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INTRODUCTION: The German Registry of central serous chorioretinopathy (CSC) collects data on CSC patients in a nationwide multicenter approach to analyze epidemiology, risk factors, clinical presentations, as well as diagnosis and treatment patterns. METHODS: In this multicenter cohort study, patients with CSC were enrolled in nine tertiary referral centers in Germany between January 2022 and June 2023. After consenting to the study, demographic data, risk factors, reported symptoms, best-corrected visual acuity (BCVA), funduscopic findings, disease severity, and diagnostic and treatment decisions were recorded and analyzed. RESULTS: A total of 539 eyes of 411 CSC patients were enrolled in this study including 308 males (75%) and 103 females (25%). Patients were predominantly of Caucasian origin and had a mean age of 55.5 years (IQR 41.0-70.0). 28% of eyes were classified as acute (<4 months duration) CSC, 28% as chronic (>4 months duration) CSC, 21% as inactive CSC, 11% as chronic atrophic CSC, and 12% as CSC with secondary CNV. 128 patients (31%) demonstrated bilateral CSC. The most common risk factors reported were psychological stress (52%), smoking (38%), arterial hypertension (38%), and a history of or current use of steroids (30%). Most frequently encountered symptoms included decreased visual acuity (76%), metamorphopsia (49%), relative scotoma (47%), blurred vision (19%), and dyschromatopsia (9%). The mean logMAR BCVA on initial examination was 0.2 (≈20/30, IQR 0.2-0.4) but showed significant variation with a tendency of lower BCVA in chronic cases. At the baseline visit, 74% of the overall cohort received no treatment, while 19% underwent local treatment and only 2% underwent systemic treatment. Of the local therapies, anti-VEGF injections were the most frequently performed procedure (33%, mainly for secondary CNV), followed by micropulse laser (28%), focal nonpulsed laser (23%), verteporfin photodynamic therapy (14%), and nonsteroidal anti-inflammatory eye drops (2%). Among intravitreal anti-VEGF agents, aflibercept was used most frequently, followed by bevacizumab and ranibizumab. CONCLUSION: This registry represents one of the largest cohorts of European patients with CSC to date. Patient age and the proportion of women were higher than expected and bilateral active disease was lower than anticipated, highlighting that neither age nor gender should be overemphasized when diagnosing CSC. Therapeutic interventions are heterogeneous and include verteporfin photodynamic therapy, micropulse laser, and anti-VEGF injections in case of secondary CNV.
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Coriorretinopatía Serosa Central , Angiografía con Fluoresceína , Sistema de Registros , Tomografía de Coherencia Óptica , Agudeza Visual , Humanos , Coriorretinopatía Serosa Central/diagnóstico , Coriorretinopatía Serosa Central/epidemiología , Coriorretinopatía Serosa Central/terapia , Persona de Mediana Edad , Masculino , Femenino , Alemania/epidemiología , Anciano , Tomografía de Coherencia Óptica/métodos , Adulto , Angiografía con Fluoresceína/métodos , Factores de Riesgo , Fondo de Ojo , Estudios Retrospectivos , Incidencia , Estudios de Seguimiento , Retina/patologíaRESUMEN
Central serous chorioretinopathy (CSC) is a relatively common disease that causes vision loss due to macular subretinal fluid leakage and it is often associated with reduced vision-related quality of life. In CSC, the leakage of subretinal fluid through defects in the retinal pigment epithelial layer's outer blood-retina barrier appears to occur secondary to choroidal abnormalities and dysfunction. The treatment of CSC is currently the subject of controversy, although recent data obtained from several large randomized controlled trials provide a wealth of new information that can be used to establish a treatment algorithm. Here, we provide a comprehensive overview of our current understanding regarding the pathogenesis of CSC, current therapeutic strategies, and an evidence-based treatment guideline for CSC. In acute CSC, treatment can often be deferred for up to 3-4 months after diagnosis; however, early treatment with either half-dose or half-fluence photodynamic therapy (PDT) with the photosensitive dye verteporfin may be beneficial in selected cases. In chronic CSC, half-dose or half-fluence PDT, which targets the abnormal choroid, should be considered the preferred treatment. If PDT is unavailable, chronic CSC with focal, non-central leakage on angiography may be treated using conventional laser photocoagulation. CSC with concurrent macular neovascularization should be treated with half-dose/half-fluence PDT and/or intravitreal injections of an anti-vascular endothelial growth factor compound. Given the current shortage of verteporfin and the paucity of evidence supporting the efficacy of other treatment options, future studies-ideally, well-designed randomized controlled trials-are needed in order to evaluate new treatment options for CSC.
