Project Summary/Abstract One of the fundamental challenges facing cardiovascular research is the treatment of atherosclerosis, the single greatest cause of cardiovascular disease worldwide. Red meat consumption has long been linked to increased atherosclerosis incidence in humans, although not in other non-human mammals. A potential human-specific mechanism proposes that atherosclerosis is partly driven by an antibody-mediated reaction to the nonhuman sialic acid Neu5Gc, a molecule that is heavily enriched in red meat. Neu5Gc is structurally similar to the human sialic acid Neu5Ac, but humans cannot produce Neu5Gc due to an evolutionary loss of the CMAH enzyme gene. However, ingested Neu5Gc can be incorporated into the glycoconjugates of human cells, including endothelial cells. Cell culture and mouse models have shown that the presence of circulating anti-Neu5Gc antibodies leads to activation of endothelial inflammation pathways and increased atherosclerosis plaque size and necrotic core volume. Removal of Neu5Gc from endothelial glycoconjugates could therefore reduce activity of the inflammatory pathways that lead to atherosclerosis. This research seeks to use novel bacterial sialidases (enzymes that can cleave Neu5Gc) in a mouse model to reduce Neu5Gc uptake and incorporation into endothelial glycoconjugates, thereby mitigating atherosclerosis development. If successful, Neu5Gc sialidases would represent a new class of atherosclerosis treatment agents to pursue. Specifically, the aims of this proposal are to: (1) to enrich bacterial species producing Neu5Gc sialidases in gut microbiota, thereby reducing intestinal Neu5Gc uptake and preventing incorporation into endothelial glycoconjugates; and (2) to inject the sialidase Sia26 to therapeutically release Neu5Gc from endothelial glycoconjugates after it has been incorporated. A humanized (Cmah-/-) mouse model of atherosclerosis will be used to provide the physiological context critical for studying Neu5Gc-associated atherosclerosis risk. Together, these aims will address the effect of Neu5Gc sialidases on atherosclerosis through Neu5Gc cleavage in the gut and in circulation. The proposed studies will clarify the role of Neu5Gc as a human-specific trigger of atherosclerosis and investigate the potential of Neu5Gc sialidases as a novel treatment strategy.