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?B crystallin is apically secreted within exosomes by polarized human retinal pigment epithelium and provides neuroprotection to adjacent cells.

ABSTRACT: ?B crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether ?B crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular ?B crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that ?B crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of ?B crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced ?B crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, ?B crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that ?B crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of ?B crystallin to the basolateral side. In normal mouse retinal sections, ?B crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous ?B crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. ?B Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for ?B crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of ?B crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for ?B crystallin in ocular diseases.

SUBMITTER: Sreekumar PG

PROVIDER: S-EPMC2951891 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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αB crystallin is apically secreted within exosomes by polarized human retinal pigment epithelium and provides neuroprotection to adjacent cells.

Sreekumar Parameswaran G PG Kannan Ram R Kitamura Mizuki M Spee Christine C Barron Ernesto E Ryan Stephen J SJ Hinton David R DR

PloS one 20101008 10

αB crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establi ...[more]

PMID: 20949024

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Project description:Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness among the elderly population in the industrialized world. One of the typical features of this pathology is the gradual death of retinal pigment epithelial (RPE) cells, which are essential for maintaining photoreceptor functions and survival. The etiology is multifactorial, and oxidative stress is clearly one of the key factors involved in disease pathogenesis (Plafker, Adv. Exp. Med. Biol. 664 (2010) 447-56; Qin, Drug Dev. Res. 68 (2007) 213-225). Recent work has revealed the presence of phosphorylated signaling proteins in the vitreous humour of patients affected by AMD or other retinal diseases. While the location of these signaling proteins is typically the cell membrane or intracellular compartments, vitreous samples were proven to be cell-free (Davuluri et al., Arch. Ophthalmol. 127 (2009) 613-21). To gain a better understanding of how these proteins can be shed into the vitreous, we used reverse phase protein arrays (RPMA) to analyze the protein and phosphoprotein content of exosomes shed by cultured ARPE-19 cells under oxidative stress conditions. Seventy two proteins were shown to be released by ARPE-19 cells and compartmentalized within exosomes. Forty one of them were selectively detected in their post-translationally modified form (i.e., phosphorylated or cleaved) for the first time in exosomes. Sets of these proteins were linked together reflecting activation of pathway units within exosomes. A subset of (phospho)proteins were altered in exosomes secreted by ARPE-19 cells subjected to oxidative stress, compared to that secreted by control/non stressed cells. Stress-altered exosome proteins were found to be involved in pathways regulating apoptosis/survival (i.e, Bak, Smac/Diablo, PDK1 (S241), Akt (T308), Src (Y416), Elk1 (S383), ERK 1/2 (T202/Y204)) and cell metabolism (i.e., AMPK?1 (S485), acetyl-CoA carboxylase (S79), LDHA). Exosomes may thus represent the conduit through which membrane and intracellular signaling proteins are released into the vitreous. Changes in their (phospho)protein content upon stress conditions suggest their possible role in mediating cell-cell signaling during physio-pathological events; furthermore, exosomes may represent a potential source of biomarkers.

Project description:Retinal ganglion cells (RGCs) undergo rapid cell death by apoptosis after injury but can be rescued by suppression of caspase-2 (CASP2) using an siRNA to CASP2 (siCASP2). Pigment epithelium-derived factor (PEDF), has neuroprotective and anti-angiogenic functions and protects RGC from death. The purpose of this study was to investigate if suppression of CASP2 is a possible mechanism of neuroprotection by PEDF in RGC. Adult rat retinal cells were treated in vitro with sub-optimal and optimal concentrations of siCASP2 and PEDF and levels of CASP2 mRNA and RGC survival were then quantified. Optic nerve crush (ONC) injury followed by intravitreal injections of siCASP2 or PEDF and eye drops of PEDF-34 were also used to determine CASP2 mRNA and protein reduction. Results showed that PEDF and PEDF-34 significantly suppressed CASP2 mRNA in culture, by 1.85- and 3.04-fold, respectively, and increased RGC survival by 63.2?±?3.8% and 81.9?±?6.6%, respectively compared to cells grown in Neurobasal-A alone. RGC survival was significantly reduced in glial proliferation inhibited and purified RGC cultures suggesting that some of the effects of PEDF were glia-mediated. In addition, intravitreal injection of PEDF and eye drops of PEDF-34 after ONC also suppressed CASP2 mRNA levels by 1.82- and 3.89-fold and cleaved caspase-2 (C-CASP2) protein levels by 4.98- and 8.93-fold compared to ONC?+?PBS vehicle groups, respectively, without affecting other executioner caspases. Treatment of retinal cultures with PEDF and PEDF-34 promoted the secretion of neurotrophic factors (NTF) into the culture media, of which brain-derived neurotrophic factor (BDNF) caused the greatest reduction in CASP2 mRNA and C-CASP2 protein. The neuroprotective effects of PEDF were blocked by a polyclonal antibody and PEDF suppressed key elements in the apoptotic pathway. In conclusion, this study shows that some of the RGC neuroprotective effects of PEDF is regulated through suppression of CASP2 and downstream apoptotic signalling molecules.

Project description:Exosomes are informative microvesicles associated with intercellular communication via the transfer of many molecular constituents such as proteins, lipids, and nucleic acids; environmental changes and the cellular status around cells greatly affect exosome components. Cells of the retinal pigment epithelium (RPE) are key players in retinal homeostasis. Transforming growth factor (TGF)-? and tumour necrosis factor (TNF)-? are increased in the vitreous and retina in several retinal diseases and activate and undergo epithelial-mesenchymal transition (EMT) in RPE cells. EMT is closely associated with mechanisms of wound healing, including fibrosis and related angiogenesis; however, whether exosome components depend on the cell status, epithelium or mesenchyme and whether these exosomes have pro- or anti-angiogenic roles in the retina are unknown. We performed this study to investigate whether these EMT inducers affect the kinds of components in exosomes secreted from RPE cells and to assess their angiogenic effects. Exosomes were collected from culture media supernatants of a human RPE cell line (ARPE-19) stimulated with or without 10 ng/ml TNF-? and/or 5 ng/ml TGF-?2. NanoSight tracking analysis and immunoblot analysis using exosome markers were used to qualify harvested vesicles. Angiogenic factor microarray analysis revealed that exosomes derived from ARPE-19 cells cultured with TNF-? alone (Exo-TNF) and co-stimulated with TNF-? and TGF-?2 (Exo-CO) contained more angiogenic factors than exosomes derived from control cells (Exo-CTL) or ARPE-19 cells cultured with TGF-?2 alone (Exo-TGF). To assess the effect on angiogenesis, we performed chemotaxis, tube formation, and proliferation assays of human umbilical vein endothelial cells (HUVECs) stimulated with or without exosomes. HUVECs migrated to RPE-derived exosomes, and exosomes derived from ARPE-19 cells accelerated HUVEC tube formation. In contrast, Exo-TNF and Exo-CO reduced HUVEC proliferation. Our findings provide insight into the mechanisms underlying the relation between angiogenesis and exosomes derived from RPE cells.

Dataset Information

?B crystallin is apically secreted within exosomes by polarized human retinal pigment epithelium and provides neuroprotection to adjacent cells.

Publications

αB crystallin is apically secreted within exosomes by polarized human retinal pigment epithelium and provides neuroprotection to adjacent cells.

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