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Coriorretinopatía Serosa Central , Fotoquimioterapia , Coriorretinopatía Serosa Central/terapia , Coriorretinopatía Serosa Central/diagnóstico , Humanos , Fotoquimioterapia/métodos , Medicina Basada en la Evidencia , Guías de Práctica Clínica como Asunto , Fármacos Fotosensibilizantes/uso terapéutico , Angiografía con Fluoresceína , Inhibidores de la Angiogénesis/uso terapéutico , Coagulación con Láser/métodosRESUMEN
Background: Retinal neovascularization (RNV) membranes can lead to a tractional retinal detachment, the primary reason for severe vision loss in end-stage disease proliferative diabetic retinopathy (PDR). The aim of this study was to characterize the molecular, cellular and immunological features of RNV in order to unravel potential novel drug treatments for PDR. Methods: A total of 43 patients undergoing vitrectomy for PDR, macular pucker or macular hole (control patients) were included in this study. The surgically removed RNV and epiretinal membranes were analyzed by RNA sequencing, single-cell based Imaging Mass Cytometry and conventional immunohistochemistry. Immune cells of the vitreous body, also known as hyalocytes, were isolated from patients with PDR by flow cytometry, cultivated and characterized by immunohistochemistry. A bioinformatical drug repurposing approach was applied in order to identify novel potential drug options for end-stage diabetic retinopathy disease. Results: The in-depth transcriptional and single-cell protein analysis of diabetic RNV tissue samples revealed an accumulation of endothelial cells, macrophages and myofibroblasts as well as an abundance of secreted ECM proteins such as SPARC, FN1 and several types of collagen in RNV tissue. The immunohistochemical staining of cultivated vitreal hyalocytes from patients with PDR showed that hyalocytes express α-SMA (alpha-smooth muscle actin), a classic myofibroblast marker. According to our drug repurposing analysis, imatinib emerged as a potential immunomodulatory drug option for future treatment of PDR. Conclusion: This study delivers the first in-depth transcriptional and single-cell proteomic characterization of RNV tissue samples. Our data suggest an important role of hyalocyte-to-myofibroblast transdifferentiation in the pathogenesis of diabetic vitreoretinal disease and their modulation as a novel possible clinical approach.
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Transdiferenciación Celular , Retinopatía Diabética/patología , Membrana Epirretinal/patología , Miofibroblastos/patología , Neovascularización Retiniana/patología , Cuerpo Vítreo/inmunología , Adulto , Anciano , Células Cultivadas , Biología Computacional , Retinopatía Diabética/complicaciones , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/metabolismo , Reposicionamiento de Medicamentos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Membrana Epirretinal/metabolismo , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Femenino , Ontología de Genes , Humanos , Mesilato de Imatinib/uso terapéutico , Factores Inmunológicos/uso terapéutico , Masculino , Persona de Mediana Edad , Neovascularización Retiniana/etiología , Neovascularización Retiniana/metabolismo , Perforaciones de la Retina/patología , Análisis de la Célula Individual , Transcriptoma , Cuerpo Vítreo/patología , Adulto JovenRESUMEN
Despite the reported low expression of the primary SARS-CoV-2 receptor ACE2 in distinct ocular tissues, some clinical evidence suggests that SARS-CoV-2 can infect the eye. In this study, we explored potential entry sites for SARS-CoV-2 by viral S protein histochemistry on various ocular tissues and compared the staining patterns with RNA and protein expression of TMPRSS2 and ACE2. Potential viral entry sites were investigated by histochemistry using tagged recombinant viral S protein on 52 ocular tissue samples including specimens of the cornea, conjunctiva, lid margin, lacrimal gland tissue, retina, choroid, and RPE. In addition, ACE2 and TMPRSS2 immunohistochemistry were performed on the same ocular tissue, each with distinct antibodies binding to different epitopes. Lung tissue samples were used as positive controls. Finally, bulk RNA sequencing (RNA-Seq) was used to determine the expression of ACE2 and its auxiliary factors in the tissues mentioned above. S protein histochemistry revealed a positive staining in lung tissue but absent staining in the cornea, the conjunctiva, eye lid samples, the lacrimal glands, the retina and the optic nerve which was supported by hardly any immunoreactivity for ACE2 and TMPRSS2 and scarce ACE2 and TMPRSS2 RNA expression. Negligible staining with antibodies targeting ACE2 or TMPRSS2 was seen in the main and accessory lacrimal glands. In contrast, ocular staining (S protein, ACE2, TMPRSS2) was distinctly present in pigmented cells of the RPE and choroid, as well as in the ciliary body and the iris stroma. S protein histochemistry revealed hardly any SARS-CoV-2 entry sites in all ocular tissues examined. Similarly, no significant ACE2 or TMPRSS2 expression was found in extra- and intraocular tissue. While this study suggest a rather low risk of ocular infection with SARS-CoV-2, it should be noted, that potential viral entry sites may increase in response to inflammation or in certain disease states.
