The proteome of central and peripheral retina with progression of age-related macular degeneration

PURPOSE: A growing understanding of the molecular events in age-related macular degeneration (AMD) has lead to targeted therapies for a select group of patients with advanced AMD. Development of therapies for the earlier stages requires further elucidation of disease mechanisms. In this study, a proteomics approach was used to identify proteins that had altered content in human donor eyes with progression of AMD. METHODS: The early molecular events associated with AMD were identified by comparing the proteome of the macular and peripheral neurosensory retina during four progressive stages of AMD. Proteins were resolved and quantified by two-dimensional gel electrophoresis. Twenty-six proteins exhibited changes in content and were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Two-dimensional (2-D) and semiquantitative one-dimensional (1-D) Western blot analyses were used to determine whether changes identified by proteomic analysis were specific for a protein subpopulation or representative of the entire protein population. RESULTS: Twenty-six proteins were identified that exhibited changes at disease onset or with progression (indicating potential causal mechanisms) and at end-stage disease (indicating potential secondary consequences). These proteins are involved in key functional pathways, such as microtubule regulation and protection from stress-induced protein unfolding. Approximately 60% of the proteins exhibited changes specific to either the macula or periphery, with the remaining 40% changing in both regions. These results imply that both the macula and periphery are affected by AMD. CONCLUSIONS: This study provides the first direct evidence of AMD stage- and region-specific changes in retinal protein levels and highlights potential novel, disease-related proteins and biochemical pathways for future studies of AMD.     

Changes in select redox proteins of the retinal pigment epithelium in age-related macular degeneration

PURPOSE: To examine changes of select reduction-oxidation (redox) sensitive proteins from human donor retinal pigment epithelium (RPE) at four stages of age-related macular degeneration (AMD). DESIGN: Experimental study. METHODS: Human donor eyes were obtained from the Minnesota Lions Eye Bank and graded using the Minnesota Grading System (MGS) into four stages that correspond to stages defined by the age-related eye disease study (AREDS). Protein content in RPE homogenates was measured using Western immunoblotting with protein-specific antibodies. RESULTS: The content of several antioxidant enzymes and specific proteins that facilitate refolding or degradation of oxidatively damaged proteins increased significantly in MGS stage 3. These proteins are involved in the primary (copper-zinc superoxide dismutase [CuZnSOD], manganese superoxide dismutase [MnSOD], and catalase) and secondary (heat shock protein [HSP] 27, HSP 90, and proteasome) defense against oxidative damage. Additionally, the insulin pro-survival receptor exhibited disease-related upregulation. CONCLUSIONS: The pattern of protein changes identified in human donor tissue graded using the MGS support the role of oxidative mechanisms in the pathogenesis and progression of AMD. The MGS uses nearly identical clinical definitions and grading criteria of AMD that are used in the AREDS, so our results apply to clinical and epidemiologic studies using similar definitions. Results from our protein analysis of human donor tissue helps to explain altered oxidative stress regulation and cell-survival pathways that occur in progressive stages of AMD.     

Age-related macular degeneration and retinal protein modification by 4-hydroxy-2-nonenal

PURPOSE: Oxidative damage to proteins, lipids, and DNA has been suggested to be a mechanism for age-related macular degeneration (AMD). The retina is particularly susceptible to lipid peroxidation due to high concentrations of easily oxidized polyunsaturated fatty acids in the presence of abundant oxygen. One of the most toxic products of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), can modify and inactivate proteins. The hypothesis was that 4-HNE-modified proteins would accumulate and serve as a marker for progressive stages of AMD. METHODS: Proteins containing HNE adducts were identified in both the macular and peripheral regions during four progressive stages of AMD. The proteins were resolved by two-dimensional (2-D) gel electrophoresis before detection of HNE-adducted proteins. Modified proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The total content of HNE adducts was compared using a slot blot immunoassay. One-dimensional Western blot analysis was used to measure levels of proteins involved in HNE detoxification. RESULTS: Nineteen proteins that were consistently modified regardless of stage of AMD or retinal region were identified. These proteins are involved in two main functions: energy production and stress response. No change in total HNE-adducted protein was observed between regions or stages. Modest increases in content of proteins involved in HNE detoxification were observed. CONCLUSIONS: Consistently modified proteins indicate preferred protein targets for oxidation by HNE. HNE-modified proteins were not different between regions or stages, suggesting that pathways for detoxification of HNE or removal of damaged proteins are adequate. Consistent levels of HNE-modified proteins suggest that HNE is not a sensitive retinal biomarker for AMD.     

