Biomechanics of an in vitro model of Descemet’s membrane in health and disease

Abstract

Purpose : Fuchs’ endothelial corneal dystrophy (FECD) is an endothelial degenerative disorder characterized by guttata or abnormal excrescences of extracellular matrix (ECM). Our laboratory recently demonstrated that the elastic modulus, or stiffness, of Descemet’s membrane (DM) is significantly decreased in human patients with versus without guttata using atomic force microscopy (AFM) at 40 and 9.8 kPa, respectively. Epithelial-mesenchymal transformation is implicated in the pathogenesis of FECD and transforming growth factor-β (TGF-β) has been demonstrated to induce a fibroblast-like phenotype in corneal endothelial cells in vitro. The purpose of this study was to create an in vitro ECM-cell derived model that mimics the biophysical attributes of normal and FECD-affected DM.

Methods : Primary bovine corneal endothelial cells (BCECs) were seeded on AFM dishes and cultured with media in the presence or absence of 1 ng/ml TGF-β1 for 6 weeks. The ECM was decellularized with ammonium hydroxide and its elastic modulus determined with AFM. Proteomics and scanning electron microscopy were performed with additional cell-derived matrices using identical experimental conditions.

Results : Elastic modulus of ECM derived for 2 cultures were 1.38 and 0.81 kPa in the absence of TGF-β1 treatment. In the presence of TGF-β1, elastic modulus was decreased to 1.07 and 0.39 kPa, respectively. Proteomic profiling revealed greater than 500 proteins (structural and soluble) associated with the ECM. The ECM deposited by untreated BCECs appear to have a compact sheet-like morphology while cells treated with TGF-β1 deposit an ECM with a greater number of fibrillar regions.

Conclusions : Treatment of primary corneal endothelial cells with TGF-β1 results in softening of the cell-derived ECM that parallels the changes observed in DM with the presence of guttata. This in vitro system holds promise for examining the interplay between soluble signaling molecules and the biophysical attributes of corneal endothelial cell derived matrices in health and disease.

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