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COVID-19/prevención & control , Conjuntiva/metabolismo , Córnea/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , Conjuntiva/virología , Córnea/virología , Perfilación de la Expresión Génica/métodos , Humanos , Inmunohistoquímica/métodos , RNA-Seq/métodos , SARS-CoV-2/fisiología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Internalización del VirusRESUMEN
This study characterizes the transcriptome and the cellular tumor microenvironment (TME) of conjunctival melanoma (CM) and identifies prognostically relevant biomarkers. 12 formalin-fixed and paraffin-embedded CM were analyzed by MACE RNA sequencing, including six cases each with good or poor clinical outcome, the latter being defined by local recurrence and/or systemic metastases. Eight healthy conjunctival specimens served as controls. The TME of CM, as determined by bioinformatic cell type enrichment analysis, was characterized by the enrichment of melanocytes, pericytes and especially various immune cell types, such as plasmacytoid dendritic cells, natural killer T cells, B cells and mast cells. Differentially expressed genes between CM and control were mainly involved in inhibition of apoptosis, proteolysis and response to growth factors. POU3F3, BIRC5 and 7 were among the top expressed genes associated with inhibition of apoptosis. 20 genes, among them CENPK, INHA, USP33, CASP3, SNORA73B, AAR2, SNRNP48 and GPN1, were identified as prognostically relevant factors reaching high classification accuracy (area under the curve: 1.0). The present study provides new insights into the TME and the transcriptional profile of CM and additionally identifies new prognostic biomarkers. These results add new diagnostic tools and may lead to new options of targeted therapy for CM.
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Neoplasias de la Conjuntiva/genética , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Recurrencia Local de Neoplasia/genética , Microambiente Tumoral/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Neoplasias de la Conjuntiva/metabolismo , Neoplasias de la Conjuntiva/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patología , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Pronóstico , TranscriptomaRESUMEN
Purpose: To decipher the transcriptional signature of macrophages of the human vitreous, also known as hyalocytes, and compare it to the profiles of other myeloid cell populations including human blood-derived monocytes, macrophages, and brain microglia. Methods: This study involves a total of 13 patients of advanced age with disorders of the vitreoretinal interface undergoing vitrectomy at the University Eye Hospital Freiburg between 2018 and 2019. Vitreal hyalocytes were analyzed by fluorescence-activated cell sorting (FACS) and isolated as CD45+CD11b+CX3CR1+Mat-Mac+ cells using a FACS-based sorting protocol. RNA extraction, library preparation and RNA sequencing were performed and the sequencing data was analyzed using the Galaxy web platform. The transcriptome of human hyalocytes was compared to the transcriptional profile of human blood-derived monocytes, macrophages and brain microglia obtained from public databases. Protein validation for selected factors was performed by immunohistochemistry on paraffin sections from three human donor eyes. Results: On average, 383 ± 233 hyalocytes were isolated per patient, resulting in 128 pg/µl ± 76 pg/µl total RNA per sample. RNA sequencing revealed that SPP1, FTL, CD74, and HLA-DRA are among the most abundantly expressed genes in hyalocytes, which was confirmed by immunofluorescence for CD74, FTL, and HLA-DRA. Gene ontology (GO) enrichment analysis showed that biological processes such as "humoral immune response," "leukocyte migration," and "antigen processing and presentation of peptide antigen" (adjusted p < 0.001) are dominating in vitreal hyalocytes. While the comparison of the gene expression profiles of hyalocytes and other myeloid cell populations showed an overall strong similarity (R2 > 0.637, p < 0.001), hyalocytes demonstrated significant differences with respect to common leukocyte-associated factors. In particular, transcripts involved in the immune privilege of the eye, such as POMC, CD46, and CD86, were significantly increased in hyalocytes compared to other myeloid cell subsets. Conclusion: Human hyalocytes represent a unique and distinct innate immune cell population specialized and adapted for the tissue-specific needs in the human vitreous. Vitreal hyalocytes are characterized by a strong expression of genes related to antigen processing and presentation as well as immune modulation. Thus, hyalocytes may represent an underestimated mediator in vitreoretinal disease and for the immune privilege of the eye.
