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PURPOSE: To determine whether the clinical and paraclinical course of Fuchs endothelial corneal dystrophy (FECD) over 1 year is related to the extent of triplet repetition in the transcription factor 4 (TCF4) gene. METHODS: A prospective study with a 1-year follow-up was conducted. A total of 104 patients (160 eyes) with FECD and an equivalent number of age- and sex-matched control subjects without FECD were included. At inclusion, the corneas were graded using the modified Krachmer grade (KG) and patients were genotyped for the number of trinucleotide repeats (TNRs) in the TCF4 gene by the short tandem repeat assay. Visual acuity, Scheimpflug tomographic features, and the Visual Function and Corneal Health Status using a visual disability instrument were measured on 2 visits at 1-year intervals. RESULTS: KGs ranged from 1 to 6, and 46% of eyes had grades 1 to 4. 71% of the patients harbored TNR expansion (>40) versus 13% in control subjects (P < 0.001). Severity at inclusion was higher in the presence of TNR expansion when considering eyes independently (mean grade ±SD, 4.08 ± 1.42) without TNR expansion and 4.66 ± 1.27 with TNR expansion (P = 0.024). In 1 year, the ETDRS score significantly decreased by -2.97 (95% confidence interval -4.69 to -1.26, P = 0.001) and the ETDRS score with glare by -4.25 (95% confidence interval -6.22 to -2.27, P < 10-5). There was no relationship between the rate of decline and TNR expansion or KG. Central corneal thickness and Visual Function and Corneal Health Status scores did not significantly vary. CONCLUSIONS: It is possible to measure a subtle progression of FECD over a period as short as 1 year. We did not find a relationship between the presence of TNR expansion and the speed of deterioration over 1 year. This work should facilitate the design of future clinical trials on FECD.Trial Registration-URL: ClinicalTrials.gov. Identifier: French Fuchs Follow-up Study (F3S): NCT03974230.
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AIM: To investigate the interest of chromatic confocal microscopy (CCM) to characterise guttae in Fuchs endothelial corneal dystrophy (FECD). METHODS: Descemet's membranes (DM) were obtained during endothelial keratoplasty in patients with FECD and pseudophakic bullous keratopathy (PBK). They were compared with healthy samples obtained from body donation to science. Samples were fixed in 0.5% paraformaldehyde and flat mounted. Surface roughness of DMs was quantified using CCM and the AltiMap software that provided the maximum peak (Sp) and valley (Sv) heights, the mean square roughness (Rq) and the asymmetry coefficient (Ssk). RESULTS: The physiological roughness of healthy samples was characterised by an Rq of 0.12±0.05 µm, which was two times rougher than in PBK (Rq=0.06±0.03 µm), but both were still flat with a symmetrical distribution between peaks and valleys (Ssk close to 0, npeaks=nvalleys), smaller than 1 µm. In FECD, the maximum peak height was 5.10±2.40 µm, up to 5.8 and 8.3 times higher than the control and PBK, respectively. The maximum valley depth was half than the peak (2.28±0.89 µm). The surface with guttae was very rough (Rq=0.45±0.14 µm) and the Ssk=1.84± 0.43 µm, greater than 0, confirms an asymmetric surface with high peaks and low valleys (npeaks>nvalleys). Moreover, the CCM provided quantitative parameters allowing to distinguish different types of guttae from different patients. CONCLUSIONS: CCM is an innovative approach to describe and quantify different morphologies of guttae. It could be useful to analyse the different stages of FECD and define subgroups of patients.
