Cholesterol Ester Metabolism Governs Intracellular Cholesterol Crystal Formation
| dc.contributor.advisor | Widenmaier, Scott B | |
| dc.contributor.committeeMember | Xiao, Changting | |
| dc.contributor.committeeMember | AbuArish, Asmahan | |
| dc.contributor.committeeMember | Fisher, Thomas | |
| dc.creator | Bairos, Jordan Anthony | |
| dc.creator.orcid | 0009-0003-2327-5808 | |
| dc.date.accessioned | 2023-10-03T16:58:35Z | |
| dc.date.available | 2023-10-03T16:58:35Z | |
| dc.date.copyright | 2023 | |
| dc.date.created | 2023-11 | |
| dc.date.issued | 2023-10-03 | |
| dc.date.submitted | November 2023 | |
| dc.date.updated | 2023-10-03T16:58:35Z | |
| dc.description.abstract | Free cholesterol (FC) accumulation underlies cholesterol crystallization, yet the mechanistic factors contributing to this process are unknown. The presence of cholesterol crystals (CC) in atherosclerotic lesions has been recognized to increase risk of thrombosis leading to major adverse cardiovascular events. Likewise, recent studies on patients with non-alcoholic steatohepatitis (NASH) indicate that CC in hepatocyte lipid droplets distinguish NASH from benign steatosis. In vitro investigations attempting to recapitulate the effects of CC on macrophages in the context of atherosclerosis demonstrate that CC activate inflammatory signaling and remodel macrophage metabolism. A wealth of literature has reported the production of intracellular CC by incubating macrophages, smooth muscle cells, and endothelial cells with modified lipoproteins. In addition, electron microscopy has provided evidence that CC nucleate intracellularly on macrophage lipid droplet surfaces. However, few studies have explored intracellular CC formation in hepatocytes, despite a strong link to NASH. Whether hepatocyte crystals actively contribute to liver disease pathogenesis and progression remains to be investigated. Due to the ability of lipid droplets to concentrate cholesterol in the form of esters, we reasoned cholesterol esterification, subsequent lipid droplet localization, and then de-esterification will concentrate FC at the lipid droplet surface to drive cholesterol nucleation and crystal formation. Hence, we hypothesized that enzymes that mediate cholesterol esterification and enzymes that de-esterify lipid droplet cholesterol ester (CE) are required for intracellular CC formation. To investigate this, human hepatocytes and mouse macrophages were loaded with cholesterol or acetylated low-density lipoprotein (acLDL), respectively, and were imaged under polarized light to detect CC. Crystals formed reliably in a time- and dose-dependent manner in both cell types. Fluorescence imaging and polarized light microscopy reveal that these crystals are associated with lipid droplets, but not lysosomes. Inhibiting acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) using pharmacologic and genetic methods diminished cholesterol crystallization and CE production. Remarkably, nonselective ACAT inhibition prevented plate-like CC cleft formation in hepatocytes. Also, blocking the de-esterification of lipid droplet CE with neutral CE hydrolase 1 (nCEH1) inhibitor JW480 blunted CC formation, but was more effective in hepatocytes than in macrophages. Collectively, we identify ACAT1 and nCEH1 as critical factors that function in series to drive intracellular CC formation. Our results provide needed mechanistic insight into cholesterol crystallization and highlight potential targets to prevent CC formation in human disease. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | https://hdl.handle.net/10388/15094 | |
| dc.language.iso | en | |
| dc.subject | Cholesterol | |
| dc.subject | Atherosclerosis | |
| dc.subject | Fatty liver disease | |
| dc.subject | Lipid droplet | |
| dc.subject | Hepatocyte | |
| dc.subject | Metabolism | |
| dc.title | Cholesterol Ester Metabolism Governs Intracellular Cholesterol Crystal Formation | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.department | Anatomy, Physiology, and Pharmacology | |
| thesis.degree.discipline | Anatomy, Physiology, and Pharmacology | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.Sc.) |