE in the absence of D2R. Results: D2R knockout (KO) mice do not show a daily burst of phagocytic activity after the onset of light. RNA sequencing revealed a total of 394 differentially expressed genes (DEGs) between ZT 23 and ZT 1 in the control mice, whereas in D2R KO mice, we detected 1054 DEGs. Pathway analysis of the gene expression data implicated integrin signaling to be one of the upregulated pathways in control but not in D2R KO mice. Consistent with the gene expression data, phosphorylation of focal adhesion kinase (FAK) did not increase significantly in KO mice at ZT 1. No difference in retinal thickness, visual function, or morphology of RPE cells was observed between wild-type (WT) and D2R KO mice at the age of 3 and 12 months. Conclusions: Our data suggest that removal of D2R prevents the burst of phagocytosis and a related increase in the phosphorylation of FAK after light onset. The pathway analysis points toward a putative role of D2R in controlling integrin signaling, which is known to play an important role in the control of the daily burst of phagocytosis by the RPE. Our data also indicate that the absence of the burst of phagocytic activity in the early morning does not produce any apparent deleterious effect on the retina or RPE up to 1 year of age.


Asunto(s)
Fagocitosis , Receptores de Dopamina D2/fisiología , Epitelio Pigmentado de la Retina/patología , Transducción de Señal/fisiología , Animales , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Integrinas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fagosomas/patología , Fosforilación/fisiología , Tomografía de Coherencia Óptica , Regulación hacia Arriba/fisiología
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