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PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: to measure endothelial viability of precut DSAEK after 3 or 10 days of storage in our ASM in a preclinical study. METHODS: Human pairs of corneas were included. The endothelial cell density (ECD in cells/mm2), and central corneal thickness (CCT in µm) were measured to ensure their initial intra pair comparability. After deswelling (CorneaJet, Eurobio) grafts preparation was performed by cutting the anterior stroma with a Moria linear microkeratome and keeping the anterior lamellae attached during storage. After randomization, one cornea was kept in the corneajet bottle (CJ) and the other was inserted into the ASM allowing a renewal or storage medium (CorneaMax, Eurobio) at 2.6 µL/min with 21 mmHg of pressure in the endothelial chamber. Both group of corneas were stored for 3 or 10 days at 31°C. The final viable ECD (vECD) was determined using the triple staining with Hoechst-Ethidium-Calcein-AM by an independent experimenter in a masked fashion. RESULTS: Initial ECDs were comparable: 2595±878 in ASM versus 2654±954 cells/mm2 in CJ for the 3-days period (n=5 pairs) and 2416±712 in ASM versus 2492±764 cells/mm2 in CJ for the 10-period (n=5 pairs). CCTs were also comparable. The anterior lamellae stayed attached in either the ASM or CJ. vECD was significantly higher in ASM than in CJ with respectively 2062±695 cells/mm2 versus 1632±633 cells/mm2 after 3 days either a cell loss of 20.5% and 38.5% respectively (p=0.0062) and 1082±649 versus 935±691 cells/mm2 for the 10-day period either a cell loss of 132% and 164% respectively (p=0.005). Grafts thickness did not differ after 3 days 219±25 µm in ASM versus 182±39 µm (p=0.063) or 10 days respectively 221±58 µm versus 189±48 µm (p=0.06). CONCLUSION: The storage of precut DSAEKs into the ASM allows a better preservation of grafts without use on deswelling storage medium. Nevertheless, the cell loss remains high after 10 days, suggesting a significant cell stress.
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Córnea , Bancos de Ojos , Humanos , Estudios de Factibilidad , Córnea/cirugía , EtidioRESUMEN
PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: To measure the endothelial viability of pre-dissected DMEK after 3 and 10 days of storage in our ASM in a preclinical study. METHODS: Pairs of human corneas were included. The endothelial cell density (ECD in cells/mm2), thickness and transparency of corneas were measured before graft preparation. Descemet's membrane (DM) was peeled using the no-touch technique leaving the graft attached to the center of the cornea (on approx. 1mm2). After randomization, one cornea was kept in organ culture (OC) and the other in the ASM (21 mmHg, 2.6 µL/min) in the same medium (CorneaMax, Eurobio). The final viable ECD was determined using the triple staining with Hoechst-Ethidium-Calcein-AM. In addition, the expression of CD166 and NCAM (lateral membranes), ZO-1 (apical junctions), Na+/K+ ATPase (endothelial pump function) and COX-IV (mitochondrial content) was studied by immunostaining to characterize endothelial cells after the storage. RESULTS: Initial ECDs were comparable: 2185±232 cells/mm2 in the ASM versus 2276±328 in OC for the 3-day period and 2680±416 cells/mm2 in the ASM versus 2644±420 in OC for the 10-day period. The DMs did not fold back in either BR or OC. The viable ECD did not differ significantly between the ASM and OC for either storage period: 2378±501 (ASM) versus 2342±503 (OC) for the 3-day period (n=8 pairs and p=0.624) and 2482±288 (ASM) versus 2579±315 (OC) for the 10-day period (n=5 pairs and p=0.176). Corneas were more transparent and thinner in the ASM than in OC after 3 days (916±86 versus 1193±136µm, p=0.0001) and 10 days (957±128 versus 1220±105µm, p=0.0625). The functional and structural markers studied were expressed in both groups after 3 and 10 days, some better preserved in the ASM. CONCLUSION: The storage of precut DMEKs is possible in ASM and OC for at least 10 days. Interestingly, a pre-dissected endothelium continues to partially exert its pump function into the ASM. In practice, this could allow the stroma to be used for DALK without further deswelling. In addition to improving the storage of whole grafts, the ASM allows the storage of precut DMEKs for up to 10 days with excellent endothelial survival.
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Córnea , Células Endoteliales , Humanos , Estudios de Factibilidad , Etidio , Bancos de Ojos , ATPasa Intercambiadora de Sodio-PotasioRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet's membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεß) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae.
