Date of Award

1-1-2003

Embargo Period

1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Craig E. Crosson

Second Advisor

Rosalie K. Crouch

Third Advisor

Donald R. Menick

Fourth Advisor

N. K. Banik

Abstract

Introduction: Vision loss resulting from retinal ischemia is associated with several systemic and ocular diseases. In the retina, ischemia is thought to lead to neuronal cell death by initiating a series of events that include oxidative stress, glutamate excitotoxicity, and intracellular Ca2+ overload. Attempts to prevent ischemic retinal degeneration have included the use of NMDA receptor antagonists, Ca2+ channel blockers, and antioxidants. These treatments have had limited success due to incomplete retinal protection and development of adverse side-effects. This has caused researchers to look toward endogenous protective mechanisms, such as ischemic preconditioning (IPC) in order to identify new strategies to prevent or ameliorate ischemic retinal damage. The cellular mechanisms responsible for IPC are unclear. It has been demonstrated that up-regulation of the small heat-shock protein Hsp27 correlates with the protection seen with retinal IPC. The goal of this dissertation is to investigate the role of Hsp27 in IPC, and to explore how IPC transcriptionally up-regulates Hsp27. Methods: The role of Hsp27 in IPC will be addressed by over-expressing the rat isoform of Hsp27 (rHsp27) in a rat retinal ganglion cell line, and measuring cell viability in response to both oxygen- and glucose-deprivation and intracellular Ca2+ overload. Studies will also be undertaken to identify which cell death mechanisms Hsp27 may mediate. The transcriptional regulation of Hsp27 after IPC will be addressed by the cloning and characterization of the Hsp27 promoter in response to a hypoxic stimulus. Results: Results from these studies show that the over-expression of rHsp27 in the RGC-5 cell line is protective against both oxygen- and glucose-deprivation and intracellular Ca2+ overload. Intracellular Ca2+ overload leads to an increase in caspase-3 activity in the RGC-5 cells. Over-expression of Hsp27 blocks this activation, but blocking caspase-3 alone does not increase cell viability. Furthermore, the hsp27 gene appears to be regulated in response to a hypoxic stimulus by interaction with the HIF-1 transcription factor. Conclusions: These data suggest that, in the retina, the initial IPC event up-regulates Hsp27 by activating HIF-1. This up-regulation of Hsp27 is able to protect retinal cells from ischemic damage by mediating several apoptotic pathways in a caspase-dependent and caspase-independent manner.

Rights

All rights reserved. All rights reserved. Copyright is held by the author.

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