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Perfilación de la Expresión Génica , Inmunidad Innata , Macrófagos/inmunología , Macrófagos/metabolismo , Transcriptoma , Cuerpo Vítreo/citología , Anciano , Anciano de 80 o más Años , Biomarcadores , Recuento de Células , Separación Celular/métodos , Biología Computacional/métodos , Femenino , Expresión Génica , Humanos , Privilegio Inmunológico/genética , Inmunohistoquímica , Inmunofenotipificación , Masculino , Anotación de Secuencia Molecular , Células Mieloides/inmunología , Células Mieloides/metabolismoRESUMEN
Recent studies deciphering the transcriptional profile of choroidal neovascularization (CNV) in body donor eyes with neovascular age-related macular degeneration are limited by the time span from death to preservation and the associated 5'-RNA degradation. This study therefore used CNV and control specimens that were formalin-fixed and paraffin-embedded immediately after surgical extraction and analyzed them by a 3'-RNA sequencing approach. Transcriptome profiles were analyzed to estimate content of immune and stromal cells and to define disease-associated gene signatures by using statistical and bioinformatics methods. This study identified 158 differentially expressed genes (DEGs) that were significantly increased in CNV compared with control tissue. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of endothelial cells, macrophages, T cells, and natural killer T cells in the CNV. Gene ontology enrichment analysis found that DEGs contributed to blood vessel development, extracellular structure organization, response to wounding, and several immune-related terms. The S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9) emerged among the top DEGs, as confirmed by immunohistochemistry on CNV tissue and protein analysis of vitreous samples. This study provides a high-resolution RNA-sequencing-based transcriptional signature of human CNV, characterizes its compositional pattern of immune and stromal cells, and reveals S100A8/A9 to be a novel biomarker and promising target for therapeutics and diagnostics directed at age-related macular degeneration.
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Neovascularización Coroidal/diagnóstico , Complejo de Antígeno L1 de Leucocito/metabolismo , Degeneración Macular/diagnóstico , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Neovascularización Coroidal/metabolismo , Células Endoteliales/metabolismo , Femenino , Humanos , Macrófagos/metabolismo , Degeneración Macular/metabolismo , Masculino , TranscriptomaRESUMEN
Background: Whether retinal microglia can maintain or restore immune homeostasis during and after inflammation is unclear. We performed single-eye mRNA-sequencing on microglia at different timepoints following a single inflammatory stimulus to characterise their transcriptome during and after resolution of endotoxin-induced uveitis (EIU). Experimental Approach:Cx3cr1CreER:R26-tdTomato (C57BL/6) male heterozygotes were administered tamoxifen via different regimes at 4-5 weeks of age. Four weeks post-tamoxifen, mice were injected intravitreally with 10 ng lipopolysaccharide (endotoxin induced uveitis, EIU). Six-hundred retinal microglia were obtained by FACS from individual naïve retinas and at 4 h, 18 h, and 2 weeks following EIU induction. Samples were sequenced to a depth of up to 16.7 million reads using the SMART-Seq v4 Ultra Low Input RNA kit. The data was analysed using Partek software and Ingenuity Pathway Analysis. Genes were considered differentially-expressed (DEG) if the FDR step-up p-value was ≤0.05 and the fold-change was ≥±2. Results: Flow cytometric analysis indicates that the Cx3cr1CreER:R26-tdTomato strain is both sensitive (>95% tagging) and specific (>95% specificity) for microglia when tamoxifen is administered topically to the eye for 3 days. During "early" activation, 613 DEGs were identified. In contrast, 537 DEGs were observed during peak cellular infiltrate and none at 2 weeks, compared to baseline controls (1,069 total unique DEGs). Key marker changes were validated by qPCR, flow cytometry, and fluorescence microscopy. C5AR1 was identified and validated as a robust marker of differentiating microglial subsets during an LPS response. Conclusion: Using EIU to provide a single defined inflammatory stimulus, mRNA-Seq identified acute transcriptional changes in retinal microglia which returned to their original transcriptome after 2 weeks. Yolk-sac derived microglia are capable of restoring their homeostatic state after acute inflammation.