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Distrofia Endotelial de Fuchs , Humanos , Distrofia Endotelial de Fuchs/metabolismo , Lámina Limitante Posterior , Proteómica , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismoRESUMEN
The retina is the neurosensitive layer of the eye. In this tissue, photoreceptors convert light into nerve signals to be relayed to the brain. Despite retinal specialization in the treatment of light, excessive exposure can cause retinal damage, called retinal phototoxicity. In recent years, lighting devices rich in wavelengths of high energy (blue light) appeared, raising new concerns about retinal protection against light damage. We focus here on light-induced ocular diseases and the possible influence on visual health of new lighting technologies.
TITLE: Les nouveaux éclairages et nos yeux. ABSTRACT: Dans la rétine, couche neurosensorielle de l'Åil, les photorécepteurs transforment le signal lumineux en influx nerveux interprétable par le cerveau. Malgré sa spécialisation dans le traitement des signaux lumineux, la rétine peut subir des dommages, à la suite d'une exposition excessive à la lumière ; on parle alors de phototoxicité rétinienne. Ces dernières années, l'apparition de dispositifs d'éclairage riches en longueurs d'onde de forte énergie (ce que l'on nomme lumière bleue), remet le problème de la phototoxicité rétinienne à l'ordre du jour. Nous discutons des pathologies oculaires induites par la lumière et de la possible influence des nouvelles technologies d'éclairage sur notre santé visuelle.
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Ojo/efectos de la radiación , Iluminación , Fenómenos Fisiológicos Oculares/efectos de la radiación , Adaptación Ocular/fisiología , Adaptación Ocular/efectos de la radiación , Ojo/fisiopatología , Humanos , Invenciones , Luz/efectos adversos , Iluminación/efectos adversos , Iluminación/métodos , Iluminación/tendenciasRESUMEN
A common allele (402H) of the complement factor H (FH) gene is the major risk factor for age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. Development and progression of AMD involves vascular and inflammatory components partly by deregulation of the alternative pathway of the complement system (AP). The loss of central vision results from atrophy and/or from abnormal neovascularization arising from the choroid. The functional link between FH, the main inhibitor of AP, and choroidal neovascularization (CNV) in AMD remains unclear. In a murine model of CNV used as a model for neovascular AMD (nAMD), intraocular human recombinant FH (recFH) reduced CNV as efficiently as currently used anti-VEGF (vascular endothelial growth factor) antibody, decreasing deposition of C3 cleavage fragments, membrane attack complex (MAC), and microglia/macrophage recruitment markers in the CNV lesion site. In sharp contrast, recFH carrying the H402 risk variant had no effect on CNV indicating a causal link to disease etiology. Only the recFH NTal region (recFH1-7), containing the CCPs1-4 C3-convertase inhibition domains and the CCP7 binding domain, exerted all differential biological effects. The CTal region (recFH7-20) containing the CCP7 and CCPs19-20 binding domains was antiangiogenic but did not reduce the microglia/macrophage recruitment. The antiangiogenic effect of both recFH1-20 and recFH-CCP7-20 resulted from thrombospondin-1 (TSP-1) upregulation independently of the C3 cleavage fragments generation. This study provides insight on the mechanistic role of FH in nAMD and invites to reconsider its therapeutic potential.
