Abstract
Purpose :
Fuchs Endothelial Corneal Dystrophy (FECD) pathogenesis has been described as a vicious cycle starting with cellular dysfunction resulting in the secretion of abnormal extracellular matrix and formation of guttae creating a toxic environment for the Corneal Endothelial Cells (CECs) and eventually leading to cellular apoptosis. In in-vitro models, guttae have be shown to induce a cellular stress response and apoptosis in a size-dependent manner. However, the molecular mechanisms underlying guttae formation and their exact composition remain unclear. The aim of this study was to investigate the proteomics composition of FECD-Descemet Membranes (FECD-DM) compared to Normal-DM (N-DM), and the guttae composition from FECD-DM in function of their diameter, using data-independent acquisition mass spectrometry (DIA-MS)-based quantitative proteomics technology.
Methods :
N-DMs were isolated by manual dissection from normal human corneas (n=3). FECD-DMs were obtained from FECD patients who underwent Descemet Membrane Endothelial Keratoplasty. All DMs were decellularized using trypsin treatment. The small (<15µm), medium (15–30µm), and large (>30µm) guttae from FECD-DM (n=9) were measured, isolated using Laser Capture Microdissection, and pooled in separate vials based on guttae diameter. The samples were used for DIA-MS-based quantitative proteomics.
Results :
In total, 1283 proteins were identified and quantified of which 296 proteins were significantly dysregulated in FECD-DM compared to N-DM (p<0.05). The gene ontology based on those 296 dysregulated proteins identified 20 different pathways including ECM organization, composition, neutrophile degranulation, oestrogen signaling, and cell substrate adhesion pathways. The levels of collagen type VIII alpha-2 chain, collagen type II alpha-1 chain, collagen type III alpha-1 chain, lumican, and were statistically higher in medium and large guttae compared to small guttae and N-DM (p<0.05). Three proteins were only found in medium and large guttae, Collagen XII alpha 1, Fibrillin1 and Matrilin-3.
Conclusions :
Comprehensive protein profiles from FECD-DM were obtained using DIA-based quantitative proteomics allowing the identification and quantification of proteins only present in guttae. Our results suggest that guttae proteomics composition might derive from a dynamic accumulation associated with changes in the dynamic reciprocity between CECs and the guttae over time.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.