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Inflamación/genética , Microglía/fisiología , ARN Mensajero/genética , Retina/fisiología , Transcriptoma/genética , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Endotoxinas/farmacología , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Neuroglía/efectos de los fármacos , Receptor de Anafilatoxina C5a/genética , Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Uveítis/inducido químicamente , Uveítis/genéticaRESUMEN
The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth.
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Permeabilidad Capilar , Neuropilina-1/fisiología , Proteínas Proto-Oncogénicas c-abl/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Semaforina-3A/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
BACKGROUND AND OBJECTIVE: The introduction of optical coherence tomography angiography (OCTA) provides new insights into the retinal vasculature. The aim of this study was to explore the value of OCTA in imaging retinal hemangioblastomas and monitoring laser treatment in patients with von Hippel-Lindau (VHL) disease. PATIENTS AND METHODS: Ten eyes of 10 patients with VHL disease were included in this retrospective case series. All patients underwent complete ophthalmological work-up including OCTA for retinal and optic nerve head hemangioblastoma. RESULTS: Two patients showed retinal scars and no recurrence of hemangioblastoma in OCTA. Three patients revealed recurrent hemangioblastomas. Two patients demonstrated a new hemangioblastoma. Three patients showed hemangioblastomas of the optic nerve head. Successful laser photocoagulation could be monitored with OCTA. CONCLUSIONS: Screening for retinal hemangioblastomas with OCTA alone is not possible. OCTA may help to distinguish hemangioblastomas and other lesions in VHL disease, especially after treatment and allows the differentiation from harmless non-vascular lesions in questionable cases. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:935-946.].
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Angiografía/métodos , Hemangioblastoma/diagnóstico , Coagulación con Láser/métodos , Neoplasias de la Retina/diagnóstico , Vasos Retinianos/diagnóstico por imagen , Tomografía de Coherencia Óptica/métodos , Enfermedad de von Hippel-Lindau/complicaciones , Adulto , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Hemangioblastoma/complicaciones , Hemangioblastoma/cirugía , Humanos , Masculino , Persona de Mediana Edad , Disco Óptico/diagnóstico por imagen , Pronóstico , Neoplasias de la Retina/complicaciones , Neoplasias de la Retina/cirugía , Estudios Retrospectivos , Adulto Joven , Enfermedad de von Hippel-Lindau/diagnósticoRESUMEN
OBJECTIVE: Ocular neovascularization (ONV) is a pathological feature of sight-threatening human diseases, such as diabetic retinopathy and age-related macular degeneration. Macrophage depletion in mouse models of ONV reduces the formation of pathological blood vessels, and myeloid cells are widely considered an important source of the vascular endothelial growth factor A (VEGF). However, the importance of VEGF or its upstream regulators hypoxia-inducible factor-1α (HIF1α) and hypoxia-inducible factor-2α (HIF2α) as myeloid-derived regulators of ONV remains to be determined. APPROACH AND RESULTS: We used 2 mouse models of ONV, choroidal neovascularization and oxygen-induced retinopathy, to show that Vegfa is highly expressed by several cell types, but not myeloid cells during ONV. Moreover, myeloid-specific VEGF ablation did not reduce total ocular VEGF during choroidal neovascularization or oxygen-induced retinopathy. In agreement, the conditional inactivation of Vegfa, Hif1a, or Epas1 in recruited and resident myeloid cells that accumulated at sites of neovascularization did not significantly reduce choroidal neovascularization or oxygen-induced retinopathy. CONCLUSIONS: The finding that myeloid cells are not a significant local source of VEGF in these rodent models of ONV suggests that myeloid function in neovascular eye disease differs from skin wound healing and other neovascular pathologies.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neovascularización Coroidal/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Mieloides/metabolismo , Neovascularización Retiniana/metabolismo , Vasos Retinianos/metabolismo , Retinopatía de la Prematuridad/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Neovascularización Coroidal/genética , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones Noqueados , Células Mieloides/patología , Oxígeno , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Vasos Retinianos/patología , Retinopatía de la Prematuridad/inducido químicamente , Retinopatía de la Prematuridad/genética , Retinopatía de la Prematuridad/patología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/deficiencia , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1(-/-) cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function.
