Shotgun proteomics identification of proteins expressed in the Descemet’s membrane of patients with Fuchs endothelial corneal dystrophy

Abstract

Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet’s membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεβ) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae.

Senescent characteristics of human corneal endothelial cells upon ultraviolet-A exposure

“…Our results also show an upregulation of proteins related to complement factors and canonical glycolysis, and SASPs in UV-A-induced senescent hCEnCs overlap with aqueous humor proteins in FECD patients [30]. Shotgun proteomics also identified 32 proteins, including 11 proteins related to ECM pathway, which are significantly upregulated in the Descemet's membrane constituents of FECD patients compared to healthy controls [31]. Amongst the 11 proteins related to ECM pathway, 6 proteins, BGN, COL6A2, COL8A1, LTBP2, MATN2, MATN3, were upregulated in UV-Ainduced senescent cells.…”

“…STC1 and GDF15 are recognized as Core SASPs, representing soluble components common to senescent cells induced by various factors [29]. To identify SASPs specific to senescent CEnCs and proteins associated with corneal endothelial diseases [30,31], we selected and heat-mapped proteins with significantly increased secretion in UV-A-induced senescent CEnCs compared to controls (Figure 7B). We detected elevated levels of CXCL1, CXCL8, MMP2, COL6A2, COL8A1, COL12A1 and other proteins that have been previously reported as SASP factors in various cell types [29].…”

Expression and Impact of Fibronectin, Tenascin-C, Osteopontin, and Type XIV Collagen in Fuchs Endothelial Corneal Dystrophy

“… 9 , 11 Tenascin-C is a glycoprotein expressed in the epithelial basement membrane and the anterior corneal stroma of FECD specimens, 40 and its presence in FECD DMs has recently been demonstrated. 41 The upregulation of the SPP1 gene raised interest because it was the only ECM-related gene overexpressed both ex vivo and in vitro in FECD CECs. Its protein, osteopontin, is a matrix structural glycophosphoprotein that acts as a cytokine and regulates the activity of resident tissue cells at sites of injury.…”