UCLA

Steroid induced ocular hypertension is a serious adverse effect of prolonged steroid therapy in patients. If left untreated, steroid induced ocular hypertension may progress to steroid induced glaucoma, involving glaucomatous optic neuropathy and permanent vision loss. Chronic steroid administration elevates intraocular pressure in approximately 40% of the general population. If this increase is of sufficient magnitude and duration, steroid induced ocular hypertension may develop. This condition is associated with the abnormal contractility of the cells residing within the trabecular meshwork, the tissue responsible for intraocular pressure regulation. Numerous studies have provided insight into the molecular mechanism changes that occur in the trabecular meshwork as a result of prolonged steroid exposure. These changes include but are not limited to alterations in extracellular matrix metabolism, cytoskeletal organization, and gene expression. However, the specific functional changes occurring at the cellular level as a result of these molecular alterations, which contribute to trabecular meshwork dysfunction and ultimately ocular hypertension, remain poorly characterized. To address the need for a comprehensive evaluation of contractile cell function, we have adapted and optimized an assay, hereinafter referred to as fluorescently labeled elastomeric contractible surfaces (FLECS), to assess the functional effects of steroids on human trabecular meshwork cells in vitro. The FLECS assay provides a flexible tool for obtaining contractility measurements of thousands of cells at the single-cell level on substrates with tunable stiffness. In this thesis, I present, for the first time, single-cell-level contractile variations in human trabecular meshwork cells in primary culture and in an in vitro model of chronic steroid exposure. Furthermore, I report alterations in single-cell contraction kinetics associated with modifications in extracellular matrix substrates and stiffness, as well as the addition of a ROCK inhibitor. These findings provide compelling supportive evidence for a model in which increased contractility, accompanied by changes in extracellular matrix components, cytoskeletal organization, and stiffness, may contribute to elevated intraocular pressure and, consequently, ocular hypertension.