Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1.

Tissues with high metabolic rates often use lipids, as well as glucose, for energy, conferring a survival advantage during feast and famine. Current dogma suggests that high-energy-consuming photoreceptors depend on glucose. Here we show that the retina also uses fatty acid ß-oxidation for energy. Moreover, we identify a lipid sensor, free fatty acid receptor 1 (Ffar1), that curbs glucose uptake when fatty acids are available. Very-low-density lipoprotein receptor (Vldlr), which is present in photoreceptors and is expressed in other tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acid. In the retinas of Vldlr(-/-) mice with low fatty acid uptake but high circulating lipid levels, we found that Ffar1 suppresses expression of the glucose transporter Glut1. Impaired glucose entry into photoreceptors results in a dual (lipid and glucose) fuel shortage and a reduction in the levels of the Krebs cycle intermediate α-ketoglutarate (α-KG). Low α-KG levels promotes stabilization of hypoxia-induced factor 1a (Hif1a) and secretion of vascular endothelial growth factor A (Vegfa) by starved Vldlr(-/-) photoreceptors, leading to neovascularization. The aberrant vessels in the Vldlr(-/-) retinas, which invade normally avascular photoreceptors, are reminiscent of the vascular defects in retinal angiomatous proliferation, a subset of neovascular age-related macular degeneration (AMD), which is associated with high vitreous VEGFA levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in macular telangiectasia, neovascular AMD and other retinal diseases.

Loss of Müller's cells and photoreceptors in macular telangiectasia type 2.

PURPOSE: To correlate postmortem histology from a patient with macular telangiectasia (MacTel) type 2 with previously recorded clinical imaging data. DESIGN: Observational clinicopathologic case report. METHODS: The distribution of retinal blood vessels was used to map the location of serial wax sections in color fundus and optical coherence tomography (OCT) images. Fluorescent immunohistochemistry was used to visualize markers for Müller's cells (vimentin and retinaldehyde-binding protein 1), photoreceptors (L-M opsin, rhodopsin, and cytochrome oxidase 2), and the outer limiting membrane (OLM) (zonula occludens 1 and occludin). MAIN OUTCOME MEASURES: Distribution of specific markers in immunohistochemistry on retinal sections through the fovea in relation to clinical data. RESULTS: The clinically recorded region of macular pigment loss in the macula correlated well with Müller's cell depletion. The OCT data showed a loss of the photoreceptor inner segment/outer segment (IS/OS) junction in the central retina, which correlated well with rod loss but not with cone loss. Markers for the OLM were lost where Müller's cells were lost. CONCLUSIONS: We have confirmed our previous finding of Müller's cell loss in MacTel type 2 and have shown that the area of Müller's cell loss matches the area of macular pigment depletion. In this patient, the IS/OS junction seen by OCT was absent in a region where rods were depleted but cones were still present.

Visualization of gene expression in whole mouse retina by in situ hybridization.

The mouse retinal vasculature provides a powerful model system for studying development and pathologies of the vasculature. Because it forms as a two-dimensional flat plexus, it is easily imaged in its entirety in whole-mount retinal preparations. In order to study molecular signaling mechanisms, it is useful to visualize the expression of specific genes in the entire vascular plexus and retina. However, in situ hybridization on whole-mount retinal preparations is problematic because isolated retinas have a tendency to curl up during hybridization and are difficult to stain. Here we provide a detailed protocol that overcomes these difficulties and visualizes the mRNA distribution of one or two genes in the context of the counterstained retinal vasculature. The protocol takes 3-4 d for single-probe stains, with an additional 2 d for immunohistochemistry co-labeling. In situ hybridization with two probes adds a further 3 d.

Characterization of antigen-binding and MHC class II-bearing T cells with suppressive activity in response to tolerogenic stimulus.

Antigen specific non-responsiveness is generally developed through clonal deletion, anergy, and suppression. The term "suppression" is being considered as a functional immune deficit that can be adoptively transferred by regulatory/suppressor T cells. Following tolerance induction protocols the aim of the present study was to characterize the T cells involved in antigen-specific suppression. After defining the immunogenic and tolerogenic protocols in vitro and in vivo, it was shown that CD90(+) cells from tolerogenic mice were able to reduce specific antibody production when adaptively transferred to immunized mice. These cells were shown to highly express CD25 and Foxp3, co-localizing with CD4 and MHC class II antigens (MHCII), while a small percentage of these cells (8-14%) was binding free antigen. Further isolation of CD90(+)MHCII(+) and CD90(+)HSA(+) from mice having received the tolerogenic treatment and adaptive transfer to immunized mice showed that CD90(+)MHCII(+) cells were able to suppress antigen-specific response only when transferred along with the second antigenic challenge, while CD90(+)HSA(+) cells were suppressive only when given along with the first antigenic challenge. The suppressive effect of these two sub-populations could also be obtained in in vitro spleen cell proliferation assays in response to the HSA antigenic stimulus. Both in vitro and in vivo tolerogenic treatments were shown to correlate with soluble MHCII production in culture supernatants or serum respectively. Increase of MHCII in the serum could only be detected upon adaptive transfer of CD90(+)HSA(+) cells, whereas transfer of CD90(+)MHCII(+) cells resulted in increased levels of IL-10 and IFN-γ in the serum. These results defined at least two different levels of suppression, one during the onset which was antigen-specific and MHC restricted, and another non-specific at the end of an immune response.