Polarized Desmosome and Hemidesmosome Shedding via Exosomes is an Early Indicator of Outer Blood-Retina Barrier Dysfunction (original) (raw)
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The Proteome of Central and Peripheral Retina with Progression of Age-Related Macular Degeneration
Investigative Opthalmology & Visual Science, 2006
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. (Invest Oph
Investigative Opthalmology & Visual Science, 2006
PURPOSE. Age-related macular degeneration (AMD) is characterized clinically by changes in the retinal pigment epithelium (RPE), formation of drusen between the RPE and the underlying vasculature, geographic atrophy, and choroidal neovascularization. Later clinical stages are accompanied by impaired central vision. A limited understanding of the molecular events responsible for AMD has constrained the development of effective treatments. A proteomics approach was used to investigate the underlying mechanisms of AMD and to identify proteins exhibiting significant changes in expression with disease onset and progression. METHODS. Human donor eyes were categorized into one of four progressive stages of AMD. Proteins from the RPE were resolved and quantified by two-dimensional (2-D) gel electrophoresis. Proteins exhibiting significant expression changes at different disease stages were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. 2-D and semiquantitative one dimensional (1-D) Western blot analyses were used to determine whether changes identified by the proteomic analysis were specific for a protein subpopulation or representative of the entire protein population. RESULTS. Proteins were identified from several critical pathways that changed at early and late disease stages, indicating potential causal mechanisms and secondary consequences of AMD, respectively. Proteins involved in protecting from stress-induced protein unfolding and aggregation, mitochondrial trafficking and refolding, and regulating apoptosis changed early in the disease process. Late-stage changes occurred in proteins that regulate retinoic acid and regeneration of the rhodopsin chromophore. CONCLUSIONS. These results provide the first direct evidence of AMD stage-specific changes in human RPE protein expression and provide a basis for functional investigation of AMD that may ultimately suggest new therapeutic strategies. (Invest
Systems pathology analysis identifies neurodegenerative nature of age-related retinal diseases
2018
Aging is a phenomenon associated with profound medical implications. Idiopathic epiretinal membrane (iEMR) and macular hole (MH) are the major vision-threatening vitreoretinal diseases affecting millions of aging people globally, making these conditions an important public health issue. The iERM is characterized by fibrous tissue developing on the surface of the macula, leading to biomechanical and biochemical macular damage. MH is a small breakage in the macula associated with many ocular conditions. Although several individual factors and pathways are suggested, a systems pathology level understanding of the molecular mechanisms underlying these disorders is lacking. Therefore, we performed mass spectrometry based label-free quantitative proteomics analysis of the vitreous proteomes from patients with iERM (n=26) and MH (n=21) to identify the key proteins as well as the multiple interconnected biochemical pathways contributing to the development of these diseases. We identified a ...
Progress in Retinal and Eye Research, 2016
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.
PLoS ONE, 2009
Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch's membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch's membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. 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 and may be associated with the pathogenesis of AMD in the elderly. Citation: Wang AL, Lukas TJ, Yuan M, Du N, Tso MO, et al. (2009) Autophagy and Exosomes in the Aged Retinal Pigment Epithelium: Possible Relevance to Drusen Formation and Age-Related Macular Degeneration. PLoS ONE 4(1): e4160.
Endoplasmic reticulum stress in age-related macular degeneration: trigger for neovascularization
Molecular medicine (Cambridge, Mass.)
Age-related macular degeneration (AMD) can be classified into two main categories: the atrophic, dry form and the exudative, wet form. The crucial difference between dry and wet AMD is the development of choroidal neovascularization in wet AMD. One fundamental cause of the neovascularization is the increased expression of VEGF (vascular endothelial growth factor) in retinal pigment epithelial cells. Progression of AMD is linked to augmentation of cellular stress, for example, oxidative stress, proteotoxic stress, inflammation and hypoxia. All these conditions can trigger stress in endoplasmic reticulum (ER), which maintains protein quality control in cells. ER stress induces the unfolded protein response (UPR) via IRE1 (inositol-requiring protein-1), PERK (protein kinase RNA-like ER kinase) and ATF6 (activating transcription factor-6) transducers. UPR signaling is a double-edged sword, that is, it can restore cellular homeostasis as far as possible, but ultimately may lead to chroni...