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Coroides/patología , Factor H de Complemento/metabolismo , Macrófagos/inmunología , Degeneración Macular/metabolismo , Alelos , Animales , Coroides/irrigación sanguínea , Neovascularización Coroidal , Activación de Complemento , Complemento C3/metabolismo , Factor H de Complemento/genética , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Endogámicas , Riesgo , Trombospondina 1/metabolismoRESUMEN
PURPOSE: This study was conducted in order to study Sostdc1 expression in rat and human developing and adult eyes. METHODS: Using the yeast signal sequence trap screening method, we identified the Sostdc1 cDNA encoding a protein secreted by the adult rat retinal pigment epithelium. We determined by in situ hybridization, RT-PCR, immunohistochemistry, and western blot analysis Sostdc1 gene and protein expression in developing and postnatal rat ocular tissue sections. We also investigated Sostdc1 immunohistolocalization in developing and adult human ocular tissues. RESULTS: We demonstrated a prominent Sostdc1 gene expression in the developing rat central nervous system (CNS) and eyes at early developmental stages from E10.5 days postconception (dpc) to E13 dpc. Specific Sostdc1 immunostaining was also detected in most adult cells of rat ocular tissue sections. We also identified the rat ocular embryonic compartments characterized by a specific Sostdc1 immunohistostaining and specific Pax6, Sox2, Otx2, and Vsx2 immunohistostaining from embryonic stages E10.5 to E13 dpc. Furthermore, we determined the localization of SOSTDC1 immunoreactivity in ocular tissue sections of developing and adult human eyes. Indeed, we detected SOSTDC1 immunostaining in developing and adult human retinal pigment epithelium (RPE) and neural retina (NR) as well as in several developing and adult human ocular compartments, including the walls of choroidal and scleral vessels. Of utmost importance, we observed a strong SOSTDC1 expression in a pathological ocular specimen of type 2 Peters' anomaly complicated by retinal neovascularization as well in the walls ofother pathological extra-ocular vessels. CONCLUSION: As rat Sostdc1 and human SOSTDC1 are dual antagonists of the Wnt/ß-catenin and BMP signaling pathways, these results underscore the potential crucial roles of these pathways and their antagonists, such as Sostdc1 and SOSTDC1, in developing and adult mammalian normal eyes as well as in syndromic and nonsyndromic congenital eye diseases.
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Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedades Hereditarias del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Epitelio Pigmentado de la Retina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Anciano , Animales , Western Blotting , Preescolar , Modelos Animales de Enfermedad , Enfermedades Hereditarias del Ojo/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratas , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/crecimiento & desarrolloRESUMEN
BACKGROUND: Alzheimer's disease (AD) and age-related macular degeneration (AMD) present similarities, particularly with respect to oxidative stress, including production of 4-Hydroxy-2- nonenal (HNE). AMD has been named the AD in the eye. The Müller cells (MC) function as a principal glia of the retina and maintain water/potassium, glutamate homeostasis and redox status. Any MC dysfunction results in retinal neurodegeneration. OBJECTIVES: We investigated the effects of HNE in human MC. RESULTS: HNE induced an increase of the reactive oxygen species associated with mitochondrial dysfunction and apoptosis. HNE induced endoplasmic reticulum (ER) stress (upregulation of GRP78/Bip, and the proapoptotic factor, CHOP). HNE also impaired expression of genes controlling potassium homeostasis (KCNJ10), glutamate detoxification (GS), and the visual cycle (RLBP1). MC adaptive response to HNE included upregulation of amyloid-ß protein precursor (AßPP). To determine the role of AßPP, we overexpressed AßPP in MC. Overexpression of AßPP induced strong antioxidant and anti-ER stress (PERK downregulation and GADD34 upregulation) responses accompanied by activation of the prosurvival branch of the unfolded protein response. It was also associated with upregulation of major genes involved in MC-controlled retinal homeostasis (KCNJ10, GS, and RLBP1) and protection against HNE-induced apoptosis. Therefore, AßPP is an ER and oxidative stress responsive molecule, and is able to stimulate the transcription of major genes involved in MC functions impaired by HNE. CONCLUSION: Our study suggests that targeting oxidative and ER stress might be a potential therapeutic strategy against glia impairment in AMD and AD, in light of the common features between the two pathologies.
