The keratoconus corneal proteome: Loss of epithelial integrity and stromal degeneration

Abstract

Keratoconus is a thinning corneal dystrophy that begins in the early teenage years and ultimately requires cornea transplantation to restore vision. Here we conducted a highly sensitive mass spectrometric analysis of the epithelium and the stroma from keratoconus and normal donor corneas. We identified a total of 932 and 1,157 proteins in the consolidated data of the epithelium and stroma, respectively. Technical replicates showed strong correlations (≥ 0.88) in levels of all common proteins, indicating very low technical variations in the data. Analysis of the most increased (≥ 1.5 fold) and decreased (≤ 0.8 fold) proteins in the keratoconus corneal epithelial protein extracts identified proteins related to dermal diseases, inflammation, epithelial stratification and mesenchymal changes. Increased proteins included keratins 6A, 16 and vimentin, while the iron transporter lactotransferrin was decreased. The keratoconus stromal proteome suggest endoplasmic reticular stress, oxidative stress and widespread decreases in many extracellular matrix proteoglycan core proteins, lumican and keratocan, collagen types I, III, V and XII. Marked increase in apoptosis and endocytosis-related proteins suggest degenerative changes in keratocytes, the resident cells of the stroma. This is the most comprehensive proteome analysis of the cornea that highlights similarities of keratoconus with other neurodegenerative diseases.

Collagen synthesis disruption and downregulation of core elements of TGF-β, Hippo, and Wnt pathways in keratoconus corneas

“…39 Recently, it was shown that one of the major corneal proteoglycans, LUM -encoding lumican, was downregulated in cell cultures derived from KTCN corneas and in the corneas. 34,40 In this study, the expression of LUM, was also diminished, which could destabilize normal corneal fibril organization.…”

“…In a previous study, a mass spectrometry assessment of protein levels in the human corneal epithelium and stroma showed that all but one of these collagens, COL5A2, had decreased. 34 Thus, we suppose that in these genes, dysregulation of their expression may have a direct impact on protein levels.…”

Keratoconus: an inflammatory disorder?

“…There are over 21 types of collagen in the human body; however, type I corresponds to 75% of the amount present in the cornea, 77 although cornea also contains collagen types III, V, and VI regularly interwoven into lamellae, and collagen type XII in epithelium basement membrane and sub-epithelial stroma. 74,78 Although the reduction in the number of lamellae within the affected region could correspond simply to a redistribution of the collagen within the cornea by slippage between the lamellae, as suggested by Polack 79 almost 40 years ago, this explanation by itself does not seem to be enough to account for the stromal thinning, especially in light of more recent studies showing that the collagen lamellae in keratoconus corneas exhibit a significant decrease in number compared with controls, 80 and that keratoconus diminishes the amount of types I, III, V, and XII collagen, as well as lumican and keratocan proteins, as determined using highly sensitive mass spectrometric analysis. 78 Additional researchers have reported the reduction of the interfibrillar distance of collagen sheets and the increase of proteoglycans with abnormalities in their configuration as the condition evolves.…”

“…74,78 Although the reduction in the number of lamellae within the affected region could correspond simply to a redistribution of the collagen within the cornea by slippage between the lamellae, as suggested by Polack 79 almost 40 years ago, this explanation by itself does not seem to be enough to account for the stromal thinning, especially in light of more recent studies showing that the collagen lamellae in keratoconus corneas exhibit a significant decrease in number compared with controls, 80 and that keratoconus diminishes the amount of types I, III, V, and XII collagen, as well as lumican and keratocan proteins, as determined using highly sensitive mass spectrometric analysis. 78 Additional researchers have reported the reduction of the interfibrillar distance of collagen sheets and the increase of proteoglycans with abnormalities in their configuration as the condition evolves. These changes allow more contact between the collagen sheets and the proteoglycans, thus altering the stroma organization where alterations in interlamellar proteoglycans might contribute to slippage of the lamellae.…”

The pathogenesis of keratoconus

“…Many extracellular matrix proteoglycans and proteins involved in proliferation, growth and migration were found to be downregulated in KC epithelium and stroma, and oxidative stress was evident. 24,25 …”