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Unique molecular signatures of microRNAs in ocular fluids and plasma in diabetic retinopathy

ABSTRACT: 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.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE140959 | GEO | 2020/06/27

REPOSITORIES: GEO

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Project description:Purpose: The purpose of this study was to identify cigarette smoke (CS) induced changes in circulatory microRNA (miRNA) in the plasma and ocular fluids of the Rhesus macaque and compare them to normal age-related changes, with the ultimate goal to develop an animal model of early dry age-related macular degeneration (AMD). AMD is a blinding disease having both genetic and environmental components, with cigarette smoke exposure (CS) as the leading environmental risk factor. Methods: All Rhesus macaques were housed at the California National Primate Research Center (CNPRC) at UC Davis. We used 6 animals of the same gender per group: Group 1 (young, 2-4 years old), Group 2 (old, 20-25 years old), Group 3 (middle aged, 9-12 years old) and Group 4 (9-12 years old, exposed to smoke for 1 month). Group 4 macaques were clinically assessed prior to and following CS exposure. Ocular fluids and plasma samples were collected, miRNA isolated, and expression data obtained from Affymetrix miRNA GeneTitan Array Plates 4.0, followed by bioinformatics analysis on Affymetrix Expression Console (EC) and Transcriptome Analysis Software (TAS), and using ANOVA. Results: Statistically significant changes were seen in miRNA populations in ocular fluids and plasma. In the plasma samples, 45 miRNAs were strongly upregulated (Fold Change >+/-1.5, p<0.05) upon CS exposure, while in aging monkeys different miRNAs in plasma were affected, and downregulated. In the vitreous, 3 miRNAs were downregulated in animals exposed to CS, intriguing enough two of them (miR-6794 and miR-6790) were the same ones downregulated with age. Retinal imaging using OCT did not show any significant changes in retinal thickness. Cotinine assays showed that animals received on average dose of 50-60 ng/ml cotinine, a marker for significant CS exposure. Conclusions: One month of CS exposure of Rhesus macaques resulted in significant changes of expression of circulatory miRNAs. We identified several CS related miRNA changes that are plasma or ocular fluid-specific. Healthy aging changes of miRNAs populations were different from the CS exposure induced changes in plasma, while the ones in vitreous were similar. This data will be utilized to develop an animal model of early dry AMD, using a monkey model exposed to CS.

Project description:Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and proliferative DR (PDR), no treatment options exist for the highly prevalent dry form of AMD. To unravel the biological processes underlying these pathologies and to find new potential biomarkers, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR (n=4), AMD (n=4) compared to idiopathic epiretinal membranes (ERM) (n=4). Post-hoc tests revealed 96 proteins capable of differentiating among the different groups, whereas 118 proteins were found differentially regulated in PDR compared to ERM and 95 proteins in PDR compared to dry AMD. The pathway analysis indicates that mediators of complement and coagulation cascades and acute phase responses are enriched in PDR vitreous, whilst proteins highly correlated to the extracellular matrix (ECM) organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development were found underexpressed. According to these results, 35 proteins were selected and validated by MRM (multiple reaction monitoring) in a larger cohort of patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and retinal detachment (n=13). Of these, 26 proteins were validated and were capable of differentiating between vitreoretinal diseases under study. Based on Partial least squares discriminant and multivariate exploratory receiver operating characteristic (ROC) analyses, a panel of 15 discriminatory biomarkers was defined, which includes complement and coagulation components (CO2 and prothrombin), acute-phase mediators (alpha-1-antichymotrypsin), adhesion molecules (e.g., myocilin, galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2).

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Unique molecular signatures of microRNAs in ocular fluids and plasma in diabetic retinopathy

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