Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors

Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD.     

HTRA1, an age‐related macular degeneration protease,processes extracellular matrix proteins EFEMP1 and TSP1

High‐temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age‐related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high‐risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high‐risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP‐1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD.     

Human ocular drusen possess novel core domains with a distinct carbohydrate composition.

Ocular drusen are extracellular deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane. Although the presence of large and/or numerous drusen in the macula is a significant risk factor for development of age-related macular degeneration (AMD), a major cause of irreversible blindness, little is known about their origin or composition. We have expanded on our previous investigations related to drusen-associated glycoconjugates by examining lectin binding patterns after removal of terminal sialic acid residues. Strikingly, intense and distinct labeling of drusen subdomains is revealed by Arachea hypogea agglutinin (PNA) after neuraminidase treatment. PNA binding is confined to discrete domains within both hard and soft drusen. These "cores" are positioned centrally within drusen and are typically juxtaposed to Bruch's membrane. Only one core per druse is observed. PNA labeling of drusen cores does not co-localize with associated lipids and is abrogated by digestion with O-glycosidase but not N-glycosidase. The association of cores with small drusen suggests that they may participate in drusen biogenesis. (J Histochem Cytochem 47:1533-1539, 1999)     

Amyloid-β(1-42) alters structure and function of retinal pigmented epithelial cells

Age-related macular degeneration (AMD) is characterized by the formation of drusen, extracellular deposits associated with atrophy of the retinal pigmented epithelium (RPE), disturbance of the transepithelial barrier and photoreceptor death. Amyloid-β (Aβ) is present in drusen but its role during AMD remains unknown. This study investigated the in vitro and in vivo effects of the oligomeric form of Aβ(1-42) – OAβ(1-42) – on RPE and found that it reduced mitochondrial redox potential and increased the production of reactive oxygen species, but did not induce apoptosis in RPE cell cultures. It also disorganized the actin cytoskeleton and halved occludin expression, markedly decreasing attachment capacity and abolishing the selectivity of RPE cell transepithelial permeability. Antioxidant pretreatment partially reversed the effects of OAβ(1-42) on mitochondrial redox potential and transepithelial permeability. Subretinally injected OAβ(1-42) induced pigmentation loss and RPE hypertrophy but not RPE cell apoptosis in C57BL/6 J mice. Rapid OAβ(1-42)-induced disorganization of cytoskeletal actin filaments was accompanied by decreased RPE expression of the tight junction proteins occludin and zonula occludens-1 and of the visual cycle proteins cellular retinaldehyde-binding protein and RPE65. The number of photoreceptors decreased by half within a few days. Our study pinpoints the role of Aβ in RPE alterations and dysfunctions leading to retinal degeneration and suggests that targeting Aβ may help develop selective methods for treating diseases involving retinal degeneration, such as AMD.     

Autophagy,exosomes and drusen formation in age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of loss of vision in developed countries. AMD is characterized by a progressive degeneration of the macula of the retina, usually bilateral, leading to a severe decrease in central vision. An early sign of AMD is the appearance of drusen, which are extracellular deposits that accumulate on Bruch’s membrane below the retinal pigment epithelium (RPE). Drusen are a risk factor for developing AMD. Some of the protein components of drusen are known, yet we know little about the processes that lead to formation of drusen. We have previously reported increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we used in vitro modeling of increased mtDNA damage. Under conditions of increased mtDNA damage, autophagy markers and exosome markers were upregulated. In addition, we found autophagy markers and exosome markers in the region of Bruch’s membrane in the retinas of old mice. Furthermore, we found that drusen in AMD donor eyes contain markers for autophagy and for exosomes. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients.     

Resveratrol attenuates CXCL11 expression induced by proinflammatory cytokines in retinal pigment epithelial cells

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). RPE cells adjacent to drusen deposits in the AMD eye are known to contain CXCL11, a chemokine involved in inflammatory cell recruitment. We investigated the CXCL11 production by the human RPE (ARPE-19) cells under inflammatory conditions and tested its response to resveratrol, a naturally occurring anti-inflammatory antioxidant. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1β and TNF-α highly increased CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells. Resveratrol substantially inhibited the proinflammatory cytokines-induced CXCL11 production while partially blocking nuclear factor-κB activation. This inhibitory action of resveratrol was also observed for the cytokines-induced expression of chemokines CXCL9, CCL2 and CCL5. Our results indicate that resveratrol could potentially attenuate RPE inflammatory response implicated in the pathogenesis of AMD.     