Disease susceptibility of the human macula: differential gene transcription in the retinal pigmented epithelium/choroid

The discoveries of gene variants associated with macular diseases have provided valuable insight into their molecular mechanisms, but they have not clarified why the macula is particularly vulnerable to degenerative disease. Its predisposition may be attributable to specialized structural features and/or functional properties of the underlying macular RPE/choroid. To examine the molecular basis for the macula's disease susceptibility, we compared the gene expression profile of the human RPE/choroid in the macula with the profile in the extramacular region using DNA microarrays. Seventy-five candidate genes with differences in macular:extramacular expression levels were identified by microarray analysis, of which 29 were selected for further analysis. Quantitative PCR confirmed that 21 showed statistically significant differences in expression. Five genes were expressed at higher levels in the macula. Two showed significant changes in the macular:extramacular expression ratio; another two exhibited changes in absolute expression level, as a function of age or AMD. Several of the differentially expressed genes have potential relevance to AMD pathobiology. One is an RPE cell growth factor (TFPI2), five are extracellular matrix components (DCN, MYOC, OGN, SMOC2, TFPI2), and six are related to inflammation (CCL19, CCL26, CXCL14, SLIT2) and/or angiogenesis (CXCL14, SLIT2, TFPI2, WFDC1). The identification of regional differences in gene expression in the RPE/choroid is a first step in clarifying the macula's propensity for degeneration. These findings lay the groundwork for further studies into the roles of the corresponding gene products in the normal, aged, and diseased macula.     

Comparative proteome analysis of native differentiated and cultured dedifferentiated human RPE cells

PURPOSE: Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells. METHODS: Differentiated native human RPE cells and monolayers of dedifferentiated cultured primary human RPE cells were processed for two-dimensional (2-D) electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3-10) and electrophoresis on 9% to 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver-stained gel spots were excised, digested in situ, and analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy (MS). The resultant peptide mass fingerprints were searched against the public domain NCBInr, MSDB, and EnsemblC databases to identify the respective proteins. RESULTS: One hundred seventy nine protein spots were analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells, including RPE65, cellular retinaldehyde-binding protein (CRALBP), and cellular retinol-binding protein (CRBP), were absent in dedifferentiated cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including cathepsin D and clathrin were still present. Dedifferentiated RPE cells displayed a strong shift toward increased expression of proteins associated with cell shape, cell adhesion, and stress fiber formation, including cytokeratin 19, gelsolin, and tropomyosins, and also acquired increased expression of factors involved in translation and tumorigenic signal transduction such as annexin I and translation initiation factor (eIF)-5A. CONCLUSIONS: Dedifferentiation of human RPE cells in vitro results in downregulation of proteins associated with highly specialized functions of the RPE and induces the differential expression of proteins related to cytoskeleton organization, cell shape, cell migration, and mediation of proliferative signal transduction. These in vitro data suggest that the dedifferentiated status of RPE cells per se may initiate PVR. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation.     

Declines in arrestin and rhodopsin in the macula with progression of age-related macular degeneration

PURPOSE: Biochemical analysis of age-related macular degeneration (AMD) at distinct stages of the disease will help further understanding of the molecular events associated with disease progression. This study was conducted to determine the ability of a new grading system for eye bank eyes, the Minnesota Grading System (MGS), to discern distinct stages of AMD so that retinal region-specific changes in rod photoreceptor protein expression from donors could be determined. METHODS: Donor eyes were assigned to a specific level of AMD by using the MGS. Expression of the rod photoreceptor proteins rhodopsin and arrestin was evaluated by Western immunoblot analysis in the macular and peripheral regions of the neurosensory retina from donors at different stages of AMD. RESULTS: A significant linear decline in both arrestin and rhodopsin content correlated with progressive MGS levels in the macula. In contrast, the peripheral region showed no significant correlation between MGS level and the content of either protein. CONCLUSIONS: The statistically significant relationship between decreasing macular rod photoreceptor proteins and progressive MGS levels of AMD demonstrates the utility of the clinically based MGS to correspond with specific protein changes found at known, progressive stages of degeneration. Future biochemical analysis of clinically characterized donor eyes will further understanding of the pathobiochemistry of AMD.     

Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate

Purpose  

The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina.     