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Precursor de Proteína beta-Amiloide/metabolismo , Supervivencia Celular/fisiología , Neuroglía/metabolismo , Estrés Oxidativo/fisiología , Transcriptoma , Respuesta de Proteína Desplegada/fisiología , Precursor de Proteína beta-Amiloide/genética , Muerte Celular/fisiología , Línea Celular , Membrana Celular/metabolismo , Chaperón BiP del Retículo Endoplásmico , Humanos , Mitocondrias/metabolismo , Neuroprotección/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética/fisiologíaRESUMEN
As a part of the central nervous system, the retina may reflect both physiological processes and abnormalities related to pathologies that affect the brain. Amyloidosis due to the accumulation of amyloid-beta (Aß) was initially regarded as a specific and exclusive characteristic of neurodegenerative alterations seen in the brain of Alzheimer's disease (AD) patients. More recently, it was discovered that amyloidosis-related alterations, similar to those seen in the brain of Alzheimer's patients, also occur in the retina. Remarkably, these alterations were identified not only in primary retinal pathologies, such as age-related macular degeneration (AMD) and glaucoma, but also in the retinas of Alzheimer's patients. In this review, we first briefly discuss the biogenesis of Aß, a peptide involved in amyloidosis. We then discuss some pathological aspects (synaptic dysfunction, mitochondrial failure, glial activation, and vascular abnormalities) related to the neurotoxic effects of Aß. We finally highlight common features shared by AD, AMD, and glaucoma in the context of Aß amyloidosis and further discuss why the retina, due to the transparency of the eye, can be considered as a "window" to the brain.
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BACKGROUND: Amyloid precursor protein knockout mice (APP-KO) have impaired differentiation of amacrine and horizontal cells. APP is part of a gene family and its paralogue amyloid precursor-like protein 2 (APLP2) has both shared as well as distinct expression patterns to APP, including in the retina. Given the impact of APP in the retina we investigated how APLP2 expression affected the retina using APLP2 knockout mice (APLP2-KO). RESULTS: Using histology, morphometric analysis with noninvasive imaging technique and electron microscopy, we showed that APLP2-KO retina displayed abnormal formation of the outer synaptic layer, accompanied with greatly impaired photoreceptor ribbon synapses in adults. Moreover, APLP2-KO displayed a significant decease in ON-bipolar, rod bipolar and type 2 OFF-cone bipolar cells (36, 21 and 63 %, respectively). Reduction of the number of bipolar cells was accompanied with disrupted dendrites, reduced expression of metabotropic glutamate receptor 6 at the dendritic tips and alteration of axon terminals in the OFF laminae of the inner plexiform layer. In contrast, the APP-KO photoreceptor ribbon synapses and bipolar cells were intact. The APLP2-KO retina displayed numerous phenotypic similarities with the congenital stationary night blindness, a non-progressive retinal degeneration disease characterized by the loss of night vision. The pathological phenotypes in the APLP2-KO mouse correlated to altered transcription of genes involved in pre- and postsynatic structure/function, including CACNA1F, GRM6, TRMP1 and Gα0, and a normal scotopic a-wave electroretinogram amplitude, markedly reduced scotopic electroretinogram b-wave and modestly reduced photopic cone response. This confirmed the impaired function of the photoreceptor ribbon synapses and retinal bipolar cells, as is also observed in congenital stationary night blindness. Since congenital stationary night blindness present at birth, we extended our analysis to retinal differentiation and showed impaired differentiation of different bipolar cell subtypes and an altered temporal sequence of development from OFF to ON laminae in the inner plexiform layer. This was associated with the altered expression patterns of bipolar cell generation and differentiation factors, including MATH3, CHX10, VSX1 and OTX2. CONCLUSIONS: These findings demonstrate that APLP2 couples retina development and synaptic genes and present the first evidence that APLP2 expression may be linked to synaptic disease.
