Thesis

Modulation of Cholesterol Homeostasis by Lipoprotein Lipase and ABCA1 in THP-1 Macrophages

Modulation of Cholesterol Homeostasis by Lipoprotein Lipase and ATP-Binding Cassette Transporter A1 in THP-1 Macrophages by Aidan Jhunghoon Minh Kim Master of Science in Biochemistry Proper regulation of cholesterol homeostasis is paramount to the overall health of the tissues within one’s body. Disruption of this regulation potentially leads to an excess buildup of lipids and cholesterol within the arteries which may lead to the formation of an atherosclerotic plaque due to macrophage involvement. An enzyme lipoprotein lipase (LPL) has been demonstrated to be proatherogenic within the atherosclerotic plaques by facilitating the cellular uptake of lipids in macrophages. Scavenger receptors also play a role as they facilitate cellular uptake of oxidized and modified lipids. ATP-binding cassette transporter A1 (ABCA1) is a cholesterol transporter and unidirectionally transports cholesterol and phospholipids out of the cell to cholesterol acceptor proteins and is crucial in preventing lipid accumulation in the macrophages. The objective of the first part of these studies was to investigate the sources of cellular cholesterol and ABCA1 mediated cholesterol efflux. The LPL gene in THP-1 macrophages was knocked down using short hairpin interfering RNA (shRNA) interference by transfecting them with a lentivirus carrying a plasmid complementary to the LPL gene. We then analyzed the gene expression levels of LPL, three different scavenger receptors (SR-AI, SR-BI, CD36), and ABCA1. Downregulation of LPL correlated with a decrease in two scavenger receptors expression levels and an increase in ABCA1 expression levels. We also demonstrated that the LPL KD macrophages exhibit lower LPL activity and a lower cholesterol content within them which suggests that the LPL enzyme may be regulating ABCA1 and scavenger receptor expression and protein levels through its lipolysis products. For the second part of our studies, we investigated if products of lipolysis such as fatty acids would decrease ABCA1 expression and protein levels and could modulate genes associated with cholesterol homeostasis. By analyzing the expression and protein levels of the scavenger receptors and ABCA1 on THP-1 WT macrophages incubated with fatty acids, we found that certain fatty acids may repress ABCA1 and SR-BI expression and protein levels. Cholesterol efflux studies showed increased ABCA1 mediated cholesterol efflux from THP-1 WT macrophages upon incubation with the fatty acids and demonstrates it is possible that lipolysis products may modulate cholesterol homeostasis.

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