Elucidating the Role of Endothelial NAD Metabolism in Atherosclerosis

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Project Summary Atherosclerosis remains a leading cause of death in the United States, even in the context of aggressive clinical and public health efforts to lower risk factors such as hyperlipidemia. Thus, there is a significant need for better mechanistic understanding of atherosclerosis pathogenesis in order to develop novel therapeutic strategies downstream of circulating risk factors. Atherosclerosis pathogenesis begins in the endothelial layer of the arterial wall: endothelial cell dysfunction (ECD) and EC inflammation precede atherosclerotic plaque formation and are associated with adverse cardiovascular events. Both ECD and EC inflammation increase with age, and age is a major non-modifiable risk factor for atherosclerosis. Importantly, activation of sirtuin (SIRT) family of nicotinamide adenine dinucleotide (NAD+) - dependent deacetylases, particularly SIRT1, has been reported to slow a variety of age-associated phenotypes. In particular, our lab and others have shown that levels of endothelial NAD+ decrease with age, and that endothelial-specific overexpression of SIRT1 is sufficient to slow ECD and progression of atherosclerosis. Furthermore, oral administration of NAD+ precursors in aged mice increases aortic SIRT1 expression and activity, and reduces ECD, suggesting that increasing NAD+ availability can promote SIRT1 activity in aged ECs. Loss of NAD+ balance thus appears to be central to atherogenesis, but little is known about how ECs maintain NAD homeostasis. Quantitative flux analysis of NAD+ precursors in live mice has recently uncovered that liver-derived nicotinamide (NAM) is the main circulating NAD+ precursor that reaches tissues. This suggests that the enzyme NAM phosphoribosyltransferase (NAMPT), which catalyzes the first and rate-limiting step in NAD+ synthesis from NAM, is critical for producing endothelial NAD+ and maintaining the activity of NAD+ - dependent enzymes. Thus, I hypothesize that endothelial NAMPT is cell-autonomously required to produce NAD+ and maintain endothelial SIRT1 activity, and that endothelial NAMPT overexpression will protect against progression of atherosclerosis. To test this hypothesis, we have generated endothelial-specific gain-of-function (GOF) and loss-of-function (LOF) NAMPT mouse models. In Aim 1, I will use cultured primary arterial ECs and stable isotope carbon tracing coupled with quantitative flux analysis to calculate rates of NAMPT NAD+ production and SIRT1 NAD+ consumption. I will also determine if NAMPT activity is required for SIRT1 deacetylation activity, and if NAMPT activation is sufficient to maintain SIRT1 activity upon oxidative stress. In Aim 2, I will test if NAMPT GOF or oral NAD+ supplementation is sufficient to slow progression of atherosclerosis, and if NAMPT LOF accelerates atherosclerosis. Ultimately, this work will improve understanding of EC NAD+ metabolism and its role in maintaining SIRT1 activity, and inform if NAMPT activation and/or NAD+ replenishment could be a therapeutic avenue for reducing the burden of atherosclerosis independently of circulating risk factors such as hyperlipidemia.