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Predisposición Genética a la Enfermedad , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Mutación/genética , Piridonas/farmacología , Pirimidinas/farmacología , Retinitis Pigmentosa/prevención & control , Rodopsina/metabolismo , Animales , Western Blotting , Células Cultivadas , Electrorretinografía , Femenino , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Genes Dominantes , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rodopsina/genética , Tomografía de Coherencia Óptica , Visión Ocular/efectos de los fármacos , Visión Ocular/fisiologíaRESUMEN
Aberrant neovascularization contributes to diseases such as cancer, blindness and atherosclerosis, and is the consequence of inappropriate angiogenic signalling. Although many regulators of pathogenic angiogenesis have been identified, our understanding of this process is incomplete. Here we explore the transcriptome of retinal microvessels isolated from mouse models of retinal disease that exhibit vascular pathology, and uncover an upregulated gene, leucine-rich alpha-2-glycoprotein 1 (Lrg1), of previously unknown function. We show that in the presence of transforming growth factor-ß1 (TGF-ß1), LRG1 is mitogenic to endothelial cells and promotes angiogenesis. Mice lacking Lrg1 develop a mild retinal vascular phenotype but exhibit a significant reduction in pathological ocular angiogenesis. LRG1 binds directly to the TGF-ß accessory receptor endoglin, which, in the presence of TGF-ß1, results in promotion of the pro-angiogenic Smad1/5/8 signalling pathway. LRG1 antibody blockade inhibits this switch and attenuates angiogenesis. These studies reveal a new regulator of angiogenesis that mediates its effect by modulating TGF-ß signalling.
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Endotelio Vascular/metabolismo , Glicoproteínas/fisiología , Neovascularización Retiniana/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Células Cultivadas , Endotelio Vascular/citología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Neovascularización Retiniana/genética , Vasos Retinianos/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
PURPOSE: Two noninvasive delivery strategies for VEGF/PDGF receptor tyrosine kinase inhibitors (RTKI) were explored that exploited uveal retention as a means for establishing an ocular drug depot: a single oral "loading" dose and topical administration. METHODS: Melanin binding was confirmed by centrifugation and mass spectrometry. Ocular retention was examined in pigmented and albino rats. Ocular release kinetics were measured 3 to 28 days postdosing in pigmented rats. Microautoradiography was used to demonstrate retention of RTKI in the uveal tract. A uveal drug depot of pazopanib was created by a single oral dose prior to induction of laser choroidal neovascularization (CNV). Choroid/retinal pigmented epithelium (RPE) retention of a related RTKI with enhanced topical bioavailability, GW771806, was confirmed by bioanalytics, and its ability to regress CNV compared with pazopanib. RESULTS: Pazopanib and GW771806 directly bound melanin and were retained within the uveal tract of pigmented rats for weeks following a single oral dose. Pazopanib was undetectable systemically following a single oral administration prior to CNV induction, and reduced CNV as well as twice daily dosing. Topical ocular delivery of GW771806 at 5 mg/mL led to high choroidal/RPE exposure and significantly regressed CNV lesions; 2 mg/mL prevented lesion progression. CONCLUSIONS: Uveal retention of drugs such as pazopanib can be used to create a sustained-release depot. Topical GW771806 regressed CNV. These data indicate that topical or infrequent oral loading dose treatment with VEGF/PDGF RTKI retained in the choroid/RPE might allow noninvasive treatments for ocular neovascular disease.
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Inhibidores de la Angiogénesis/administración & dosificación , Neovascularización Coroidal/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Indazoles/administración & dosificación , Pirimidinas/administración & dosificación , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , Sulfonas/administración & dosificación , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Administración Oral , Administración Tópica , Inhibidores de la Angiogénesis/farmacocinética , Animales , Autorradiografía , Neovascularización Coroidal/diagnóstico , Neovascularización Coroidal/metabolismo , Femenino , Angiografía con Fluoresceína , Semivida , Indazoles/farmacocinética , Melaninas/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Pirimidinas/farmacocinética , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Sulfonamidas/farmacocinética , Sulfonas/farmacocinética , Úvea/metabolismoRESUMEN