Retinal pigment epithelial expression of complement regulator CD46 is altered early in the course of geographic atrophy

In geographic atrophy (GA), the non-neovascular end stage of age-related macular degeneration (AMD), the macular retinal pigment epithelium (RPE) progressively degenerates. Membrane cofactor protein (MCP, CD46) is the only membrane-bound regulator of complement expressed on the human RPE basolateral surface. Based on evidence of the role of complement in AMD, we hypothesized that altered CD46 expression on the RPE would be associated with GA development and/or progression. Here we report the timeline of CD46 protein expression changes across the GA transition zone, relative to control eyes, and relative to events in other chorioretinal layers. Eleven donor eyes (mean age 87.0 ± 4.1 yr) with GA and 5 control eyes (mean age 84.0 ± 8.9 yr) without GA were evaluated. Macular cryosections were stained with PASH for basal deposits, von Kossa for calcium, and for CD46 immunoreactivity. Internal controls for protein expression were provided by an independent basolateral protein, monocarboxylate transporter 3 (MCT3) and an apical protein, ezrin. Within zones defined by 8 different semi-quantitative grades of RPE morphology, we determined the location and intensity of immunoreactivity, outer segment length, and Bruch’s membrane calcification. Differences between GA and control eyes and between milder and more severe RPE stages in GA eyes were assessed statistically. Increasing grades of RPE degeneration were associated with progressive loss of polarity and loss of intensity of staining of CD46, beginning with the stages that are considered normal aging (grades 0–1). Those GA stages with affected CD46 immunoreactivity exhibited basal laminar deposit, still-normal photoreceptors, and concomitant changes in control protein expression. Activated or anteriorly migrated RPE (grades 2–3) exhibited greatly diminished CD46. Changes in RPE CD46 expression thus occur early in GA, before there is evidence of morphological RPE change. At later stages of degeneration, CD46 alterations occur within a context of altered RPE polarity. These changes precede degeneration of the overlying retina and suggest that therapeutic interventions be targeted to the RPE.     

Receptor for advanced glycation end products and age-related macular degeneration

PURPOSE: Advanced glycation end products (AGE) exacerbate disease progression through two general mechanisms: modifying molecules and forming nondegradable aggregates, thus impairing normal cellular/tissue functions, and altering cellular function directly through receptor-mediated activation. In the present study receptor for AGE (RAGE)-mediated cellular activation was evaluated in the etiology of human retinal aging and disease. METHODS: The maculas of human donor retinas from normal eyes and eyes with early age-related macular degeneration (AMD) and advanced AMD with geographic atrophy (GA) were assayed for AGE and RAGE by immunocytochemistry. Cultured ARPE-19 cells were challenged with known ligands for RAGE, AGE, and S100B, to test for activation capacity. Immunocytochemistry, real-time RT-PCR, immunoblot analysis, and the TUNEL assay were used to determine the consequences of RPE cellular activation. RESULTS: Little to no immunolabeling for AGE or RAGE was found in photoreceptor and RPE cell layers in normal retinas. However, when small drusen were present, AGE and RAGE were identified in the RPE or both the RPE and photoreceptors. In early AMD and GA, the RPE and remnant photoreceptor cells showed intense AGE and RAGE immunolabeling. Both AGE and S100B activated cultured RPE cells, as revealed by upregulated expression of RAGE, NFkappaB nuclear translocation, and apoptotic cell death. CONCLUSIONS: Immunolocalization of RAGE in RPE and photoreceptors coincided with AGE deposits and macular disease in aged, early AMD, and GA retinas. Further, AGE stimulated RAGE-mediated activation of cultured ARPE-19 cells in a dose-dependent fashion. AGE accumulation, as occurs with normal aging and in disease, may induce receptor-mediated activation of RPE/photoreceptor cells, contributing to disease progression in the aging human retinas.     

Phenotypic variation of retinal pigment epithelium in age-related macular degeneration.

PURPOSE: To determine whether retinal pigment epithelium (RPE) in eyes with age-related macular degeneration (ARMD) express vimentin and alpha smooth muscle actin (alphaSMA), two cytoskeletal proteins associated with phenotypic variation in culture. METHODS: Six eyes with late ARMD and three age-matched control eyes were preserved in buffered 4% paraformaldehyde and cryosectioned at 10 microm. Stages of RPE morphology and pigmentation were assessed by the Alabama Age-Related Macular Degeneration Grading System. Vimentin, alphaSMA, and glial fibrillary acidic protein (GFAP) expression was detected by indirect immunofluorescence. These results were compared with regional variations in disease severity. RESULTS: RPE changes in ARMD included acquired expression of vimentin, but alphaSMA-positive cells were rare. GFAP expression increased in Müller cells in the neural retina in association with RPE changes and photoreceptor degeneration. CONCLUSIONS: The initial stages of RPE changes in eyes with ARMD mimic those reported for cultured RPE cells. The absence of alphaSMA-positive cells in regions of RPE atrophy suggests that RPE are lost rather than persist in a dedifferentiated state.     

