Abstract
Introduction: This is a basic science research proteomic study. The study took place at the 1st University Ophthalmology Department, ‘G.Gennimatas’ General Hospital of Athens in collaboration with B.S.R.C “Alexander Fleming” in Vari, Attiki. The study was conducted from March 2022 until February 2025. Objectives: The aim of the present study is to investigate and identify proteomic changes and corresponding signaling pathways involved in the formation of age-related, diabetic, and post-vitrectomy cataracts by analyzing three distinct sample types from each participant: the aqueous humour, the anterior capsule, and the content of the phaco cassette, which contains the lens fragments. First, proteomic changes and the associated signaling pathways in the aqueous humour will be compared across the three cataract groups (age-related, diabetic, and post-vitrectomy), with the objective of identifying both common and distinct cataract-specific mechanisms. Next, the proteomic alterations and as ...
Introduction: This is a basic science research proteomic study. The study took place at the 1st University Ophthalmology Department, ‘G.Gennimatas’ General Hospital of Athens in collaboration with B.S.R.C “Alexander Fleming” in Vari, Attiki. The study was conducted from March 2022 until February 2025. Objectives: The aim of the present study is to investigate and identify proteomic changes and corresponding signaling pathways involved in the formation of age-related, diabetic, and post-vitrectomy cataracts by analyzing three distinct sample types from each participant: the aqueous humour, the anterior capsule, and the content of the phaco cassette, which contains the lens fragments. First, proteomic changes and the associated signaling pathways in the aqueous humour will be compared across the three cataract groups (age-related, diabetic, and post-vitrectomy), with the objective of identifying both common and distinct cataract-specific mechanisms. Next, the proteomic alterations and associated signaling pathways in the anterior capsule will be examined and compared between the different cataract types. Finally, a novel analytical method will be developed to examine the contents of the phaco cassette, which contains lens fragments collected during cataract surgery. This method will allow for the identification of proteomic changes and relevant signaling pathways in the lens across the three cataract types. In addition to the proteomic analyses, demographic data from each participant will be collected and analyzed to investigate potential risk factors and correlations with the significant pathways identified. Material and methods: All patients presented with cataract at the 1st University Ophthalmology Department in ‘G.Gennimatas’ General Hospital of Athens were eligible to the study. Three cataract groups were organized and each participant was assigned to a specific group based on certain criteria. The three cataract groups were the age-related cataract (ARC) group (No history of diabetes mellitus and age older than 75 years); the diabetic cataract (DC) group (History of diabetes mellitus and age younger than 65 years); and the post-vitrectomy cataract (PVC) group (Previous vitrectomy surgery for retinal detachment repair with gas tamponade in the past 12 months and no history of diabetes mellitus). Every participant included in the study filled in a questionnaire. Three samples were collected during routine cataract surgery, performed by the same experienced surgeon under local anesthesia; the aqueous humour, the anterior capsule and the content of the phaco cassette bag. Sp3 protocol (Single-Pot, Solid-Phase, Sample-Preparation) was used for the sample preparation. The recognition and quantification of proteins were carried out with liquid chromatography online with tandem mass spectrometry, using a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer. The DIA-NN software was applied for identification and quantification of peptides/proteins. The software Perseus (Version 1.6.15.0) was used for statistical analysis (defined groups, t-test) and data visualization. Two-sample t-tests were conducted to compare ARC-DC and ARC-PVC groups. Volcano plots were generated to visualize the t-test results. ANOVA tests were performed for comparisons across all groups, with the results visualized in heatmaps. For gene enrichment analysis ShinyGO (Version 0.77) was used. All analyses of demographic data were carried out using the R programming language and the RStudio IDE. Results: A very rich atlas of the lens and aqueous humour proteome has been generated. In this study, a total of 1639 proteins were identified in the aqueous humour samples, 2815 proteins in the anterior capsule samples, and 2975 proteins in the phaco cassette content samples. Glycosaminoglycan biosynthesis and non-canonical Wnt receptor signaling pathway were significantly differentially expressed in the ARC group compared to both DC and PVC groups. In the PVC group, complement activation was differentially expressed in the aqueous humour samples, while glutathione metabolism and oxidoreductase activity were differentially expressed in the anterior capsule samples. Microfilament motor activity, microtubule cytoskeleton organization, and microtubule binding were differentially expressed in DC and PVC groups in both aqueous humour and anterior capsule samples. In the phaco cassette content samples, proteins with significantly different expression in the ARC group compared to DC and PVC groups were involved in pathways, including actin binding, actin cytoskeleton reorganization, actin filament capping, cortical actin cytoskeleton organization, and small GTPase-mediated signal transduction pathways. In the phaco cassette content samples, pathways significantly differentially expressed in the ARC group to the DC and PVC groups included the glycolipid metabolic, glycosphingolipid biosynthetic, and glycosphingolipid metabolic processes, with GLA being among the most significant proteins in the ARC group. Similarly, in the anterior capsule samples, the main significantly differentially expressed pathways in the ARC group compared to the DC and PVC groups were the glycolipid metabolic, glycosphingolipid biosynthetic, and glycosphingolipid metabolic processes, with ST3GAL5 being among the most significant proteins in the ARC group. Conclusions: The results of this study expand the existing knowledge on pathways involved in the pathophysiology of cataract. In this study, key pathways involved in cataract formation in age-related, diabetic, and post-vitrectomy cataracts were identified. Differential expression of the non-canonical Wnt receptor signaling pathway in the age-related cataract may promote abnormal proliferation and migration of lens epithelial cells, contributing to cataract formation. Similarly, the differential expression of glycosaminoglycan and glycosphingolipid pathways in the ARC group suggests potential therapeutic targets for preventing cell proliferation and repairing damaged lens tissue. In the PVC group, the significant differential expression of oxidation-reduction pathways in anterior capsule samples suggests a protective role of lens epithelial cells against oxidative stress. However, post-vitrectomy cataract formation may occur when oxidative stress surpasses protective mechanisms. This is the first study to use phaco cassette content samples to investigate cataract formation, opening new avenues for research. Our results highlight novel pathways involved in cataract pathophysiology and suggest druggable targets for slowing or preventing cataract progression. Future research should focus on identifying diagnostic biomarkers and developing a grading system for cataract severity.
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