Keratoconus: Cellular and molecular characterization of a novel human Corneal Collagen Cross-linking 3-D in vitro model

Abstract

Purpose : Keratoconus (KC) is a progressive eye condition, represented by biomechanical corneal instability, and severe vision impairment. Despite the large number of studies, but no existing animal model, the exact underlying KC pathobiology remains unclear. We utilized our established 3-D in vitro KC model to study the human corneal stroma, and matrix assembly following radiation induced corneal collagen crosslinking (CXL). This 2016 FDA approved application is the only current aetiopathogenic mechanism that promises to halt KC progression. In this study we take the lead in determining the cellular and molecular effects on human corneal stroma pre/post CXL, which unfortunately, have not been previously investigated.

Methods : Human corneal fibroblasts (HCF) and keratoconus (HKC) cells were seeded on a poly-carbonate membrane culture plates, at initial density of 1×10⁶ cells/well and cultured in a 10% FBS EMEM medium supplemented with 0.5 mM of Vitamin C. Cultures were grown for 4 weeks, and fresh media was supplied every other day. At the end of week 4 all cultures were exposed to UVA light (wavelength 365 nm) at a 3 cm distance for 3, and 5 minutes with an irradiation dose of 3mW/cm². Cell viability was determined using live/dead cell staining technique. All samples were analyzed using RT–PCR and western blotting to quantify changes in the expressions of key proteins LOX, COL-I,-III,-V, and the transcriptional SMAD proteins, between HCFs and HKCs.

Results : Our data indicates small but significant percentage of apoptotic keratocytes following in vitro CXL. Upon measurement of matrix hydration percentage, HKC-CXL showed a quick water loss transition when compared to controls resulting in compaction of corneal stroma. Western blot analysis revealed a reduced expression of the regulatory SMAD 6 (p<0.0001) in untreated HKCs, contrary to SMAD 3 that showed to be downregulated (p=0.02) post KC.CXL, suggesting a malfunction in the SMAD regulated downstream signaling pathway following CXL. We observed a significant increase in LOX expression (p< 0.0001) post-CXL compared to controls.

Conclusions : Our 3-D model provided excellent grounds to study CXL effects on the corneal stromal cellular and molecular environment. In addition, this in vitro CXL system is promising to establish optimized treatment modalities in patients suffering from KC.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.