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Proteome and Metabolome of Subretinal Fluid in Central Serous Chorioretinopathy and Rhegmatogenous Retinal Detachment: a Pilot Case Study

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Proteome and Metabolome of Subretinal Fluid in Central Serous Chorioretinopathy and Rhegmatogenous Retinal Detachment: a Pilot Case Study https://doi.org/10.1167/tvst.7.1.3 Article Proteome and Metabolome of Subretinal Fluid in Central Serous Chorioretinopathy and Rhegmatogenous Retinal Detachment: A Pilot Case Study Laura Kowalczuk1,*, Alexandre Matet1,*, Marianne Dor2, Nasim Bararpour3, Alejandra Daruich1, Ali Dirani1, Francine Behar-Cohen4,5, Aurelien´ Thomas3,4,†, and Natacha Turck2,† 1 Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland 2 OPTICS Laboratory, Department of Human Protein Science, University of Geneva, Geneva, Switzerland 3 Unit of Toxicology, CURML, Lausanne-Geneva, Switzerland 4 Faculty of Biology and Medicine, Lausanne University Hospital, University of Lausanne, Lausanne, Switzerland 5 Inserm, U1138, Team 17, From physiopathology of ocular diseases to clinical development, Universite´ Paris Descartes Sorbonne Paris Cite,´ Centre de Recherche des Cordeliers, Paris, France Correspondence: Francine Behar- Purpose: To investigate the molecular composition of subretinal fluid (SRF) in central Cohen, Inserm U1138, Team 17, serous chorioretinopathy (CSCR) and rhegmatogenous retinal detachment (RRD) Centre de Recherche des Cordeliers, using proteomics and metabolomics. 15 rue de l’Ecole de Medecine,´ 75006 Paris, France. e-mail: francine. Methods: SRF was obtained from one patient with severe nonresolving bullous CSCR [email protected] requiring surgical subretinal fibrin removal, and two patients with long-standing RRD. Proteins were trypsin-digested, labeled with Tandem-Mass-Tag and fractionated Received: 5 July 2017 according to their isoelectric point for identification and quantification by tandem Accepted: 2 November 2017 mass spectrometry. Independently, metabolites were extracted on cold methanol/ Published: 18 January 2018 ethanol, and identified by untargeted ultra-high performance liquid chromatography Keywords: subretinal space; retinal and high-resolution mass spectrometry. Bioinformatics analyses were conducted. pigment epithelium; retinal de- Results: In total, 291 proteins and 651 metabolites were identified in SRF samples. tachment; retinal metabolism; vit- Compared with RRD, 128 proteins (77 downregulated; 51 upregulated) and 76 reoretinal surgery metabolites (43 downregulated; 33 upregulated) differed in the SRF from CSCR. Citation: Kowalczuk L, Matet A, Dor Protein and metabolites notably deregulated in CSCR were related to glycolysis/ M, Bararpour N, Daruich A, Dirani A, gluconeogenesis, inflammation (including serum amyloid P component, versican), Behar-Cohan F, Thomas A, Turck N. alternative complement pathway (complement factor H and complement factor H– Proteome and metabolome of sub- related protein), cellular adhesion, biliary acid metabolism (farnesoid X receptor/ retinal fluid in central serous cho- retinoid X receptor), and gluco- and mineralocorticoid systems (aldosterone, rioretinopathy and rhegmatogenous angiotensin, and corticosteroid-binding globulin). retinal detachment: a pilot case study. Trans Vis Sci Tech. 2018;7(1):3, Conclusions: Proteomics and metabolomics can be performed on SRF. A unique SRF https://doi.org/10.1167/tvst.7.1.3 sample from CSCR exhibited a distinct molecular profile compared with RRD. Copyright 2018 The Authors Translational Relevance: This first comparative multiomics analysis of SRF improved the understanding of CSCR and RRD pathophysiology. It identified pathways potentially involved in the better photoreceptor preservation in CSCR, suggesting neuroprotective targets that will require additional confirmation. epithelial detachments, increased choroidal thickness Introduction and choroidal vascular hyperpermeability. Severe CSCR can present as bullous exudative retinal Central serous chorioretinopathy (CSCR) is char- detachment with persistence of subretinal material,1 acterized by serous detachments of the neurosensory that may exceptionally require surgery.2 During the retina, frequently affecting the macula, focal pigment procedure, subretinal fluid (SRF) can be collected, 1 TVST j 2018 j Vol. 7 j No. 1 j Article 3 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded From: http://tvst.arvojournals.org/pdfaccess.ashx?url=/data/journals/tvst/936672/ on 01/22/2018 Kowalczuk et al. but to the best of our knowledge, the composition of patients underwent vitrectomy for long-standing SRF from a CSCR patient has never been analyzed. macula-off RRD repair: an 82-year-old Caucasian Mechanisms of SRF accumulation in CSCR are still female (Patient RRD-1), and a 58-year-old African uncertain. Several theories have been proposed to male (Patient