New Biomarkers in the Retina and RPE Under Oxidative Stress
Ocular Diseases, 2012
Early Biosignature of Retina Degeneration 123 are available, but most of these models do not represent the full spectrum of pathological changes observed in human AMD. Animal models that mimic the complex and progressive characteristics of AMD are extremely valuable for studying the pathogenesis of AMD and testing different treatment modalities. Phosphoproteins have been studied using chemical and affinity-based methods [Zhou et al., 2008; Tao et al., 2005]. However, the rapid and dynamic nature of the underlying changes and the low abundance of phosphoproteins reflecting their substoichiometry present challenges to the quantitative study of the phosphoproteome. Thus, the isolation of the phosphoproteome or phosphopeptidome represents a potentially advanced step in this analysis. We introduced a system-wide, unbiased, and high-throughput approach to investigate global phosphoproteome of oxidative stress-induced or aged RPE and retinal cells using phosphoproteome enrichment and labeling method. Phosphoprotein-enriched extracts from human RPE cells under stress were separated by two-dimensional (2D) electrophoresis. Serine, threonine, and tyrosine phosphorylation were visualized by 2D phospho-Western blotting and specific phosphorylation sites were analyzed by tandem mass spectrometry. We examined phosphoproteome changes under oxidative stress in vitro and aging-induced phosphoproteome in vivo. Our results suggest a positive correlation between early biomarkers of phosphoproteome under oxidative stress and RPE proteins from AMD patients. The outcome of the current work is the initial delineation of the underlying physiology of oxidative stress-mediated phosphorylation signaling in RPE apoptosis. In addition, our study suggests a stimulus for understanding oxidative stress-induced cytoskeletal changes and the aggregate formation mechanism by phosphorylations. As a consequence, an effective therapeutic approach and animal model based on the modulation of phosphorylations are expected to result.
Proteomics Characterization of Cell Membrane Blebs in Human Retinal Pigment Epithelium Cells
Molecular & Cellular Proteomics, 2009
Abbreviations: AMD, age-related macular degeneration; BrM, Bruch's membrane; GFP, green fluorescent protein; HQ, hydroquinone; MMP, matrix metalloproteinase; RPE, retinal pigment epithelium; ECM, extracellular matrix; LC MS/MS, liquid chromatography tandem mass spectrometry; KEGG, Kyoto encyclopedia of genes and genomes; PBS, phosphate buffered saline MCP Papers in Press. SUMMARY Age-related macular degeneration (AMD) is the leading cause of legal blindness among elderly population in the industrialized world, affecting about 14 million people in the US alone. Smoking is a major environmental risk factor for AMD and hydroquinone (HQ) is a major component in cigarette smoke. HQ induces the formation of cell membrane blebs in human retinal pigment epithelium (RPE). Blebs may accumulate and eventually contribute first to sub-RPE deposits and then drusen formation, which is a prominent histopathologic feature in eyes with AMD. As an attempt to better understand the mechanisms involved in early AMD, we sought to investigate the proteomic profile of RPE blebs. Isolated blebs were subjected to SDS-PAGE fractionation and in-gel trypsin digested peptides were analyzed by LC-MS/MS that lead to identification of a total of 314 proteins. Identified proteins are predominantly involved in oxidative phosphorylation, cell junction, focal adhesion, cytoskeleton regulation, and immunogenic processes. Importantly, basigin and matrix metalloproteinase-14, key proteins involved in extracellular matrix remodeling, were identified in RPE blebs and shown to be more prevalent in AMD patients. Altogether, our findings suggest, for the first time, the potential involvement of RPE blebs in eye disease, and shed light into the implication of cell-derived microvesicles in human pathology.
Emerging Role of Exosomes in Retinal Diseases
Frontiers in Cell and Developmental Biology, 2021
Retinal diseases, the leading causes of vison loss and blindness, are associated with complicated pathogeneses such as angiogenesis, inflammation, immune regulation, fibrous proliferation, and neurodegeneration. The retina is a complex tissue, where the various resident cell types communicate between themselves and with cells from the blood and immune systems. Exosomes, which are bilayer membrane vesicles with diameters of 30–150 nm, carry a variety of proteins, lipids, and nucleic acids, and participate in cell-to-cell communication. Recently, the roles of exosomes in pathophysiological process and their therapeutic potential have been emerging. Here, we critically review the roles of exosomes as possible intracellular mediators and discuss the possibility of using exosomes as therapeutic agents in retinal diseases.