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Protein composition of the subretinal fluid suggest selective diffusion of vitreous proteins in retinal detachment

ABSTRACT: Purpose: To study the proteome of the subretinal fluid (SRF) from rhegmatogenous retinal detachment (RRD) in search for novel markers for improved diagnosis and prognosis of RRD. Methods: Human undiluted SRF obtained during vitrectomy for primary RRD using a 41G needle (n = 24) was analyzed and compared to vitreous humor from 2-days post-mortem eyes (n = 20). Sample preparation underwent nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). Label-free quantification (LFQ) using MaxQuant was used to determine differential expressed proteins between SRF and vitreous humor. The intensity-based absolute quantification (iBAQ) was used to rank proteins according to their molar fractions within groups. Identification of proteins beyond the quantitative level was performed using the Mascot search engine. Results: The protein concentration of the control vitreous humor was lower and more consistent (1.2 ± 0.4 mg) than that of the SRF (17.9 ± 22 mg). The iBAQ analysis showed high resemblance between SRF and vitreous humor, except for crystallins solely identified in vitreous humor. The LFQ analysis found 37 protein misregulations between SRF and vitreous humor of which the blood coagulation pathway was found to be enriched using the PANTHER Classification System. Combined, the iBAQ, LFQ and Mascot analysis found an overlap only in chitinase-3-like protein 1 and galectin-3-binding protein unique to the SRF. Conclusions: The proteome of the SRF was highly represented by proteins involved in proteolysis. Such proteins can possibly serve as targets in modulating the effects of SRF in RD.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Vitreous Humor

DISEASE(S): Retinal Detachment

SUBMITTER: Ebbe Toftgaard Poulsen

LAB HEAD: Jan J. Enghild

PROVIDER: PXD019159 | Pride | 2020-10-20

REPOSITORIES: Pride

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Project description:PURPOSE: To investigate the circulatory microRNA (miRNA) profiles of aqueous, vitreous, and plasma in order to identify biomarkers in aqueous humor or plasma that are reflecting changes in vitreous of patients with diabetes. METHODS: Aqueous, vitreous and plasma samples were collected from a total of 27 patients - 11 controls (macular pucker or macular hole patients) and 16 patients with diabetes mellitus (DM) undergoing vitreoretinal surgery: DM-Type I with proliferative diabetic retinopathy (PDR) (DMI-PDR), DM Type II with PDR (DMII-PDR) and DM Type II with nonproliferative DR (DMII-NPDR). MiRNAs were isolated using Qiagen microRNeasy kit, quantified on BioAnalyzer, labeled with FlashTag kit, and profiled on Affymetrix GeneChip miRNA 3.0 microarrays. Data analysis was done using Expression Console (EC), Transcriptome Analysis Console (TAC), and Ingenuity Pathway Analysis (IPA) software. RESULTS: Our comparison of circulatory miRNA population of aqueous and vitreous humor and plasma showed that out of total of 847 human miRNA probes on the Affymetrix GeneChip miRNA 3.0 we found common miRNAs for both aqueous and vitreous samples, as well as larger number of unique miRNA, dependent on the DM type and presence of retinopathy. Most of the dysregulated miRNAs in aqueous and vitreous of DM patients were upregulated, while in plasma, most of the DM-specific miRNAs were downregulated. Dysregulation of miRNAs in aqueous generally do not appear to be a good representative of the miRNA abundance in vitreous, or plasma, although we did identify a few candidates for common biomarkers: let-7b, miR-320b, miR-762 and miR-4488. Additionally, each of the DR subtypes showed a set of miRNA that is uniquely dysregulated in each fluid, for example in aqueous samples for DMII-NPDR it was miR-455-3p, for DMII-PDR was miR-296, and for DMI-PDR it was miR-3202. Pathway analysis identified TGF-beta and VEGF pathways as the common targets for miRNAs dysregulated in DR aqueous and vitreous. CONCLUSIONS: The comparative profiling of circulatory miRNAs in aqueous, vitreous, and plasma showed that a small number of circulatory miRNAs displayed differential presence in controls vs. diabetic retinopathy. A pattern is emerging of sets of miRNA that are common or uniquely dysregulated in the blood plasma or ocular fluids of DR subtypes, offering promise for the use of ocular fluids and plasma for identifying diagnostic and therapeutic targets.

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Protein composition of the subretinal fluid suggest selective diffusion of vitreous proteins in retinal detachment

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