RRD-2). In all cases, 23-G trocars were explain fluid entry from the choroid toward the inserted at the pars plana and SRF was collected subretinal space: dilated and hyperpermeable choroi- using a back flush cannula connected to a syringe, dal vessels favoring trans- or interretinal pigment through retinal tears in RRD, and through a epithelium (RPE) entry flow,3 changes in RPE cell retinotomy in the CSCR case, as described in the polarity altering hydroionic pumping direction,4 uni- Results section. Samples were directly frozen and or multifocal rupture of the RPE barrier,5 or active stored at À808C in our institutional biobank, a reverse flow by unknown triggering mechanisms.6 platform similar to the biorepository described by Although serous macular detachments form rap- Skeie at al.13 Coded samples and their associated idly and last for 3 to 6 months in most cases,7 visual clinical data were then sent to the laboratories in acuity is usually preserved, suggesting a good charge of the proteomics and metabolomics analyses. preservation of photoreceptors function and struc- The CSCR patient underwent multimodal retinal ture.8 This differs strikingly with macula-off rhegma- imaging at regular intervals, consisting in spectral- togenous retinal detachment (RRD), in which domain optical coherence tomography (SD-OCT), detachments lasting more than 3 to 5 days lead to fundus autofluorescence, fluorescein, and indocya- irreversible visual impairment.9 Mechanisms of pho- nine green (ICG) angiography on Spectralis (Heidel- toreceptor cell death after RRD are multiple, berg Engineering, Heidelberg, Germany). triggered by oxidative and metabolic stress, comple- ment alternative pathway activation, immune re- Proteomic Analysis 10,11 sponse, and inflammation. In RRD, SRF The proteome of the SRF was compared between originates mainly from liquefied vitreous, which samples from the CSCR patient and the two patients diffuses through a retinal tear under the neuroretina. with chronic RRD. The total protein concentration in In response to neuroretinal detachment, RPE cells 12 each sample was determined using a Bradford protein proliferate and migrate into the subretinal space, assay (Protein assay Dye reagent concentrate, Bio- which may contribute to SRF accumulation. Whether Rad, Hercules, CA) according to the manufacturer’s SRF composition influences the differential photore- instructions. ceptor survival and visual prognosis in different types of neuroretinal detachment is not known. Sample Preparation The aim of this study was to investigate the The two control RRD samples were pooled to molecular composition of subretinal fluid in CSCR homogenize experimental conditions and filter small and RRD using proteomics and metabolomics. We interindividual differences, while increasing the con- compared the SRF profile from one case with severe centration of proteins or metabolites related to the CSCR and two cases with chronic RRD. Due to the pathogenic process, as previously described for the rarity of SRF samples from CSCR, we opted for an omics analysis of ocular fluids.14 In order to reduce untargeted proteomic approach, with independent the impact of potential blood contamination during cross-assessment by metabolomics analysis. sample collection, 100 lg of SRF from the CSCR patient and 100 lg of SRF pooled from the two RRD Methods patients (a 1:1 vol/vol ratio) were filtered using a commercial resin according to the manufacturer’s Study Subjects recommendations (Proteome Purify 12 Human Serum Protein Immunodepletion Resin; R&D Systems, This study involving human subjects adhered to Abingdon Science Park, UK) to deplete the classical the tenets of the Declaration of Helsinki, and was top-12 most abundant serum proteins (alpha-1-acid approved by the local Ethics Committee of the Swiss glycoprotein, alpha-1-antitrypsin, alpha-2-macro- Department of Health on research involving human globulin, albumin, apolipoprotein A-I, apolipopro- subjects (CER-VD N8340/15 and CER-VD N819/15). tein A-II, fibrinogen, haptoglobin, immunoglobulin Patients signed an informed consent. A 48-year-old (Ig)A, IgG, IgM, transferrin). Using the Bradford male patient with persistent bullous CSCR underwent method, the total protein concentration after deple- vitrectomy for subretinal fibrinous clot removal. Two tion was estimated at 9.5 lg/mL in the CSCR sample 2 TVST j 2018 j Vol. 7 j No. 1 j Article 3 Downloaded From: http://tvst.arvojournals.org/pdfaccess.ashx?url=/data/journals/tvst/936672/ on 01/22/2018 Kowalczuk et al. and 9.6 lg/mL in the pooled RRD samples. Pro (Thermo Fisher Instrument, Steinhaussen, Swit- Subsequently, 5 lg of samples (CSCR and RRD) zerland) coupled to a nanoflow high pressure liquid were reduced using 33 uL of 6-M urea (Merk, BoisIˆle- chromatography (HPLC, nanoaquity system; Waters, de-France, France) and 2 uL of
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