Graduation Year

2025

Document Type

Thesis

Degree

M.S.

Degree Name

Master of Science (M.S.)

Degree Granting Department

Pharmacy

Major Professor

Manas R. Biswal, Ph.D.

Committee Member

Mark Kindy, Ph.D.

Committee Member

Christopher Passaglia, Ph.D.

Committee Member

Vijaykumar Sutariya, Ph.D.

Keywords

ARPE-19, Erythropoietin, Ferroptosis, Retinal Degeneration

Abstract

Age-Related Macular Degeneration (AMD) causes dysfunction and loss of Retinal pigment epithelium (RPE). The degeneration and death of RPE cells causes irreversible visual impairment. Oxidative stress is a widely accepted triggering factor for RPE dysfunction. A hormone called Erythropoietin (EPO) is responsible to facilitate the erythropoiesis process, prevents progenitor cells from apoptosis and protects photoreceptors downregulating retinal degeneration. In this study, we aim to elucidate the role of EPO in downregulating oxidative stress in RPE. It is hypothesized that EPO will have a positive impact on suppressing oxidative stress and ferroptosis-mediated RPE degeneration due to its vital role in apoptosis prevention. For our experiment, we have used a modified version of Erythropoietin, EPO-R76E which has been shown to have neuroprotective activity. For the delivery of EPO-R76E to the ARPE-19 cells, we cloned EPOR76E into lentiviral plasmid and prepared lentivirus to develop a stable ARPE cell lines for further experiments. Oxidative stress was introduced to the ARPE-19 cells using Paraquat. In EPO-R76E treated ARPE-19 cells with paraquat, we observed protection against ferroptosis and increased cell viability under cell toxicity assay. Western blot analysis revealed reduced levels of ferroptosis biomarkers ferritin, p62, and p-AMP and an improved GPX4 level in cells expressing EPO-R76E suggesting that EPO-R76E enhances the cellular antioxidant defense mechanism. The intracellular iron content was found lower in EPO-R76E expressing cells indicating a potential protective effect of EPO-R76E against ferroptosis and reduction of oxidative stress. This finding will be a significant groundbreaking approach to combat retinal degeneration in AMD.

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