Exposure to the complement C5b-9 complex sensitizes 661W photoreceptor cells to both apoptosis and necroptosis.

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease

Retinal changes precede visual dysfunction in the complement factor h knockout mouse

We previously reported that aged mice lacking complement factor H (CFH) exhibit visual defects and structural changes in the retina. However, it is not known whether this phenotype is age-related or is the consequence of disturbed development. To address this question we investigated the effect of Cfh gene deletion on the retinal phenotype of young and mid-age mice. Cfh (-/-) mouse eyes exhibited thickening of the retina and reduced nuclear density, but relatively normal scotopic and photopic electroretinograms. At 12 months there was evidence of subtle astroglial activation in the Cfh (-/-) eyes, and significant elevation of the complement regulator, decay-accelerating factor (DAF) in Müller cells. In the retinal pigment epithelium (RPE) of young control and Cfh (-/-) animals mitochondria and melanosomes were oriented basally and apically respectively, whereas the apical positioning of melanosomes was significantly perturbed in the mid-age Cfh (-/-) RPE. We conclude that deletion of Cfh in the mouse leads to defects in the retina that precede any marked loss of visual function, but which become progressively more marked as the animals age. These observations are consistent with a lifelong role for CFH in retinal homeostasis

Functional Evaluation of AMD-Associated Risk Variants of Complement Factor B

Purpose: The 32W and 32Q variants of complement factor B (CFB) are associated with reduced risk of developing neovascular age-related macular degeneration (AMD) compared with the common 32R allele. The objective of this study was to determine if the most protective R32Q variant affects the neovascular process in a manner consistent with the reported reduced disease association. Methods: The 32R, 32W, and 32Q human CFB variants were expressed in human embryonic kidney 293T cells and purified from culture supernatant. The ex vivo mouse fetal metatarsal explant model was used to investigate the effect of these three human CFB variants on angiogenesis. Metatarsal bones were isolated from mouse embryos and cultured in the presence of the three CFB variants, and angiogenesis was measured following immunostaining of fixed samples. ELISAs were used to quantify C3 and VEGF protein levels in metatarsal culture and quantitative PCR to measure Cfb, C3, and Vegf expression. Results: We show here that the three CFB variants have different biological activities in the mouse metatarsal assay, with CFBR32 exhibiting significantly greater angiogenic activity than CFBQ32 or CFBW32, which were broadly similar. We also observed differences in macrophage phenotype with these two variants that may contribute to their activities in this experimental model. Conclusions: We have demonstrated that the biological activities of CFBR32, CFBW32, and CFBQ32 are consistent with their AMD risk association, and we provide functional evidence of roles for these variants in angiogenesis that may be relevant to the pathogenesis of the neovascular form of AMD

In vitro characterization of a spontaneously immortalized human Muller cell line (MIO-M1)

purpose. To characterize a spontaneously immortalized human Müller cell line and to determine whether it retains the characteristics of primary isolated cells without undergoing differentiation in vitro. methods. An immortalized cell line obtained from human retina was investigated for the expression of known markers of Müller cells, including cellular retinaldehyde binding protein (CRALBP), glutamine synthetase, epidermal growth factor receptor (EGF-R), α-smooth muscle actin (α-SMA), and glial fibrillary acidic protein (GFAP). Also examined were the morphologic features of these cells, by scanning and transmission electron microscopy, and their functional characteristics, by electrogenic responses to glutamate. In addition, comparative studies were made of these cells with primary cultures of freshly isolated human Müller cells. results. The cells expressed CRALBP, EGF-R, glutamine synthetase, and α-SMA, as judged by confocal microscopy and Western blot analysis of cell lysates. Western blot analysis did not detect GFAP in cell lysates, but confocal microscopy showed that occasional cells expressed GFAP after detachment from the monolayer. The morphologic features of the cells examined, as judged by scanning and transmission electron microscopy, resemble those of cells derived from primary cell cultures. They possess villous projections on their apical surfaces and contain loose bundles of microtubules aligned parallel to one another and the long axis of the cell process. Characteristically, they contain abundant deposits of glycogen particles that do not differ from those seen in primary isolated cells. Preliminary recordings with intracellular electrodes revealed that these cells have properties similar to those described for mammalian Müller cells and depolarize in response to l-glutamate without significant change in membrane resistance, consistent with the well-established electrogenic uptake of this amino acid. conclusions. A spontaneously immortalized Müller cell line was characterized that retains the characteristics of primary isolated cells in culture. To the authors’ knowledge, it constitutes the first human Müller cell line reported in the literature. It has been named MIO-M1 (Moorfields/Institute of Ophthalmology-Müller 1) after the authors’ institution. Availability of this human cell line will facilitate studies designed to obtain a better understanding of the role of Müller cells in normal and pathologic conditions