PURPOSE: Malignant tumors metabolize glucose to lactate even in the presence of oxygen via the pentose-phosphate pathway. The metabolic switch from oxidative glycolysis to nonoxidative fermentation in tumors has been associated with overexpression of the transketolase-like-1-gene (TKTL1), which encodes an essential and rate-limiting enzyme in the nonoxidative part of the pentose-phosphate pathway. This study investigates the role of TKTL1 in ocular adnexal tumors and analyzes how its expression correlates with the clinical outcomes against the background of tumor thickness and mitotic rate. DESIGN: Comparative case studies. PARTICIPANTS: We included 89 subjects with malignant tumors of the ocular adnexa (44 squamous cell carcinomas, 26 lymphomas, 19 malignant melanomas) who had been treated at the University Eye Hospital Freiburg from 1994 to 2008. Sixteen subjects with conjunctival nevi, 19 with conjunctival papilloma, and 2 with conjunctival-reactive lymphoid hyperplasia were included as controls. METHODS: TKTL1 expression was assessed by reverse transcriptase-polymerase chain reaction and immunohistochemistry and semiquantitatively analyzed using an established immunoreactive score (IRS). The tumor recurrence rate, metastasis occurrence, and survival time of each patient were assessed retrospectively and correlated with the TKTL IRS using Kaplan-Meier and Cox regression analyses. MAIN OUTCOME MEASURES: TKTL1 expression, mitotic rate within the tumor mass, and tumor thickness and its association with clinical outcome. RESULTS: We identified increased TKTL1 protein levels in malignant conjunctival tumors compared with control samples and detected an average IRS of 1.78 (standard deviation [SD], ± 0.46) for melanomas, 1.3 for lymphomas (SD, ± 0.79), and 1.22 for squamous cell carcinomas (SD, ± 0. 97) compared with 0.86 for conjunctival nevi (SD, ± 0.57) and 0.5 for conjunctival papilloma (SD, ± 0.83). Multifactorial survival analysis showed that TKTL1 overexpression correlated with the patient outcomes in malignant tumors (P = 0.045). In the squamous cell carcinomas, tumor thickness and mitotic rate correlated more strongly with prognosis compared with TKTL1 overexpression (P = 0.0061, P = 0.015, and P = 0.061, respectively). CONCLUSIONS: TKTL1 is dysregulated in malignant tumors of the ocular adnexa, and enhanced expression seems to predict clinical outcome, especially the tumor recurrence rate.
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Carcinoma de Células Escamosas/genética , Neoplasias de la Conjuntiva/genética , Regulación Neoplásica de la Expresión Génica , Linfoma/genética , Melanoma/genética , Recurrencia Local de Neoplasia , Transcetolasa/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Niño , Neoplasias de la Conjuntiva/metabolismo , Neoplasias de la Conjuntiva/patología , Humanos , Linfoma/metabolismo , Linfoma/patología , Melanoma/metabolismo , Melanoma/patología , Persona de Mediana Edad , Índice Mitótico , Reacción en Cadena de la Polimerasa , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Adulto JovenRESUMEN
Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.
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Ojo/crecimiento & desarrollo , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Epitelio Pigmentado de la Retina/crecimiento & desarrollo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología , Animales , Aniridia/genética , Aniridia/patología , Apoptosis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/análisis , Proliferación Celular , Electrorretinografía , Eritropoyetina/metabolismo , Ojo/irrigación sanguínea , Ojo/citología , Eliminación de Gen , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microftalmía/genética , Microftalmía/patología , Epitelio Pigmentado de la Retina/citología , Factor A de Crecimiento Endotelial Vascular/análisis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived CCl2(-/-)/Crb1(Rd8/RD8), Cx3cr1(-/-)/Crb1(Rd8/RD8) and CCl2(-/-)/Cx3cr1(-/-)/Crb1(Rd8/RD8) mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling.
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Quimiocina CCL2/inmunología , Receptores de Quimiocina/inmunología , Retina/patología , Degeneración Retiniana/inmunología , Degeneración Retiniana/patología , Animales , Receptor 1 de Quimiocinas CX3C , Quimiocina CCL2/genética , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteínas del Tejido Nervioso/genética , Receptores de Quimiocina/genética , Retina/inmunología , Retina/metabolismo , Degeneración Retiniana/genéticaRESUMEN
Because retinal ischemia is a common cause of vision loss, we sought to determine the effects of ischemia on neuroretinal function and survival in murine oxygen-induced retinopathy (OIR) and to define the role of endogenous erythropoietin (EPO) in this model. OIR is a reproducible model of ischemia-induced retinal neovascularization; it is used commonly to develop antiangiogenic strategies. We investigated the effects of ischemia in murine OIR on retinal function and neurodegeneration by electroretinography and detailed morphology. OIR was associated with significant neuroretinal dysfunction, with reduced photopic and scotopic ERG responses and reduced b-wave/a-wave ratios consistent with specific inner-retinal dysfunction. OIR resulted in significantly increased apoptosis and atrophy of the inner retina in areas of ischemia. EPO deficiency in heterozygous Epo-Tag transgenic mice was associated with more profound retinal dysfunction after OIR, indicated by a significantly greater suppression of ERG amplitudes, but had no measurable effect on the extent of retinal ischemia, preretinal neovascularization, or neuroretinal degeneration in OIR. Systemic administration of recombinant EPO protected EPO-deficient mice against this additional suppression, but EPO supplementation in wild-type animals with OIR did not rescue neuroretinal dysfunction or degeneration. Murine OIR offers a valuable model of ischemic neuroretinal dysfunction and degeneration in which to investigate adaptive tissue responses and evaluate novel therapeutic approaches. Endogenous EPO can protect neuroretinal function in ischemic retinopathy.
