Blue-violet light decreases VEGFa production in an in vitro model of AMD

International audienceBlue light is an identified risk factor for age-related macular degeneration (AMD). The production of vascular endothelial growth factor (VEGF), leading to neovascularization, is a major complication of the wet form of this disease. We investigated how blue light affects VEGF expression and secretion using A2E-loaded retinal pigment epithelium (RPE) cells, a cell model of AMD. Incubation of RPE cells with A2E resulted in a significant increase in VEGF mRNA and, intracellular and secreted VEGF protein levels, but not mRNA levels of VEGFR1 or VEGFR2. Blue light exposure of A2E-loaded RPE cells resulted in a decrease in VEGF mRNA and protein levels, but an increase in VEGFR1 levels. The toxicity of 440 nm light on A2E-loaded RPE cells was enhanced by VEGF supplementation. Our results suggest that age-related A2E accumulation may result in VEGF synthesis and release. This synthesis of VEGF, which enhances blue light toxicity for the RPE cells, is itself suppressed by blue light. Anti-VEGF therapy may therefore improve RPE survival in AMD

Bird fancier's lung serodiagnosis by automated r-IgLL1 ELISA.

International audienceBird fancier's lung (BFL) is the most prevalent form of hypersensitivity pneumonitis (HP) worldwide. The current techniques used for the serological diagnosis of BFL all use crude extracts from feathers, droppings, and blooms as test antigens, which is associated with a lack of standardization and variability of the results. An antigenic protein, immunoglobulin lambda-like polypeptide-1 (IgLL1), isolated from pigeon droppings, was recently identified to be associated with BFL. We used genetic engineering to produce IgLL1 as a recombinant antigen

Transferrin Non-Viral Gene Therapy for Treatment of Retinal Degeneration

International audienceDysregulation of iron metabolism is observed in animal models of retinitis pigmentosa (RP) and in patients with age-related macular degeneration (AMD), possibly contributing to oxidative damage of the retina. Transferrin (TF), an endogenous iron chelator, was proposed as a therapeutic candidate. Here, the efficacy of TF non-viral gene therapy based on the electrotransfection of pEYS611, a plasmid encoding human TF, into the ciliary muscle was evaluated in several rat models of retinal degeneration. pEYS611 administration allowed for the sustained intraocular production of TF for at least 3 and 6 months in rats and rabbits, respectively. In the photo-oxidative damage model, pEYS611 protected both retinal structure and function more efficiently than carnosic acid, a natural antioxidant, reduced microglial infiltration in the outer retina and preserved the integrity of the outer retinal barrier. pEYS611 also protected photoreceptors from N-methyl-N-nitrosourea-induced apoptosis. Finally, pEYS611 delayed structural and functional degeneration in the RCS rat model of RP while malondialdehyde (MDA) ocular content, a biomarker of oxidative stress, was decreased. The neuroprotective benefits of TF non-viral gene delivery in retinal degenerative disease models further validates iron overload as a therapeutic target and supports the continued development of pEY611 for treatment of RP and dry AMD

Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF-α

International audienceOrthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14 + blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human-induced pluripotent stem cell-derived RPE cells, we show that activated Mos markedly inhibit RPE OTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF-α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others. Our data show how subretinal, chronic inflammation and in particular TNF-α can affect RPE function, which might contribute to the visual dysfunctions in diseases such as age-related macular degeneration (AMD) where subretinal macrophages are observed. Our findings provide important mechanistic insights into the regulation of OTX2 under inflammatory conditions. Therapeutic restoration of OTX2 expression might help revive RPE and visual function in retinal diseases such as AMD

Phototoxic Action Spectrum on a Retinal Pigment Epithelium Model of Age-Related Macular Degeneration Exposed to Sunlight Normalized Conditions

International audienceAmong the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye

Taurine deficiency damages retinal neurones: cone photoreceptors and retinal ganglion cells

