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2017 Cardiovascular Research Day Abstract Book

49 Endocytosis Mediates

49 Endocytosis Mediates Platelets' Responses to Viruses In The Vasculature Meenakshi Banerjee, PhD 1 • Sidney Whiteheart, PhD 1 1Molecular and Cellular Biochemistry, University of Kentucky Postdoc Platelet endocytosis is essential for Fibrinogen (Fg) uptake, receptor trafficking of integrins (αIIbβ3, αvβ3) and other surface receptors. However, the mechanistic underpinnings of endocytosis, its importance in platelets, and the molecular machinery required and possible trafficking routes are understudied, in part due to a lack of viable experimental tools. Previously we showed the importance of ADP-ribosylation factor 6 (Arf6), which regulates αIIbβ3-mediated Fg uptake/storage and affects acute platelet functions e.g., clot retraction and spreading. To further identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing Soluble N-ethylmaleimide Factor Attachment Protein Receptor (v-SNARE) called Cellubrevin/Vesicle Associated Membrane Protein-3 (VAMP-3) in platelet function. VAMP-3 KO platelets have defective uptake and accumulation of Fg, which led to enhanced platelet spreading on Fg and faster clot retraction. Using both Arf6 KO and VAMP-3 KO mutants as tools to probe the importance of endocytosis in platelets, we posited that platelet endocytosis could potentially be critical for actively sensing pathogenic damage in the vascular microenvironments and allowing platelets to act as immune cells. Previous reports show that platelets do endocytose viruses such as HIV-1 but the molecular machinery is ill-defined. In nucleated cells, responses to HIV-1 are mediated by virus phagocytosis/endocytosis, degradation to release Toll-like Receptor ligands, and subsequent receptor activation. Is this process recapitulated in platelets? Here we show that platelets indeed use VAMP-3 and Arf6-dependent pathways to endocytose HIV-1 virions, degrade retroviral particles to release TLR ligands, which initiate platelet activation and secretion. Consequently, HIV-1 uptake and subsequent activation is abolished in VAMP-3 and Arf6 KO mice. Collectively, our studies shed light on how platelets act at the early stage of pathogen recognition and are able to process them to initiate an immune response. 65

50 Association between Plasma Cholesterol Levels and the Development of Xanthomas, Malaise, and Inappetance in Cynomlgus Monkeys Morgan Kelly 1 • Courtney Burkett 1 • Sierra Paxton 1 • Lei Cai 1 • Ryan Temel, PhD 1 1Cardiovascular Research Center, University of Kentucky Undergraduate Cardiovascular disease is the leading cause of death in the United States. There is a positive correlation between coronary heart disease (CHD) and LDL cholesterol (LDL-C) levels. Statins are used to lower LDL-C and CHD risk but do not completely eliminate CHD events caused by atherosclerotic lesion rupture. Therefore, finding a therapy capable of regressing or stabilizing atherosclerotic lesions is a priority for academic and pharmaceutical researchers. By stimulating macrophage cholesterol efflux and promoting anti-inflammatory macrophage polarization, antagonism of microRNA-33a (miR-33a) in mice causes aortic atherosclerotic lesions to acquire a more stable composition. However, the preclinical mouse studies have limited translational value since mice do not develop coronary artery atherosclerosis and express only one of the two miR-33 family members found in humans. In contrast, nonhuman primates (NHPs) have miR-33a and miR- 33b and develop coronary artery atherosclerosis and thus are a good model to determine the therapeutic potential of miR-33 antagonism in humans. In order to determine whether miR-33a/b antagonism can regress or stabilize atherosclerotic lesions, 36 male cynomolgus monkeys were fed for 20 months a diet high in fat and cholesterol, which increased LDL-C and drove atherosclerosis formation. During this period, the animals developed xanthomas and callosities due to cholesterol deposition in the skin. In addition, some of the monkeys went through prolonged periods of malaise and inappetance. The objectives of this project were to review the veterinary records and determine 1) the number of days on atherogenic diet needed to first observe xanthomas and callosities and 2) the number of incidences of malaise and prolonged inappetance per monkey. This data was then correlated to the average total plasma cholesterol (TPC) concentration during the 20 months on atherogenic diet. A statistically significant, negative correlation was observed between the number of days to initial callosity formation and TPC (p= 0.049 and = 0.19). There were no statistically significant correlations between TPC and the other clinical observations. We are in the process of data collection but hypothesize that coronary artery atherosclerotic lesion size will be greater in animals that went through periods of malaise and inappetance. In addition, we hypothesize that the days for xanthoma/callosity formation will be inversely associated with plaque size. This information may allow our lab to predict atherosclerosis severity in monkeys based upon clinical observations. 66

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