SOD2 knockdown mouse model of early AMD

PURPOSE: To test the hypothesis that oxidative injury to the retinal pigment epithelium (RPE) may lead to retinal damage similar to that associated with the early stages of age-related macular degeneration (AMD). METHODS: A ribozyme that targets the protective enzyme manganese superoxide dismutase (MnSOD) was expressed in RPE-J cells, and adeno-associated virus (AAV) expressing the ribozyme gene was injected beneath the retinas of adult C57BL/6 mice. The RPE/choroid complex was examined for SOD2 protein levels and protein markers of oxidative damage using immunoblot analysis and LC MS/MS-identification of proteins and nitration sites. Lipids were extracted from retinal tissue and analyzed for the bis-retinoid compounds A2E and iso-A2E. The mice were analyzed by full-field electroretinography (ERG) for light response. Light and electron microscopy were used to measure cytological changes in the retinas. RESULTS: The treatment of RPE-J cells with Rz432 resulted in decreased MnSOD mRNA and protein as well as increased levels of superoxide anion and apoptotic cell death. When delivered by AAV, Rz432 reduced MnSOD protein and increased markers of oxidative damage, including nitrated and carboxyethylpyrrole-modified proteins in the RPE-choroid of mice. Ribozyme delivery caused a progressive loss of electroretinograph response, vacuolization, degeneration of the RPE, thickening of Bruch's membrane, and shortening and disorganization of the photoreceptor outer and inner segments. Progressive thinning of the photoreceptor outer nuclear layer resulted from apoptotic cell death. Similar to the eyes of patients with AMD, ribozyme-treated eyes exhibited increased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin. CONCLUSIONS: These results support the hypothesis that oxidative damage to the RPE may play a role in some of the key features of AMD.     

Functional and structural implications of the complement factor H Y402H polymorphism associated with age-related macular degeneration

PURPOSE: A Tyr-to-His (Y402H) sequence variant in the factor H (FH) and factor H-like protein (FHL-1) gene is strongly associated with an increased susceptibility for age-related macular degeneration (AMD). The purpose of this study was to understand how the Y402H variant in FH/FHL-1 contributes to the pathogenesis of AMD and, in particular, whether interactions mediated by FH/FHL-1, including binding to C-reactive protein (CRP), group A streptococcal M protein (GAS M6), heparin, and retinal pigment epithelial cells (RPE), are affected. METHODS: FH was purified from sera of patients homozygous for FH(Y402) or (H402), and recombinant FH fragments representing FHL-1 were generated. Proteins were analyzed for binding to CRP, GAS M6, heparin, and RPE cells. RESULTS: Binding of the FH and FH1 to seven polymorphic variants to CRP and M protein was reduced. The variant did not influence the interaction of FH with heparin but did reduce binding of FHL-1. Binding of the FH and FHL-1 polymorphic variant to RPE cells was not affected. CONCLUSIONS: The FH Y402H polymorphism associated with AMD causes a reduction in binding of FH and FHL-1 to CRP and M protein. Both variants show comparable binding to RPE cells, indicating that AMD is unlikely to manifest as a result of impaired host cell-surface recognition. The decreased interaction between FH and CRP, which is essential for the anti-inflammatory function of CRP, provides a possible pathophysiological explanation for the association of the Y402H variant with AMD.     

Thrombospondin-1 Expression in RPE and Choroidal Neovascular Membranes

Introduction Age-related macular degeneration (AMD) s the leading cause of blindness in the West or individuals more than 50 years of age[1-3].Severe visual loss in the late stages of AMD most commonly results from choroidal neovas- cularization (CNV), a process characterized by the growth of new vessels from the choriocapil- laris through Bruch′s membrane. These new vessels are prone to leakage and bleeding and may be associated with detachment of the retinal pigment epithelium (RPE). …     

