Human in vitro Model Reveals the Effects of Collagen Cross-linking on Keratoconus Pathogenesis

Rabab Sharif,Dimitrios Karamichos,Jesper Hjortdal,Henrik Sejersen,Garett Frank

doi:10.1038/s41598-017-12598-8

Abstract

Keratoconus (KC) is a corneal thinning disorder that leads to severe vision impairment As opposed to corneal transplantation; corneal collagen crosslinking (CXL) is a relatively non-invasive procedure that leads to an increase in corneal stiffness. In order to evaluate the effect of CXL on human corneal stromal cells in vitro, we developed a 3-D in vitro CXL model, using primary Human corneal fibroblasts (HCFs) from healthy patients and Human Keratoconus fibroblasts (HKCs) from KC patients. Cells were plated on transwell polycarbonate membranes and stimulated by a stable vitamin C. CXL was performed using a mixed riboflavin 0.1% PBS solution followed by UVA irradiation. Our data revealed no significant apoptosis in either HCFs or HKCs following CXL. However, corneal fibrosis markers, Collagen III and α-smooth muscle actin, were significantly downregulated in CXL HKCs. Furthermore, a significant downregulation was seen in SMAD3, SMAD7, and phosphorylated SMADs -2 and -3 expression in CXL HKCs, contrary to a significant upregulation in both SMAD2 and Lysyl oxidase expression, compared to HCFs. Our novel 3-D in vitro model can be utilized to determine the cellular and molecular effects on the human corneal stroma post CXL, and promises to establish optimized treatment modalities in patients with KC.

Highlights

In the last decade CXL has become a fundamental treatment for progressive ectasias including KC, and has recently been approved by the US Food and Drug Administration (FDA)
transforming growth factor-β (TGF-β) has been identified as an important growth factor involved in the development of corneal fibrosis and scarring, as it activates corneal keratocytes, and promotes fibrosis represented by an increase in collagen type III and α-smooth muscle actin (α-SMA) expression[35]
The inhibitory SMAD7 is known to bind to TGF-β receptor competitively and interferes with the activation of SMAD2 and SMAD3 leading to an inhibition in the TGF-β signal transduction[10]

Summary

Introduction

In the last decade CXL has become a fundamental treatment for progressive ectasias including KC, and has recently been approved by the US Food and Drug Administration (FDA). UV irradiation excites the fluorescent molecule to a triplet state, with consequent generation of a singlet oxygen and superoxide radical[19] These radical products are able to strengthen the corneal stromal collagen bonds and increase resistance to enzymatic degradation forming covalent bonds between the amino acids of the adjacent collagen fibers[23]. CXL without epithelial debridement (epi-on) technique has been attempted and aims to reduce the risk of infection and post-operative pain, associated with epi-off[26]. Both techniques have been proven to be effective studies regarding safety and long term adverse effects are inconclusive[27]. The long term implications of our study are important for KC patients since it could establish optimized treatment modalities in these patients

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