Date of Award

1-1-2016

Embargo Period

1-1-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Zsolt Ablonczy

Second Advisor

Craig E. Crosson

Third Advisor

Donald Menick

Fourth Advisor

Lauren Ball

Fifth Advisor

Steven Rosenzweig

Abstract

Diabetic retinopathy is the foremost cause of blindness of working-age adults in the developed world. Diabetic macular edema (DME), accumulation of fluid within the retina, is the most common blindness-causing lesion in this population. Edema arises with dysfunction of the dual blood-retina barriers (BRB). The outer BRB, comprised of tight junctions between retinal pigment epithelium (RPE) cells plays a significant role in fluid homeostasis in the retina but is less well-studied than the inner BRB. Recent work in the RPE shows advanced glycation end-products (AGEs), which accumulate due to elevated glucose levels, induce barrier dysfunction in a vascular endothelial growth factor (VEGF) dependent manner. Anti-VEGF pharmacotherapeutics are currently the mainstay of treatment; however, the cost and risks of this therapy leave room for improvement in treatment of DME. Acetylation is a post-translational modification regulating the actions of many proteins. Histone deacetylases (HDACs), which remove this modification, are altered in diabetes prompting study into HDAC contribution to diabetic sequelae. In this body of work, I tested the hypothesis that retinal pigment epithelium protein acetylation plays a central role in the development of diabetic macular edema. The first specific aim tested the ability of HDAC inhibition to reverse the acute AGE effects. Here, the RPE exposure to glycated-albumin (gAlb) was measured by transepithelial electrical resistance (TEER) in vitro. Subretinal bleb resorption in rabbit eyes confirmed that gAlb acutely disrupted RPE barrier function. Additionally, Trichostatin A (TSA), a broad spectrum HDAC inhibitor, prevented barrier breakdown with pretreatment in vitro and cotreatment in vivo. Additional in vitro studies indicated significant decreases in acetyl-α-tubulin with gAlb treatment were reversed with TSA. HDAC6 activity increased with gAlb exposure, and loss of TEER was blocked with the HDAC6 inhibitor Tubastatin A. The second specific aim established a streptozotocin-induced hyperglycemic rat model measuring RPE health via subretinal bleb resorption. RPE dysfunction occurred independently of vascular leakage. A four-day treatment at 8.5 weeks with systemic TSA was able to reverse RPE dysfunction. Taken together, my data show that HDAC inhibition preserves RPE integrity in response to AGE and hyperglycemia, making it an alternative approach for treatment of DME.

Rights

All rights reserved. Copyright is held by the author.

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