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Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Enfermedades Hereditarias del Ojo/genética , Eliminación de Gen , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Miopía/genética , Ceguera Nocturna/genética , Envejecimiento/patología , Células Amacrinas/metabolismo , Precursor de Proteína beta-Amiloide/deficiencia , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular , Proteínas del Sistema Complemento/metabolismo , Dendritas/metabolismo , Enfermedades Hereditarias del Ojo/patología , Enfermedades Hereditarias del Ojo/fisiopatología , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Miopía/patología , Miopía/fisiopatología , Neurogénesis , Ceguera Nocturna/patología , Ceguera Nocturna/fisiopatología , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/ultraestructura , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Bipolares de la Retina/metabolismo , Células Bipolares de la Retina/patología , Células Bipolares de la Retina/ultraestructura , Transmisión Sináptica , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Retinal Müller glial cells have already been implicated in age-related macular degeneration (AMD). AMD is characterized by accumulation of toxic amyloid-ß peptide (Aß); the question we raise is as follows: is P2X7 receptor, known to play an important role in several degenerative diseases, involved in Aß toxicity on Müller cells? Retinal Müller glial cells were incubated with Aß for 48 h. Cell viability was assessed using the alamarBlue assay and cytotoxicity using the lactate dehydrogenase (LDH) release assay. P2X7 receptor expression was highlighted by immunolabeling observed on confocal microscopy and its activation was evaluated by YO-PRO-1 assay. Hoechst 33342 was used to evaluate chromatin condensation, and caspases 8 and 3 activation was assessed using AMC assays. Lipid formulation rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) used in Age-Related Eye Disease Study 2 was incubated on cells for 15 min prior to Aß incubation. For the first time, we showed that Aß induced caspase-independent apoptosis through P2X7 receptor activation on our retinal model. DHA and EPA are polyunsaturated fatty acids recommended in food supplement to prevent AMD. We therefore modulated Aß cytotoxicity using a lipid formulation rich in DHA and EPA to have a better understanding of the results observed in clinical studies. We showed that fish oil rich in EPA and DHA, in combination with a potent P2X7 receptor antagonist, represents an efficient modulator of Aß toxicity and that P2X7 could be an interesting therapeutic target to prevent AMD.
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Péptidos beta-Amiloides/fisiología , Apoptosis/fisiología , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Aceites de Pescado/química , Receptores Purinérgicos P2X7/fisiología , Línea Celular Transformada , Células Cultivadas , Humanos , Microscopía FluorescenteRESUMEN
Imatinib mesylate is used in targeted therapy of cancer to inhibit type III tyrosine kinase receptors, such as KIT and platelet-derived growth factor receptors (PDGFRs). Expression of KIT in uveal melanoma (UM) suggests that this receptor may be the target of imatinib mesylate therapy. However, phase II multicenter clinical studies have shown no effect of imatinib mesylate in patients with unresectable liver metastases of UM. We therefore investigated which molecular mechanisms promote imatinib mesylate-resistance in metastatic UM. Expression of KIT, stem cell factor (SCF), PDGFRα and PDGFRß, was analyzed by RT-PCR, immunostaining, and Western blot in twenty-four samples of UM liver metastases, as well as UM primary tumor and metastatic cell lines. Soluble SCF was quantified in UM cell lines using enzyme-linked immunosorbent assay. Cell viability of UM cell lines treated with imatinib mesylate and grown in SCF-supplemented medium or in microvascular endothelial cells-conditioned medium was studied by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays. UM liver metastases and cell lines expressed KIT and SCF, but not the PDGFRs. Ninety-five percent of liver metastases expressed KIT at the protein level, but PDGFRs were not detected in these samples. Imatinib mesylate reduced the viability of UM metastatic cell lines in a concentration-dependent manner, but an increased resistance to this drug was observed when cells were incubated in SCF-supplemented or microvascular endothelial cells-conditioned medium. This study provides evidence that tumor microenvironment cytokines such as SCF may promote resistance to imatinib mesylate in metastatic UM.
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Antineoplásicos/farmacología , Benzamidas/farmacología , Resistencia a Antineoplásicos/genética , Melanoma/genética , Melanoma/patología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Pirimidinas/farmacología , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/patología , Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Línea Celular Tumoral , Expresión Génica , Humanos , Mesilato de Imatinib , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Melanoma/tratamiento farmacológico , Metástasis de la Neoplasia , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/uso terapéutico , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Células Madre/genética , Resultado del Tratamiento , Neoplasias de la Úvea/tratamiento farmacológicoRESUMEN
In normal retinas, amyloid-ß (Aß) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aß remain inadequately elucidated. We previously showed that subretinal injection of Aß(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aß(1-42) on RMG cells and of Aß(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aß(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aß(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aß(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aß of specific RMG cell functions.
