(Major Center-Driven Research Project) UC Davis: S Fore, T Huser, L Higgins, J Connolly-Rohrbach,J Rutledge; LLNL: J Chan  Atherosclerotic cardiovascular disease is the single largest cause of morbidity and mortality in the U.S. Triglyceride-rich lipoproteins, in particular very low density lipoproteins (VLDL) that facilitate interactions with cells and the uptake of fats by vascular cells are believed to be central to the initial stages of atherosclerosis. The mechanisms by which VLDL cause atherosclerosis, however, remain unclear. Our lack of understanding of the mechanisms of triglyceride-induced artery disease severely limits our ability to attack this very important health care problem and develop therapeutic interventions to attenuate and prevent arterial disease. This project aims at providing this critical information through a careful study of VLDL interaction with vascular cells, i.e. endothelial cells and monocytes. The general goal of this project is to better characterize these interactions and develop new biomarkers for identifying individuals predisposed to atherosclerosis. Specifically we are testing the hypotheses related to the study of lipid-monocyte interactions and to biochemcially examine lipoproteins (TGRL) and their lipolysis products from human subjects using Laser Trapping Raman Spectroscopy (LTRS) and coherent anti-Stokes Raman scattering (CARS) imaging. Previous studies suggest that TGRL and their lipolysis products injure endothelial cells, monocytes and platelets, yielding a proinflammatory state. Cells and TGRL can be probed in a more native environment, using a method that is label-free, non-invasive, and non-destructive. Our group was the first to analyze single lipoproteins by spontaneous Raman spectroscopy. This is a significant achievement because of the relatively low sensitivity of spontaneous Raman spectroscopy, which typically requires micromolar concentrations in the focus volume of the laser beam to obtain spectra with high signal-to-noise ratio. The analysis of saturated fatty acid content in VLDLs by Raman spectroscopy provides a direct, nondestructive means of analyzing VLDL biochemical composition, and their distribution for large numbers of VLDL. This is information that cannot be obtained by any other means. Numerous studies have been performed this past year including 1) effects of meals on TGRL composition and structure, 2) Raman spectroscopy to characterize monocytes, effects of lipolysis products on THP-1 cultured monocytes, 3) studies of the role of fatty acids in lipid droplet formation in cells, and 4) dynamic imaging studies of lipid body formation. Using laser trapping Raman spectroscopy and CARS imaging, we have developed new approaches to analyze single lipoproteins and single monocytes and their interactions. These new approaches will enable a unique, translational view of vascular biology. Future studies will aim to identify the mechanisms responsible for the lipid body formation and to investigate possible fatty acid uptake in monocytes and other cells. We plan to continue to characterize the interactions of lipoproteins with vascular cells, such as endothelial cells, monocytes, but also stem cells by CARS and fluorescence microscopy. Understanding how lipoproteins modulate cellular properties, such as membrane fluidity, cell signaling, and cell-cell interactions will enable us to suggest solutions for novel interventions at this fundamental level. |
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