In this closed MASc oral exam, Myagmartsend Enkhbat will present their research.
Thesis title:
In Vitro Modelling of Fuchs Endothelial Dystrophy for Corneal Endothelial Cell Apoptosis and Characterization of Human Descemet’s Membrane
Abstract:
The corneal endothelium is a terminally differentiated tissue that is typically considered nonproliferative in vivo. Corneal endothelial dystrophies, including Fuchs' endothelial dystrophy (FED), are a significant cause of dysfunction, ultimately leading to corneal edema. Corneal transplantation is a well-established and effective treatment for corneal endothelial dysfunction. This condition is particularly prominent post-cataract surgery, which increases the risk of corneal transplant failure. However, the global shortage of human donor corneas has prompted ongoing research into alternative treatment approaches. Utilizing engineering solutions to model Fuch’s endothelial dystrophy and investigating the mechanical properties of Descemet’s membrane (DM) are pivotal for advancing our understanding and improving treatment options.
This research has two aims to advance our understanding of FED. The first aim was to investigate how human corneal endothelial cells (HCEC-B4G12) respond to synthetic guttata (Sguttata) pillars, with a specific emphasis on cell apoptosis, gene expression, and cytoskeletal changes. Subsequent examination of B4G12 cell responses to these pillars revealed induction of early and late apoptosis, with significantly higher rates observed on 20×20×20 (20 μm diameter, 20 μm spacing, and 20 μm height) and 40×80×20 (40 μm diameter,
80 μm spacing 20 μm, and height) pillars compared to unpatterned tissue culture polystyrene (TCPS) controls, respectively. Flow cytometry analysis confirmed enhanced early apoptosis on day 2, particularly on the 20×20×20 pillars. The α1 type V collagen (Col5A1) coating, which was shown to be highly expressed on large guttata in FED patients’ DM, on S-guttata enhanced the cell apoptosis level on the larger 40×80×20 pillars. Moreover, the results demonstrated cytoskeletal stress induction in B4G12 cells in contact with S-guttata pillars, as evidenced by the increased alpha smooth muscle actin (α-SMA) and phosphorylated-myosin light chain 2 (pMLCK) expression levels, suggesting a potential mechanism underlying the observed apoptotic response. The RT-qPCR results revealed differential modulation of oxidative stress-related gene expression in B4G12 cells on S-guttata pillars, with upregulation of NQO1 and SOD2 on 40 μm pillars and downregulation of
SOD2 on 20×20×20 pillars. The second aim was to characterize and develop a method to measure the mechanical properties of human DM across distinct regions, including the central (CE), peripheral (PE), and transition zone (TZ). Variations in the thickness were observed, with TZ being the thickest region, followed by CE and PE. Furthermore, the stiffness analysis revealed that the TZ exhibited the lowest stiffness compared to the CE and PE regions. Our developed approach can be used to noninvasively measure DM thickness, and these findings will contribute to advancing our knowledge of corneal endothelial dystrophy and have the potential to improve current therapies, ultimately benefiting clinical outcomes and patient well-being.