Retinal pigment epithelium is protected against apoptosis by alphaB-crystallin

PURPOSE: The degeneration of retinal pigment epithelial (RPE) cells is considered to be a crucial event in the pathophysiology of age-related macular degeneration (AMD). Cumulative oxidative damage has been implicated in the development of the changes seen in AMD. The present study was undertaken to evaluate the expression of the small heat shock protein alphaB-crystallin in the RPE in response to oxidative stress and to explore whether alphaB-crystallin expression confers an antiapoptotic cytoprotective effect on RPE cells. METHODS: Native human RPE cells from the macula and retinal periphery were analyzed by RT-PCR and Western blot analysis for expression of alphaB-crystallin. Monolayer cultures of human RPE cells were stressed by heat shock (42 degrees C for 20 minutes) or oxidant-mediated injury (50-300 micro M H(2)O(2) for 1 hour). Induction of alphaB-crystallin and the corresponding mRNA was assessed by Western and Northern blot analyses. To study the cytoprotective effect of alphaB-crystallin, human RPE cells were transfected with either a neomycin-selectable expression vector containing alphaB-crystallin cDNA or a control vector without alphaB-crystallin cDNA. Caspase-3 activity was determined by observing the cleavage of a colorimetric peptide substrate. Cell viability was quantified by combined propidium iodide and Hoechst 33342 staining. RESULTS: alphaB-crystallin is constitutively expressed in RPE under in vivo and in vitro conditions. Western blot analysis of freshly isolated RPE showed greater baseline expression levels in RPE derived from the macular area than in that from the more peripheral regions. Heat shock treatment and oxidative stress caused a significant increase in alphaB-crystallin mRNA and protein. Oxidant-mediated injury in RPE cells with baseline expression levels of alphaB-crystallin resulted in apoptotic cell death, as measured by caspase-3 activity, whereas RPE cells that had been stably transfected with alphaB-crystallin were more resistant to H(2)O(2)-induced cellular injury. CONCLUSIONS: alphaB-crystallin may function as a stress-inducible antiapoptotic protein in human RPE and is inducible by oxidative stress, a condition implicated in the pathogenesis of AMD. Overexpression of alphaB-crystallin may be an important mechanism for the RPE to prevent apoptotic cell death in response to cellular stress.     

Defects in retinal pigment epithelial cell proteolysis and the pathology associated with age-related macular degeneration

Maintenance of protein homeostasis, also referred to as “Proteostasis”, integrates multiple pathways that regulate protein synthesis, folding, translocation, and degradation. Failure in proteostasis may be one of the underlying mechanisms responsible for the cascade of events leading to age-related macular degeneration (AMD). This review covers the major degradative pathways (ubiquitin-proteasome and lysosomal involvement in phagocytosis and autophagy) in the retinal pigment epithelium (RPE) and summarizes evidence of their involvement in AMD. Degradation of damaged and misfolded proteins via the proteasome occurs in coordination with heat shock proteins. Evidence of increased content of proteasome and heat shock proteins in retinas from human donors with AMD is consistent with increased oxidative stress and extensive protein damage with AMD. Phagocytosis and autophagy share key molecules in phagosome maturation as well as degradation of their cargo following fusion with lysosomes. Phagocytosis and degradation of photoreceptor outer segments ensures functional integrity of the neural retina. Autophagy rids the cell of toxic protein aggregates and defective mitochondria. Evidence suggesting a decline in autophagic flux includes the accumulation of autophagic substrates and damaged mitochondria in RPE from AMD donors. An age-related decrease in lysosomal enzymatic activity inhibits autophagic clearance of outer segments, mitochondria, and protein aggregates, thereby accelerating the accumulation of lipofuscin. This cumulative damage over a person’s lifetime tips the balance in RPE from a state of para-inflammation, which strives to restore cell homeostasis, to the chronic inflammation associated with AMD.     