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Péptidos beta-Amiloides/administración & dosificación , Gliosis/patología , Inflamación/patología , Fragmentos de Péptidos/administración & dosificación , Degeneración Retiniana/patología , Péptidos beta-Amiloides/toxicidad , Animales , Apoptosis/efectos de los fármacos , Barrera Hematorretinal/efectos de los fármacos , Barrera Hematorretinal/patología , Barrera Hematorretinal/fisiopatología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Homeostasis/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/toxicidad , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/fisiología , Retina/efectos de los fármacos , Retina/patología , Retina/fisiopatología , Degeneración Retiniana/genética , Degeneración Retiniana/fisiopatologíaRESUMEN
Amyloid precursor protein (APP) is a transmembrane glycoprotein frequently studied for its role in Alzheimer's disease. Our recent study in APP knockout (KO) mice identified an important role for APP in modulating normal neuronal development in the retina. However the role APP plays in the adult retina and whether it is required for vision is unknown. In this study we evaluated the role of APP in retinal function and morphology comparing adult wildtype (WT) and APP-KO mice. APP was expressed on neuronal cells of the inner retina, including horizontal, cone bipolar, amacrine and ganglion cells in WT mice. The function of the retina was assessed using the electroretinogram and although the rod photoreceptor responses were similar in APP-KO and WT mice, the post-photoreceptor, inner retinal responses of both the rod and cone pathways were reduced in APP-KO mice. These changes in inner retinal function did not translate to a substantial change in visual acuity as assessed using the optokinetic response or to changes in the gross cellular structure of the retina. These findings indicate that APP is not required for basic visual function, but that it is involved in modulating inner retinal circuitry.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Células Fotorreceptoras de Vertebrados/fisiología , Retina/fisiología , Transducción de Señal , Precursor de Proteína beta-Amiloide/genética , Animales , Electrorretinografía , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/citología , Retina/metabolismo , Factores de TiempoAsunto(s)
Péptidos beta-Amiloides/metabolismo , Degeneración Macular/metabolismo , Degeneración Macular/patología , Retinitis/metabolismo , Retinitis/patología , Animales , Proteínas del Sistema Complemento/inmunología , Humanos , Degeneración Macular/inmunología , Ratones , Drusas del Disco Óptico/inmunología , Drusas del Disco Óptico/metabolismo , Drusas del Disco Óptico/patología , Retinitis/inmunologíaRESUMEN
RASSF1A gene, found at the 3p21.3 locus, is a tumor suppressor gene frequently hypermethylated in human cancers. In this study, we report that compared with melanocytes in normal choroid, RASSF1A is downregulated in uveal melanoma samples and in uveal melanoma cell lines. LOH at 3p21.3 was detected in 50% of uveal melanoma. Moreover, methylation of the RASSF1A promoter was detected in 35 of 42 tumors (83%) and RASSF1A was also weakly expressed at the mRNA level. These data indicate that LOH at the RASSF1A locus or RASSF1A promoter methylation may partly account for the suppression of RASSF1A expression observed in uveal melanoma. Furthermore, following ectopic expression in three RASSF1A-deficient melanoma cell lines (OCM-1, Mel270, and 92.1), RASSF1A weakly reduces cell proliferation and anchorage-independent growth of uveal melanoma cells without effect on ERK1/2 activation, cyclin D1 and p27(Kip1) expression. This study explored biological functions and underlying mechanisms of RASSF1A in the ERK1/2 pathway in normal uveal melanocytes. We showed that siRNA-mediated depletion of RASSF1A increased ERK1/2 activation, cyclin D1 expression, and also decreased p27(Kip1) expression in normal uveal melanocytes. Moreover, that the depletion of RASSF1A induced senescence-associated ß-galactosidase activity and increased p21(Cip1) expression suggests that RASSF1A plays a role in the escape of cellular senescence in normal uveal melanocytes. Interestingly, we found that RASSF1A was epigenetically inactivated in long-term culture of uveal melanocytes. Taken together, these data show that depletion of RASSF1A could be an early event observed during senescence of normal uveal melanocytes and that additional alterations are acquired during malignant transformation to uveal melanoma.