An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice

The study and treatment of age-related macular degeneration (AMD), a leading cause of blindness, has been hampered by a lack of animal models. Here we report that mice deficient either in monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1) or its cognate C-C chemokine receptor-2 (Ccr-2) develop cardinal features of AMD, including accumulation of lipofuscin in and drusen beneath the retinal pigmented epithelium (RPE), photoreceptor atrophy and choroidal neovascularization (CNV). Complement and IgG deposition in RPE and choroid accompanies senescence in this model, as in human AMD. RPE or choroidal endothelial production of Ccl-2 induced by complement C5a and IgG may mediate choroidal macrophage infiltration into aged wild-type choroids. Wild-type choroidal macrophages degrade C5 and IgG in eye sections of Ccl2(-/-) or Ccr2(-/-) mice. Impaired macrophage recruitment may allow accumulation of C5a and IgG, which induces vascular endothelial growth factor (VEGF) production by RPE, possibly mediating development of CNV. These models implicate macrophage dysfunction in AMD pathogenesis and may be useful as a platform for validating therapies.     

The human retina and retinal pigment epithelium are abundant sources of vitronectin mRNA.

Vitronectin (Vn), a multifunctional plasma protein synthesized primarily in the liver, is often present as a component of the extracellular plaques and deposits that accompany various age-related human diseases. Recently, we reported that Vn is also a prominent molecular constituent of drusen, the extracellular deposits associated with age-related macular degeneration (AMD) (1). The cellular source(s) of the Vn in drusen, as well as in these other plaques and deposits, remains uncertain. In this study, we used real-time quantitative RT-PCR to measure the relative levels of Vn mRNA in the cells and tissues that lie in close proximity to drusen. The results confirm that the human liver is an abundant source of Vn mRNA. Levels of Vn mRNA in kidney, lung, and fetal or adult brain are <3% of those in liver. Remarkably, mean Vn mRNA levels in the neural retina significantly exceed those in brain and represent close to 40% of the Vn mRNA value measured in human liver. Substantial levels of Vn mRNA are also present in the adjacent retinal pigment epithelium (RPE). These results identify the neural retina, for the first time, as an abundant source of Vn mRNA. They also suggest that both the neural retina and RPE are potent biosynthetic sources of Vn in humans, and potentially significant local contributors to the Vn that accumulates in drusen.     

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.     

Amyloid‐β up‐regulates complement factor B in retinal pigment epithelial cells through cytokines released from recruited macrophages/microglia: Another mechanism of complement activation in age‐related macular degeneration

One of the earliest signs of age‐related macular degeneration (AMD) is the formation of drusen which are extracellular deposits beneath the retinal pigmented epithelium (RPE). To investigate the relationship between drusen and AMD, we focused on amyloid β (Aβ), a major component of drusen and also of senile plaques in the brain of Alzheimer's patients. We previously reported that Aβ was accumulated in drusen‐like structure in senescent neprilysin gene‐disrupted mice. The purpose of this study was to investigate the influence of Aβ on factor B, the main activator of the complement alternative pathway. The results showed that Aβ did not directly modulate factor B expression in RPE cells, but increased the production of monocyte chemoattractant protein‐1 (MCP‐1). Aβ also increased the production of IL‐1β and TNF‐α in macrophages/microglia, and exposure of RPE cells to IL‐1β and TNF‐α significantly up‐regulated factor B. Co‐cultures of RPE cells and macrophages/microglia in the presence of Aβ significantly increased the expression of factor B in RPE. These findings indicate that cytokines produced by macrophages/microglia that were recruited by MCP‐1 produced in RPE cells stimulated by Aβ up‐regulate factor B in RPE cells. Thus, a combined mechanism exists for Aβ‐induced for the activation of the complement alternative pathway in the subretinal space; cytokine‐induced up‐regulation of activator factor B and dysfunction of the inhibitor factor I by direct binding to Aβ as suggested in our earlier study. J. Cell. Physiol. 220: 119–128, 2009. © 2009 Wiley‐Liss, Inc.     

Basal laminar drusen caused by compound heterozygous variants in the CFH gene

Age-related macular degeneration (AMD) is a multifactorial disease that is strongly associated with the Tyr402His variant in the complement factor H (CFH) gene. Drusen are hallmark lesions of AMD and consist of focal-inflammatory and/or immune-mediated depositions of extracellular material at the interface of the retinal pigment epithelium (RPE) and the Bruch membrane. We evaluated the role of CFH in 30 probands with early-onset drusen and identified heterozygous nonsense, missense, and splice variants in five families. The affected individuals all carried the Tyr402His AMD risk variant on the other allele. This supports an autosomal-recessive disease model in which individuals who carry a CFH mutation on one allele and the Tyr402His variant on the other allele develop drusen. Our findings strongly suggest that monogenic inheritance of CFH variants can result in basal laminar drusen in young adults, and this can progress to maculopathy and severe vision loss later in life.     

Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of polarized cells located between retinal photoreceptors and blood vessels of the choroid. The basal surface of RPE cells rests on Bruch's membrane, a complex extracellular matrix structure which becomes abnormal in several disease processes, including age-related macular degeneration (AMD). Ruptures or abnormalities in Bruch's membrane are frequently accompanied by choroidal neovascularization. Disturbed interaction of RPE cells with their extracellular matrix (ECM) could play a role in this process. The present study was undertaken to examine the complex interactions between hypoxia, integrin, and ECM in the regulation of RPE functions. Antibody blocking experiments demonstrated that RPE cell adhesion to vitronectin is mediated primarily through alphavbeta5 and adhesion to fibronectin occurs through alpha5beta1. RPE adhesion to immobilized laminin demonstrated highest level of non-RGD-mediated adhesion as compared to that with collagen IV or the RGD matrices such as vitronectin (alphavalpha5) , fibronectin (alpha5beta1), or thrombospondin (alpha5beta1 + alphavbeta5). Addition of soluble vitronectin, or fibrinogen to RPE cell cultures resulted in a small to moderate increase in VEGF and FGF2 in the media, while each of these growth factors was dramatically increased after addition of thrombospondin 1 (TSP1). In contrast, soluble fibronectin resulted in differential upregulation of VEGF but not FGF2. Similarly, immobilized TSP1 resulted in differential greater upregulation in VEGF but not FGF2 release from RPE as compared to other ECMs under either normoxic or hypoxic conditions. Additionally, hypoxia resulted in a time-dependent increase in VEGF, but not FGF2 release in the media. RPE cells grown on TSP1-coated plates showed increased VEGF and FGF2 in their media compared to cells grown on plates coated with type IV collagen, laminin, vitronectin, or fibronectin. The TSP1-induced increase in secretion of growth factors was partially blocked by anti-alpha5beta1, anti-alphavbeta3, and anti-alphavbeta5 antibodies indicating that it may be mediated in part by TSP1 binding to those integrins. These data suggest that alterations in oxygen levels (hypoxia/ischemia) and ECM of RPE cells, a prominent feature of AMD, can cause increased secretion of angiogenic growth factors that might contribute to the development of choroidal neovascularization. These data also suggest the potential modulatory role of VEGF release from RPE by ECM and alphavbeta5 and alpha5beta1 integrins.     

Regulated expression of apolipoprotein E by human retinal pigment epithelial cells

In early age-related macular degeneration (AMD), lipid-containing deposits (drusen) accumulate in Bruch's membrane underlying the retinal pigment epithelium (RPE). Recent studies indicate that apolipoprotein E (apoE) may play a role in lipid trafficking in AMD. Compared with the apoE3 allele, the apoE4 and apoE2 alleles are associated with decreased and increased risk for AMD, respectively; drusen contain high levels of apoE, and apoE null mice develop lipid deposits in Bruch's membrane similar to those observed in AMD. Primary cultures of human RPE cells expressing the apoE3 allele were grown on Transwell culture plates. Western blotting, ELISA assay, and mass spectrometry confirmed that apoE3 was secreted into the apical and basal chambers and that secretion was upregulated by thyroid hormone, 9-cis-retinoic acid, and 22(R)-hydroxycholesterol. In addition, basally secreted apoE associated with exogenously added HDL. These results indicate that apoE secretion can be regulated by specific hormones and that apoE associates with HDL. The findings are consistent with a role for apoE in lipid trafficking through Bruch's membrane and may be relevant to AMD.     