LRG1 as a novel therapeutic target in eye disease

Retinal and choroidal diseases are major causes of blindness and visual impairment in the developed world and on the rise due to an ageing population and diabetes epidemic. Standard of care is centred around blockade of vascular endothelial growth factor (VEGF), but despite having halved the number of patients losing sight, a high rate of patient non-response and loss of efficacy over time are key challenges. Dysregulation of vascular homoeostasis, coupled with fibrosis and inflammation, are major culprits driving sight-threatening eye diseases. Improving our knowledge of these pathological processes should inform the development of new drugs to address the current clinical challenges for patients. Leucine-rich α-2 glycoprotein 1 (LRG1) is an emerging key player in vascular dysfunction, inflammation and fibrosis. Under physiological conditions, LRG1 is constitutively expressed by the liver and granulocytes, but little is known about its normal biological function. In pathological scenarios, such as diabetic retinopathy (DR) and neovascular age-related macular degeneration (nvAMD), its expression is ectopically upregulated and it acquires a much better understood pathogenic role. Context-dependent modulation of the transforming growth-factor β (TGFβ) pathway is one of the main activities of LRG1, but additional roles have recently been emerging. This review aims to highlight the clinical and pre-clinical evidence for the pathogenic contribution of LRG1 to vascular retinopathies, as well as extrapolate from other diseases, functions which may be relevant to eye disease. Finally, we will provide a current update on the development of anti-LRG1 therapies for the treatment of nvAMD

Annexin A8 regulates Wnt signaling to maintain the phenotypic plasticity of retinal pigment epithelial cells

Wnt signalling mediates complex cell-cellinteractions during development and proliferation. Annexin A8 (AnxA8), a calcium-dependent phospholipid-binding protein, and canonical Wnt signalling mechanisms have both been implicated in retinal pigment epithelial (RPE) cell differentiation. The aim here was to examine the possibility of cross-talk between AnxA8 and Wnt signalling, as both are down-regulated upon fenretinide (FR)-mediated RPE transdifferentiation. AnxA8 suppression in RPE cells via siRNA or administration of FR induced neuronal-like cell transdifferentiation and reduced expression of Wnt-related genes, as measured by real-time PCR and western blotting. AnxA8 gene expression, on the other hand, remained unaltered upon manipulating Wnt signalling, suggesting Wnt-related genes to be downstream effectors of AnxA8. Co-immunoprecipitation revealed an interaction between AnxA8 and β-catenin, which was reduced in the presence of activated TGF-β1. TGF-β1 signalling also reversed the AnxA8 loss-induced cell morphology changes, and induced β-catenin translocation and GSK-3β phosphorylation in the absence of AnxA8. Ectopic over-expression of AnxA8 led to an increase in active β-catenin and GSK-3β phosphorylation. These data demonstrate an important role for AnxA8 as a regulator of Wnt signalling and a determinant of RPE phenotype, with implications for regenerative medicine approaches that utilise stem cell-derived RPE cells to treat conditions such as age-related macular degeneration

Regulation of C3 Activation by the Alternative Complement Pathway in the Mouse Retina

The purpose of this study was to examine the retinas of mice carrying hemizygous and null double deletions of Cfb-/- and Cfh-/-, and to compare these with the single knockouts of Cfb, Cfh and Cfd. Retinas were isolated from wild type (WT), Cfb-/-/Cfh-/-, Cfb-/-/Cfh+/-, Cfh-/-/Cfb+/-, Cfb-/-, Cfh-/- Cfd-/-, and Cfd+/- mice. Complement proteins were evaluated by western blotting, ELISA and immunocytochemistry, and retinal morphology was assessed using toluidine blue stained semi-thin sections. WT mice showed staining for C3 and its breakdown products in the retinal vasculature and the basal surface of the retinal pigment epithelium (RPE). Cfb-/- mice exhibited a similar C3 staining pattern to WT in the retinal vessels but a decrease in C3 and its breakdown products at the basal surface of the RPE. Deletion of both Cfb and Cfh restored C3 to levels similar to those observed in WT mice, however this reversal of phenotype was not observed in Cfh-/-/Cfb+/- or Cfb-/-/Cfh+/- mice. Loss of CFD caused an increase in C3 and a decrease in C3 breakdown products along the basal surface of the RPE. Overall the retinal morphology and retinal vasculature did not appear different across the various genotypes. We observed that C3 accumulates at the basal RPE in Cfb-/-, Cfb-/-/Cfh-/-, Cfb-/-/Cfh+/-, Cfd-/- and WT mice, but is absent in Cfh-/- and Cfh-/-/Cfb+/- mice, consistent with its consumption in the serum of mice lacking CFH when CFB is present. C3 breakdown products along the surface of the RPE were either decreased or absent when CFB, CFH or CFD was deleted or partially deleted

Retinal pigment epithelial cells mitigate the effects of complement attack by endocytosis of C5b-9

Retinal pigment epithelial (RPE) cell death is a hallmark of age-related macular degeneration. The alternative pathway of complement activation is strongly implicated in RPE cell dysfunction and loss in age-related macular degeneration; therefore, it is critical that RPE cells use molecular strategies to mitigate the potentially harmful effects of complement attack. We show that the terminal complement complex C5b-9 assembles rapidly on the basal surface of cultured primary porcine RPE cells but disappears over 48 h without any discernable adverse effects on the cells. However, in the presence of the dynamin inhibitor dynasore, C5b-9 was almost completely retained at the cell surface, suggesting that, under normal circumstances, it is eliminated via the endocytic pathway. In support of this idea, we observed that C5b-9 colocalizes with the early endosome marker EEA1 and that, in the presence of protease inhibitors, it can be detected in lysosomes. Preventing the endocytosis of C5b-9 by RPE cells led to structural defects in mitochondrial morphology consistent with cell stress. We conclude that RPE cells use the endocytic pathway to prevent the accumulation of C5b-9 on the cell surface and that processing and destruction of C5b-9 by this route are essential for RPE cell survival

Regulation of retinal pigment epithelial cell phenotype by Annexin A8

The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre- and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype