Year of Publication

2008

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Graduate School

Department

Toxicology

First Advisor

Dr. Bernhard Hennig

Abstract

Atherosclerosis is a chronic inflammatory disease thought to be initiated by endothelial cell dysfunction. Research described in this dissertation is focused on the role of zinc deficiency in endothelial cell activation with an emphasis on the function of the transcription factors nuclear factor-κB (NF-κB), peroxisome proliferator activated receptor (PPAR), and the aryl hydrocarbon receptor (AhR), which all play critical roles in the early pathology of atherosclerosis. Cultured porcine aortic vascular endothelial cells were deprived of zinc by the zinc chelator TPEN and/or treated with the NF-κB inhibitor CAPE or the PPARγ agonist rosiglitazone, followed by measurements of PPARα expression, cellular oxidative stress, NF-κB and PPAR DNA binding, COX-2 and E-selectin expression, and monocyte adhesion. Cellular labile zinc deficiency increased oxidative stress and NF-κB DNA binding activity, and induced COX-2 and Eselectin gene expression, as well as monocyte adhesion in endothelial cells. CAPE significantly reduced the zinc deficiency-induced COX-2 expression, suggesting regulation through NF-κB signaling. PPAR can inhibit NF-κB signaling. Zinc deficiency down-regulated PPARα expression and PPAR DNA binding activity in endothelial cells. Zinc deficiency compromised PPARγ transactivation activity in PPARγ and PPRE co-transfected rat aortic vascular smooth muscle cells. Furthermore, rosiglitazone was unable to inhibit the adhesion of monocytes to endothelial cells during zinc deficiency. Most of these effects of zinc deficiency could be reversed by zinc supplementation. An in vivo study utilizing the atherogenic LDL-R-/- mouse model generally supported the importance of PPAR dysregulation during zinc deficiency. LDLR-/- mice were maintained for four weeks on either zinc deficient or zinc adequate diets. Half of the mice within each zinc group were gavaged daily with rosiglitazone during the last stage of the study. Selected inflammation and lipid parameters were measured. The anti-inflammatory properties of rosiglitazone were compromised during zinc deficiency. Specifically, rosiglitazone induced inflammatory genes (MCP-1) in abdominal aorta only during zinc deficiency, and adequate zinc was required for rosiglitazone to down-regulate pro-inflammatory markers such as iNOS in abdominal aorta of the mice. Rosiglitazone significantly up-regulated liver IκBα protein expression only in zinc adequate mice.

Plasma data also suggest an overall pro-inflammatory environment during zinc deficiency and support the concept that zinc is required for proper anti-inflammatory or protective functions of PPAR. Zinc deficiency also altered PPAR-regulated lipid metabolism in LDL-R-/- mice. Specifically, zinc deficiency increased plasma total cholesterol, and non- HDL (VLDL, IDL and LDL)-cholesterol. Plasma total fatty acids tended to be increased during zinc deficiency, and rosiglitazone treatment resulted in similar changes in fatty acid profile in zinc deficient mice. FAT/CD36 expression in abdominal aorta was upregulated by rosiglitazone only in zinc-deficient mice. In contrast, rosiglitazone treatment markedly increased LPL expression only in zinc-adequate mice. These data suggest that in this atherogenic mouse model treated with rosiglitazone, lipid metabolism can be compromised during zinc deficiency. AhR is another transcription factor involved in the development and homeostasis of the cardiovascular system. Cultured porcine aortic endothelial cells were exposed to the AhR ligands PCB77 or beta-naphthoflavone (β-NF) alone or in combination with the zinc chelator TPEN, followed by measurements of the AhR responsive cytochrome P450 enzymes CYP1A1 and 1B1. Zinc deficiency significantly reduced PCB77- induced CYP1A1 activity and mRNA expression, as well as PCB77 or β-NF-induced CYP1A1 protein expression, which could be restored by zinc supplementation. These data suggest that adequate zinc is required for the activation of the AhR-CYP1A1 pathway. Impairment of the AhR pathway presents an additional mechanism by which zinc deficiency negatively affects transcription factor function and homeostasis of the vascular system. Taken together, zinc nutrition can markedly modulate the pathogenesis of inflammatory diseases such as atherosclerosis.

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