Immunohistochemical localization of complement regulatory proteins in the human retina

Age-related macular degeneration (AMD) is a complex disease. Genetic studies have found strong associations between AMD and variants of several complement pathway-associated genes. The regulation of the complement cascade seems to be critical in the pathogenesis of AMD. In 45 human donor eyes immunohistochemistry was performed using antibodies directed against major regulators of the complement system: complement factor H (CFH), decay accelerating factor (DAF/CD55), complement receptor 1 (CR1/CD35), and membrane cofactor protein (MCP/CD46). All eyes were classified in AMD and controls. 11 eyes were graded as early AMD. 34 eyes were controls. In all eyes staining was found in intercapillary pillars of choroid adjacent to Bruch's membrane for CFH, at the basal surface of RPE cells for MCP, and at the apical side of the retinal pigment epithelium for CR1. DAF immunoreactivity was increased along the inner segments of rod and cone photoreceptor cells at the level of the external limiting membrane Labeling of soft drusen was found for CFH and CR1. In addition, DAF and CR1 showed staining of ganglion cells in all eyes. CFH and particularly MCP showed decreased or absent staining in eyes with early AMD adjacent to Bruch's membrane. The overlapping expression of regulators at the level of Bruch's membrane and the retinal pigment epithelium shows the importance of this site for control of the complement system. Decreased and therefore unbalanced expression of regulators, as shown in this study for CFH and MCP, may ultimately lead to AMD.     

Pluripotent human stem cells for the treatment of retinal disease

Despite advancements made in our understanding of ocular biology, therapeutic options for many debilitating retinal diseases remain limited. Stem cell-based therapies are a potential avenue for treatment of retinal disease, and this mini-review will focus on current research in this area. Cellular therapies to replace retinal pigmented epithelium (RPE) and/or photoreceptors to treat age-related macular degeneration (AMD), Stargardt's macular dystrophy, and retinitis pigmentosa are currently being developed. Over the past decade, significant advancements have been made using different types of human stem cells with varying capacities to differentiate into these target retinal cell types. We review and evaluate pluripotent stem cells, both human embryonic stem cells and human induced pluripotent stem cells, as well as protocols for differentiation of ocular cells, and culture and transplant techniques that might be used to deliver cells to patients.     

Programmed necrosis,not apoptosis,is a key mediator of cell loss and DAMP-mediated inflammation in dsRNA-induced retinal degeneration

There is no known treatment for the dry form of an age-related macular degeneration (AMD). Cell death and inflammation are important biological processes thought to have central role in AMD. Here we show that receptor-interacting protein (RIP) kinase mediates necrosis and enhances inflammation in a mouse model of retinal degeneration induced by dsRNA, a component of drusen in AMD. In contrast to photoreceptor-induced apoptosis, subretinal injection of the dsRNA analog poly(I : C) caused necrosis of the retinal pigment epithelium (RPE), as well as macrophage infiltration into the outer retinas. In Rip3^−/− mice, both necrosis and inflammation were prevented, providing substantial protection against poly(I : C)-induced retinal degeneration. Moreover, after poly(I : C) injection, Rip3^−/− mice displayed decreased levels of pro-inflammatory cytokines (such as TNF-α and IL-6) in the retina, and attenuated intravitreal release of high-mobility group box-1 (HMGB1), a major damage-associated molecular pattern (DAMP). In vitro, poly(I : C)-induced necrosis were inhibited in Rip3-deficient RPE cells, which in turn suppressed HMGB1 release and dampened TNF-α and IL-6 induction evoked by necrotic supernatants. On the other hand, Rip3 deficiency did not modulate directly TNF-α and IL-6 production after poly(I : C) stimulation in RPE cells or macrophages. Therefore, programmed necrosis is crucial in dsRNA-induced retinal degeneration and may promote inflammation by regulating the release of intracellular DAMPs, suggesting novel therapeutic targets for diseases such as AMD.     

Lipofuscin-formation in cultured retinal pigment epithelial cells is related to their melanin content

Age-related macular degeneration (AMD), the leading cause of blindness in the developed world, is accompanied by degeneration of the retinal pigment epithelial (RPE) cells. There is an inverse correlation between the melanin content of the eye and the incidence of AMD. Lipofuscin (LF)-accumulation in RPE cells accompanies the process of aging, and may also be related to AMD. This study was designed to evaluate the effect of melanin/melanosomes on the rate of LF formation in cultured rabbit and bovine RPE cells subjected to oxidative stress (40% normobaric O(2)) and daily supplementation with photoreceptor outer segments for 4 weeks. The LF content was measured at 0, 2, and 4 weeks in RPE cells from pigmented and albino rabbits, as well as in pigment-rich and pigment-poor bovine cells. Albino rabbit and pigment-poor bovine cells accumulated significantly higher amounts of LF than pigmented rabbit cells and pigment-rich bovine RPE cells after both 2 and 4 weeks of exposure. Autometallography of melanin-containing cells, without previous exposure to ammonium sulfide, showed a positive outcome, indicating either the occurrence of pre-existing iron-sulphur clusters or an extremely high intrinsic reducing capacity. These results suggest that melanin acts as an efficient antioxidant, perhaps by interacting with transition metals.