Transplantation of the RPE in AMD

The retinal pigment epithelium (RPE) maintains retinal function as the metabolic gatekeeper between photoreceptors (PRs) and the choriocapillaries. The RPE and Bruch's membrane (BM) suffer cumulative damage over lifetime, which is thought to induce age-related macular degeneration (AMD) in susceptible individuals. Unlike palliative pharmacologic treatments, replacement of the RPE has a curative potential for AMD. This article reviews mechanisms leading to RPE dysfunction in aging and AMD, laboratory studies on RPE transplantation, and surgical techniques used in AMD patients. Future strategies using ex vivo steps prior to transplantation, BM prosthetics, and stem cell applications are discussed. The functional peculiarity of the macular region, epigenetic phenomena leading to an age-related shift in protein expression, along with the accumulation of lipofuscin may affect the metabolism in the central RPE. Thickening of BM with age decreases its hydraulic conductivity. Drusen are deposits of extracellular material and formed in part by activation of the alternative complement pathway in individuals carrying a mutant allele of complement factor H. AMD likely represents an umbrella term for a disease entity with multifactorial etiology and manifestations. Presently, a slow progressing (dry) non-neovascular atrophic form and a rapidly blinding neovascular (wet) form are discerned. No therapy is currently available for the former, while RPE transplantation and promising (albeit non-causal) anti-angiogenic therapies are available for the latter. The potential of RPE transplantation was demonstrated in animal models. Rejection of allogeneic homologous transplants in patients focused further studies on autologous sources. In vitro studies elucidated cell adhesion and wound healing mechanisms on aged human BM. Currently, autologous RPE, harvested from the midperiphery, is being transplanted as a cell suspension or a patch of RPE and choroid in AMD patients. These techniques have been evaluated from several groups. Autologous RPE transplants may have the disadvantage of carrying the same genetic information that may have led to AMD manifestation. An intermittent culturing step would allow for in vitro therapy of the RPE, its rejuvenation and prosthesis of BM to improve the success RPE transplants. Recent advances in stem cell biology when combined with lessons learned from studies of RPE transplantation are intriguing future therapeutic modalities for AMD patients.     

A human apoB100 transgenic mouse expresses human apoB100 in the RPE and develops features of early AMD

apoB100 lipoprotein particles have been found to accumulate in Bruch membrane prior to the development of age-related macular degeneration (AMD). This work was performed to determine whether mice that overexpress apoB100 in the RPE/choroid and liver develop landmarks of early AMD over time. Mice transgenic for a human genomic fragment encoding the full length human apoB (“apoB100” mice) and litter-mate control mice were given a normal chow or high-fat diet for 12 months. Mice were evaluated for human apoB mRNA expression in the RPE/choroid and liver by RT-qPCR. Phenotypic changes associated with early AMD were evaluated by ultrastructural analysis using transmission electron microscopy. Changes were semi-quantified using linear regression analysis. Both the RPE/choroid and liver of apoB100 mice expressed both human and mouse apoB mRNA. Transmission electron microscopy showed ultrastructural changes consistent with early human AMD including loss of basal infoldings and accumulation of cytoplasmic vacuoles in the RPE, and basal laminar deposits containing long-spacing collagen and heterogeneous debris in Bruch membrane of apoB100 mice. In apoB100 mice given a high-fat diet, basal linear-like deposits were identified in 12-month-old mice. Linear regression analysis showed that the genotype (human apoB transgene) was a stronger influencing factor than high-fat diet in producing AMD-like lesions used in this study. Human apoB100 transgenic mice overexpress apoB in RPE and, with time, develop validated phenotypic changes that are seen in early human AMD. The phenotypic changes were aggravated by feeding a high-fat diet. The apoB100 mouse model could be valuable in determining the role of apoB-containing lipoproteins in triggering the onset of early AMD.     

Differential and Altered Spatial Distribution of Complement Expression in Age-Related Macular Degeneration

PurposeDysregulation of the alternative complement pathway is a major pathogenic mechanism in age-related macular degeneration. We investigated whether locally synthesized complement components contribute to AMD by profiling complement expression in postmortem eyes with and without AMD.MethodsAMD severity grade 1 to 4 was determined by analysis of postmortem acquired fundus images and hematoxylin and eosin stained histological sections. TaqMan (donor eyes n = 39) and RNAscope/in situ hybridization (n = 10) were performed to detect complement mRNA. Meso scale discovery assay and Western blot (n = 31) were used to measure complement protein levels.ResultsThe levels of complement mRNA and protein expression were approximately 15- to 100-fold (P < 0.0001–0.001) higher in macular retinal pigment epithelium (RPE)/choroid tissue than in neural retina, regardless of AMD grade status. Complement mRNA and protein levels were modestly elevated in vitreous and the macular neural retina in eyes with geographic atrophy (GA), but not in eyes with early or intermediate AMD, compared to normal eyes. Alternative and classical pathway complement mRNAs (C3, CFB, CFH, CFI, C1QA) identified by RNAscope were conspicuous in areas of atrophy; in those areas C3 mRNA was observed in a subset of IBA1⁺ microglia or macrophages.ConclusionsWe verified that RPE/choroid contains most ocular complement; thus RPE/choroid rather than the neural retina or vitreous is likely to be the key site for complement inhibition to treat GA or earlier stage of the disease. Outer retinal local production of complement mRNAs along with evidence of increased complement activation is a feature of GA.