Asunto(s)
Melanocitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Pérdida de Heterocigocidad , Sistema de Señalización de MAP Quinasas , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genéticaRESUMEN
Age-related macular degeneration is characterized by the formation of drusen containing amyloid-ß (Aß) and the degeneration of photoreceptors. To explore the largely unknown role of Aß in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aß(1-42). Subretinal injection of the Aß peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aß did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aß-induced photoreceptor apoptosis. Subretinal injection of Aß was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aß in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aß-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aß-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aß-injected retinas. Our study pinpoints the roles of Aß and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Apoptosis/fisiología , Quimiocina CCL2/genética , Citoprotección , Inflamación/metabolismo , Fragmentos de Péptidos/toxicidad , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Receptores CCR2/genética , Animales , Quimiocina CCL2/deficiencia , Citoprotección/genética , Humanos , Inflamación/genética , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR2/deficienciaRESUMEN
Very few studies have examined expression and function of amyloid precursor protein (APP) in the retina. We showed that APP mRNA and protein are expressed according to the different waves of retinal differentiation. Depletion of App led to an absence of amacrine cells, a 50% increase in the number of horizontal cells and alteration of the synapses. The retinas of adult APP(-/-) mice showed only half as many glycinergic amacrine cells as wild-type retinas. We identified Ptf1a, which plays a role in controlling both amacrine and horizontal cell fates, as a downstream effector of APP. The observation of a similar phenotype in sorLA knockout mice, a major regulator of APP processing, suggests that regulation of APP functions via sorLA controls the determination of amacrine and horizontal cell fate. These findings provide novel insights that indicate that APP plays an important role in retinal differentiation.
Asunto(s)
Precursor de Proteína beta-Amiloide/fisiología , Diferenciación Celular/fisiología , Retina/embriología , Retina/crecimiento & desarrollo , Envejecimiento/fisiología , Células Amacrinas/citología , Células Amacrinas/fisiología , Animales , Proliferación Celular , Ratones , Ratones Noqueados , Modelos Animales , Retina/citología , Células Horizontales de la Retina/citología , Células Horizontales de la Retina/fisiología , Sinapsis/fisiología , Factores de Transcripción/fisiologíaRESUMEN
PURPOSE: Activated B-Raf alone cannot induce melanoma but must cooperate with other signaling pathways. The phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR)/p70S6K pathways are critical for tumorigenesis. The authors investigated the role of these pathways in uveal melanoma cells. METHODS: The effects of PI3K and mTOR activation and inhibition on the proliferation of human uveal melanoma cell lines expressing either activated (WT)B-Raf or (V600E)B-Raf were investigated. Interactions among PI3K, mTOR, and B-Raf/ERK were studied. RESULTS: Inhibition of PI3K deactivated P70S6 kinase, reduced cell proliferation by 71% to 84%, and increased apoptosis by a factor of 5.0 to 8.4 without reducing ERK1/2 activation, indicating that ERK plays no role in mediating PI3K in these processes. In contrast, rapamycin-induced inhibition of mTOR did not significantly affect cell proliferation because it simultaneously stimulated PI3K/Akt activation and cyclin D1 expression. Regardless of B-Raf mutation status, cotreatment with the PI3K inhibitor effectively sensitized all melanoma cell lines to the B-Raf or ERK1/2 inhibition-induced reduction of cell proliferation. B-Raf/ERK and PI3K signaling, but not mTOR signaling, converged to control cyclin D1 expression. Moreover, p70S6K required the activation of ERK1/2. These data demonstrate that PI3K/Akt and mTOR/P70S6K interact with B-Raf/ERK. CONCLUSIONS: Activated PI3K/Akt attenuates the inhibitory effects of rapamycin on cell proliferation and thus serves as a negative feedback mechanism. This finding suggests that rapamycin is unlikely to inhibit uveal melanoma growth. In contrast, targeting PI3K while inhibiting B-Raf/ERK may be a promising approach to reduce the proliferation of uveal melanoma cells.