Date of Award

Fall 2023

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Don M Wojchowski

Second Advisor

Rick H Cote

Third Advisor

Xuanmao Chen

Abstract

ABSTRACTELUCUDATING MECHANISMS OF ACTION OF EICOSAPENTAENOIC ACID IN MODELS OF ATHEROSCLEROTIC CARDIOVASCULAR DISEASE by Samuel C.R. Sherratt, B.S. University of New Hampshire Omega-3 fatty acids (O3FAs) possess beneficial properties for cardiovascular (CV) health and elevated O3FA levels are associated with lower incident risk for CV disease (CVD). Yet, treatment of at-risk patients with various O3FA formulations has produced disparate results in large, well-controlled and well-conducted clinical trials. The formulation of the O3FAs used, specifically monotherapy of eicosapentaenoic acid (EPA) or mixtures of EPA and docosahexaenoic acid (DHA), appears to dictate clinical efficacy, with benefits only observed with EPA monotherapy. The mechanism of action of EPA remains poorly understood, but may involve its effects on membrane stability, fatty acid and protein expression, and endothelial function. I set out to characterize novel mechanisms of action of EPA that may explain its clinical benefits. Small-angle X-ray scattering (SAXS) analysis was used to compare the separate and combined effects of phospholipid-linked EPA (PL-EPA) and PL-DHA on electron density distribution in model membranes. PL-EPA adopted a stable, extended conformation in membranes that was dependent on the surrounding bulk lipid environment, while PL-DHA rapidly isomerizes resulting in increased membrane fluidity. The stabilizing effects of PL-EPA were attenuated when combined with PL-DHA. I then analyzed the effects of EPA on endothelial cell (EC) function, fatty acid expression, and global protein expression. Human umbilical vein EC (HUVEC) and pulmonary EC were used as the model systems, EC function was assessed using tandem porphyrinic nanosensors following calcium stimulation, global fatty acid expression was analyzed using gas chromatography with flame ionization detection (GC-FID), and global protein expression was measured using liquid chromatograph/mass spectrometry (LC/MS)-based proteomics. Under normal conditions, EPA significantly improved eNOS coupling efficiency in HUVECs compared to other fatty acids, including DHA and arachidonic acid (AA). This correlated with large, significant increases in the EPA/AA ratio with EPA treatment. In pulmonary ECs, inflammation was triggered using multiple air pollution particulate matter (PM) isolates of varying chemical composition and particle diameter size. EPA reversed air pollution PM-induced eNOS uncoupling, decreased inflammatory adhesion molecule expression, and modulated inflammatory biological pathways. EPA also increased expression of the cytoprotective protein heme oxygenase-1. Finally, I compared the effects of EPA, DHA, and different placebo oils (mineral and corn oil) on lipoprotein and model membrane oxidation, a study spurred by speculation that differences in placebo oil choice in large clinical trials were responsible for divergent outcomes due to pro-atherogenic effects of mineral oil. Small dense low-density lipoprotein (sdLDL) and very low-density lipoprotein (VLDL) were isolated from healthy volunteers and subjected to copper induced oxidation in the absence or presence of equimolar EPA, DHA, mineral oil, or corn oil. Model membranes were prepared as binary mixtures of POPC and cholesterol at a 0.6 cholesterol-to-phospholipid (C/P) mole ratio in the absence or presence of each agent and exposed to atmospheric conditions. EPA significantly inhibited the rate of oxidation of sdLDL, VLDL, and membranes more than DHA, and neither placebo oil had any significant effect on lipoprotein or membrane oxidation. Together, these results may help elucidate various mechanisms of atheroprotection with EPA and explain the positive outcomes in clinical trials using EPA monotherapy.

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