Ahmetaj-Shala, Blerina (2013) The role of nitric oxide synthase inhibitors on macrophage function. (PhD thesis), Kingston University, .
Abstract
Asymmetrically methylated forms of arginine (asymmetric dimethylarginine, ADMA and L-N-monomethylarginine, L-NMMA) are competitive inhibitors of all three isoforms of nitric oxide synthase (NOS). These molecules are produced endogenously in all cells by a process that involves protein arginine methyltransferase (PRMT)-catalysed methylation of certain arginine residues in proteins and the subsequent proteolysis of these methylated proteins. Free methylarginines accumulate in the cytosol where they are actively metabolized to citrulline and methylamines by the enzyme dimethylarginine dimethylaminohydrolase (DDAH1 and 2). ADMA and L-NMMA have been shown to be elevated in patients with cardiovascular and renal disease, however, their role in immune cells and in particular macrophages is yet to be confirmed. The aim of this project was to determine the effect of increased ADMA, both endogenous and exogenous, on macrophage function. U937-derived macrophages (a human monocytic cell line) and primary peritoneal macrophages extracted from wild type (DDAH2+/+), global DDAH2 knock-out (DDAH2-/-) and macrophage-specific DDAH2 knock-out (DDAH2flox/flox LysM-Cre) mice were used. Their NO production, motility, phagocytosis, chemotaxis, and cytokine levels was determined using the Griess assay, real-time imaging, a fluorescently labelled E-coli phagocytosis assay, a transwell migration assay and RNA-sequencing respectively. Here we show that, in contrast to endothelial cells, DDAH2 is the only DDAH isoform expressed in primary murine macrophages basally or following cytokine stimulation of the cells. Results showed that DDAH2 metabolises methylarginines in macrophages through a VEGF-independent mechanism and significantly attenuates cytokine-stimulated NO synthesis. Both the pharmacological addition of ADMA and genetic deletion of DDAH2 in macrophages results in impaired macrophage function (as assessed by motility, phagocytosis and cytokine production). These data identify DDAH2 as a key regulator of macrophage NO synthesis and demonstrate the potential therapeutic utility of DDAH2-selective inhibitors.
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