International audienceIn 1970s, taurine deficiency was reported to induce photoreceptor degeneration in cats and rats. Recently, we found that taurine deficiency contributes to the retinal toxicity of vigabatrin, an antiepileptic drug. However, in this toxicity, retinal ganglion cells were degenerating in parallel to cone photoreceptors. The aim of this study was to re-assess a classic mouse model of taurine deficiency following a treatment with guanidoethane sulfonate (GES), a taurine transporter inhibitor to determine whether retinal ganglion cells are also affected. GES treatment induced a significant reduction in the taurine plasma levels and a lower weight increase. At the functional level, photopic electroretinograms were reduced indicating a dysfunction in the cone pathway. A change in the autofluorescence appearance of the eye fundus was explained on histological sections by an increased autofluorescence of the retinal pigment epithelium. Although the general morphology of the retina was not affected, cell damages were indicated by the general increase in glial fibrillary acidic protein expression. When cell quantification was achieved on retinal sections, the number of outer/inner segments of cone photoreceptors was reduced (20 %) as the number of retinal ganglion cells (19 %). An abnormal synaptic plasticity of rod bipolar cell dendrites was also observed in GES-treated mice. These results indicate that taurine deficiency can not only lead to photoreceptor degeneration but also to retinal ganglion cell loss. Cone photoreceptors and retinal ganglion cells appear as the most sensitive cells to taurine deficiency. These results may explain the recent therapeutic interest of taurine in retinal degenerative pathologies

Characterization of the autofluorescence in A2E-loaded RPE cells.

<p><b>A.</b> Absorbance of RPE cells treated with 40 µM A2E (A2E+RPE (a), solid line) or A2E-untreated (RPE (b), dashed line). The curve ((a)–(b), dot line) representing the difference of absorption spectra between A2E-loaded RPE cells (a) and A2E-untreated RPE cells (b), shows absorption peaks at 335 nm and 440 nm. <b>B.</b> Absorbance spectra of free A2E in pure ethanol (solid line) or in modified DMEM (dashed line). Spectra are similar in both media and A2E displays maxima of absorbance at 335 nm and 440 nm. <b>C.</b> Emission spectra of RPE cells treated with 40 µM of A2E (A2E+RPE (a), solid line) or untreated (RPE (b), dashed line) under a 440 nm excitation. The curve ((a)–(b), dot line) representing the difference between the emission spectra in A2E-loaded RPE cells (a) and A2E-untreated RPE cells (b) shows a peak at 620 nm. <b>D.</b> Emission spectra of free A2E in pure ethanol (solid line) or in modified DMEM (dashed line) with a 440 nm excitation. Spectra are similar in both media and A2E displays a maximum of emission at 640 nm. <b>E.</b> A2E contents quantified by UPLC in RPE cells after 6 hours of incubation in various A2E concentrations (0, 5, 15, 20, 30 and 40 µM) in culture medium. The A2E content in RPE cells increases in a linear way according to the incubated A2E concentration. (n = 4, r² = 0.9955). (RFU: Relative fluorescence unit).</p

Light-induced morphological changes in A2E-loaded RPE cells.

<p>Images of RPE cells were obtained 6 hours after 18 hour light exposure with a 10 nm illumination band centered at 440 nm (<b>E–H</b>), at 480 nm (<b>I–L</b>) or maintained in darkness (<b>A–D</b>). RPE cells were incubated with A2E at 0 µM (<b>A, E, I</b>), 12.5 µM (<b>B, F, J</b>), 20 µM (<b>C, G, K</b>) or 40 µM (<b>D, H, L</b>). Note the yellow tint of A2E-loaded RPE cells maintained in darkness (<b>B–D</b>). RPE cells treated with A2E at 20 µM (<b>G</b>) or 40 µM (H) became round and lost their confluence after their exposure to a 10 nm band centered at 440 nm. By contrast, A2E-loaded RPE cells appeared healthy after their exposure to a 10 nm band centered at 480 nm (<b>J–L</b>) similarly as cells maintained in darkness (<b>B–D</b>) or A2E-untreated (<b>A, I</b>). Scale bar in A represents 20 µm.</p

A2E dose response curves of the A2E-elicited phototoxicity on RPE cells.

<p>Cell viability (<b>A</b>) and cell apoptosis (<b>B</b>) were quantified with the ApoTox-Glo™ assay according to the A2E concentrations (0, 12.5, 20 and 40 µM) for RPE cells exposed to the 10 nm illumination bands centered at 440 or 480 nm. Cell viability and apoptosis were normalized with the experimental value obtained for RPE cells maintained in darkness without A2E treatment. The <i>P</i>-value was calculated using t-test. For the two illumination bands, statistically significant differences are indicated with respect to the illuminated conditions but in the absence of A2E (^#p<0.05, ^##p<0.01, ^###p<0.001) and when considering two A2E concentrations (*p<0.05, **p<0.01, ***p<0.001).</p