Date of Award

4-3-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Edward Carlson

Abstract

Diabetes mellitus increases the risk for cardiovascular disease due, in part, to hyperglycemia-induced oxidative stress. To investigate the potential for antioxidant protection of the vasculature, a transgenic mouse (Jtmt) that overexpresses the antioxidant metallothionein (MT) specifically in endothelial cells (EC) was produced. A 16kb transgene consisting of the murine Tie2 promoter and enhancer ligated to the human MTII gene was microinjected into FVB mouse embryos. Two founder mice, Jtmt1 and Jtmt4, produced transgenic progeny identified by PCR using human MTII primers. At 60 days of age, there were no distinguishable differences in phenotype, behavior, body and organ weights, and glucose levels in transgenic animals. Importantly, immunocytological methods confirmed the MT overexpression was specific to the endothelium of both the micro- and macrovasculature of kidney, heart, brain, and tail tissues. A MT competitive ELISA assay determined the amount of overexpression in renal tissues to be three fold in the Jtmt4 line. In cardiac tissues, the Jtmt1 had double the amount of MT and Jtmt4 three times that of controls. Transgenic animals did not exhibit elevated levels of urinary albumin excretion suggesting that the function of the glomerular EC was not disrupted by MT overexpression. Transmission electron microscopic (TEM) stereometric techniques also demonstrated that the MT overexpression did not adversely affect renal glomerular EC or underlying glomerular basement membrane (GBM) structure. Future studies include crossing Jtmt and OVE26 transgenic diabetic mice in an effort to determine the potential benefits of endothelial-specific antioxidant protection of the diabetic vasculature.

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