Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Neuroscience

Abstract

Primary open-angle glaucoma (POAG) is a progressive optic neuropathy

characterized by loss of peripheral vision secondarily associated with elevated intraocular pressure (IOP). Transforming growth factor (TGF)-β2 is markedly elevated in the aqueous humor (AH) of patients with POAG. It has been previously shown that TGF-β2 increases IOP, in part, by inducing expression and release of endothelin-1 (ET-1) and connective tissue growth factor (CTGF) within the trabecular meshwork (TM). ET-1 and CTGF may ultimately lead to increases in IOP by enhancing TM cell contractility and extracellular matrix (ECM) deposition. Here we confirm that TGF-β2 enhances mRNA content of ET-1, CTGF, and various ECM components in cultured human TM cells. In addition, we demonstrate a dose- and time-dependent decrease in the bradykinin B2 receptor expression in response to exogenous TGF-β2. This finding represents a novel mechanism by which TGF-β2 may elevate IOP through perturbation of the kallikrein- kinin system. Despite these advancements, the direct effect of elevated IOP on TM cell responsiveness remains unknown.

To accurately model and characterize the effect of elevated IOP on the TM in vitro, we designed and built a custom hydrostatic pressure chamber. Cultured human TM cells exposed to elevated hydrostatic pressure exhibit decreased viability, accumulation of F-actin, and selective changes in the expression of matricellular and structural ECM genes associated with the pathogenesis of POAG including TGF-β2, ET-1, and CTGF. Furthermore, secreted ET-1, in response to elevated hydrostatic pressure, acts in an autocrine manner through the ETB receptor to enhance TGF-β2 and ppET-1 mRNA, but not CTGF content. The mechanism(s) by which TM cells sense and respond to elevated hydrostatic pressure remain unknown, but findings presented here suggest that neither activation of mechanosensitive TRPV channels nor oxidative stress is involved. Finally, we demonstrate that a second form of mechanical stimuli, fluid sheer stress as a result of increased perfusion flow rate in the anterior segment perfusion assay, can also influence both CTGF and ppET-1 mRNA content in the TM in situ.

Taken together, these results support the hypothesis that pressure-dependent changes in TM cell gene expression represent a feed-forward mechanism that exacerbates TGF-β2 associated increases in TM cell contractility and altered ECM synthesis and deposition in affected POAG patients.

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