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Eritropoyetina/fisiología , Isquemia/fisiopatología , Neuronas Retinianas/fisiología , Vasos Retinianos/patología , Animales , Apoptosis/fisiología , Hipoxia de la Célula/fisiología , Modelos Animales de Enfermedad , Electrorretinografía/métodos , Eritropoyetina/biosíntesis , Eritropoyetina/deficiencia , Eritropoyetina/uso terapéutico , Isquemia/complicaciones , Riñón/metabolismo , Ratones , Ratones Transgénicos , Oxígeno , Proteínas Recombinantes/uso terapéutico , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/etiología , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Neuronas Retinianas/patología , Regulación hacia ArribaRESUMEN
Retinal pathologies are frequently accompanied by retinal vascular responses, including the formation of new vessels by angiogenesis (neovascularization). Pathological vascular changes may also include less well characterized traits of vascular remodeling that are non-neovascular, such as vessel pruning and the emergence of dilated and tortuous vessel phenotypes (telangiectasis). The molecular mechanisms underlying neovascular growth versus non-neovascular remodeling are poorly understood. We therefore undertook to identify novel regulators of non-neovascular remodeling in the retina by using the dystrophic Royal College of Surgeons (RCS) rat and the retinal dystrophy 1 (RD1) mouse, both of which display pronounced non-neovascular remodeling. Gene expression profiling of isolated retinal vessels from these mutant rodent models and wild-type controls revealed 60 differentially expressed genes. These included the genes for apelin (Apln) and for its receptor (Aplnr), both of which were strongly up-regulated in the mutants. Crossing RD1 mice into an Apln-null background substantially reduced vascular telangiectasia. In contrast, Apln gene deletion had no effect in two models of neovascular pathology [laser-induced choroidal neovascularization and the very low density lipoprotein receptor (Vldlr)-knockout mouse]. These findings suggest that in these models apelin has minimal effect on sprouting retinal angiogenesis, but contributes significantly to pathogenic non-neovascular remodeling.
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Péptidos y Proteínas de Señalización Intercelular/fisiología , Degeneración Retiniana/fisiopatología , Vasos Retinianos/metabolismo , Adipoquinas , Animales , Apelina , Neovascularización Coroidal/genética , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/fisiopatología , Expresión Génica , Silenciador del Gen , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Microvasos/metabolismo , Mutación/genética , Ratas , Retina , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Telangiectasia Retiniana/prevención & control , Regulación hacia ArribaRESUMEN
The retina has a uniquely high metabolic demand for oxygen that is normally met by a highly efficient vascular supply. Oxygen plays an essential role in oxidative phosphorylation as an electron acceptor in the mitochondrial respiratory chain in the synthesis of adenosine triphosphate required to support the metabolic demand, including that of the visual cycle. Maintenance of normal retinal function depends on a continuous supply of oxygen and on the capability to detect and respond rapidly to local oxygen deficiency (hypoxia). The functional reserve of oxygen is small and retinal hypoxia can cause neuroretinal dysfunction and degeneration that lead directly to vision loss. Local oxygen sensing mechanisms control adaptive responses that can help protect against ischaemic injury. In the retina, powerful oxygen sensing mechanisms rapidly detect alterations in intracellular oxygen tension and respond with adaptive changes that redress the balance between oxygen supply and demand. These responses include rapid changes in blood flow, protective metabolic adaptations and angiogenesis. In the eye, however, the angiogenic response to hypoxia is typically associated with oedema, haemorrhage and fibrosis that can exacerbate hypoxic neuroretinal injury, causing severe vision loss. This aberrant response is the target of novel therapies including inhibitors of vascular endothelial growth factor. However, non-specific angiostatic agents fail to consider appropriate beneficial adaptive responses to hypoxia, and risk compromising neuroprotective mechanisms. In this review, we discuss the current understanding of retinal oxygenation and oxygen sensing in health and disease, focussing on the central role of hypoxia-inducible transcription factors, and suggest that therapeutic strategies may be improved by considering more targeted interventions.