In vivo angiogenic gene regulation by inhalant nitrite
Tran, Doanh C
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Inhalant nitrites are volatile organic nitrites that were used for medical treatment of angina pectoris, but are now mainly drugs of abuse. Nitrite exposure has been shown to enhance tumor growth in mice and associated with an increased risk of Kaposi's sarcoma. In vitro studies have shown that nitric oxide (NO) can stimulate the expression of vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis. Because nitrites produce NO, we hypothesized that their toxicological effects might be partly mediated via regulation of angiogenic factors. In this thesis, we examined the effects of in vivo nitrite exposure on the expression of genes related to angiogenesis and tumor growth. We found that acute in vivo exposure to 1400 parts-per-million (ppm) of ISBN for 4 hours in C57BL/6 mice led to alteration of several cancer- and angiogenesis-related genes in the liver. In particular, VEGF mRNA and protein expression were significantly elevated following nitrite exposure. Interestingly, no significant effect on gene expression was observed in the lung. Follow-up studies revealed that a lower exposure concentration of 900 ppm ISBN also increased hepatic VEGF mRNA and protein expression, although to a smaller extent. When this inhalant exposure dose was delivered as 6 daily short exposures of 45 minutes each, a significantly greater increase in VEGF mRNA expression was observed. Examination of gene expression following this multiple exposure protocol revealed several angiogenesis related genes were significantly up-regulated in the liver, namely FGF1, FGF4 (fibroblast growth factor 1 and 4 respectively), HO-1 (heme oxygenase-1), Hspa4 (heat shock 70kDa protein 4), and PF4 (platelet factor 4). These results thus showed that in vivo nitrite exposure could affect the expression of genes involved in the regulation of angiogenic pathways, including the up-regulation of VEGF expression. Additional studies found that the majority of the nitrite-induced VEGF protein expression was found in the cytoplasm of hepatocytes and that eNOS absence did not significantly affect nitrite-induced VEGF regulation. Nitrite exposure also appeared to enhance the growth of implanted B16-F1 tumors. The work in this thesis demonstrated that in vivo nitrite exposure could affect the angiogenic pathways.