Abstract 208: Isolevuglandin, a Highly Reactive γ-ketoaldehyde Formed from the Isoprostane Pathway, Induces Deleterious Structural and Functional Consequences to High-density Lipoprotein

Abstract

Recent evidence suggest that cardiovascular disease (CVD) risk depends on levels of functional HDL particles, not HDL-cholesterol. In CVD, increased oxidative stress generates reactive lipid species that alter HDL function. Isolevuglandins (isoLGs), generated in parallel to isoprostanes, are extremely reactive to lysine residues of proteins and headgroups of phosphatidylethanolamine (PE). Importantly, IsoLG protein and PE adducts are elevated in atherosclerosis. Recently, our group observed a 42% reduction of atherosclerotic lesion size when salicylamine (SAM), a small molecule scavenger of reactive dicarbonyls including IsoLG, was administered to LDLr -/- mice. Little is known about the consequences of IsoLG to HDL function. The aim of this study is to compare effects of IsoLG on apolipoprotein crosslinking, morphology and size of HDL to its functions: cholesterol efflux, apoA-I exchange and anti-inflammation. Human HDL was incubated overnight at 37°C with IsoLG. Thioglycolate-induced intraperitoneal macrophages were harvested from apoE -/- mice. IsoLG crosslinked structural apolipoproteins, apoA-I and apoA-II, starting at 0.3 mol IsoLG per mol apoA-I (0.3 eq). HDL modified with 3 eq IsoLG formed subpopulations of two distinct sizes, 6-13 nm and 16-23 nm. A 40.6±0.04% decrease in 3 H-cholesterol efflux from macrophages was observed at 1 eq IsoLG compared to unmodified control HDL. At this IsoLG concentration, HDL-ApoA-I exchange was reduced (P<0.01, n=4), from 47.4±2.8% with control HDL to only 24.8±5.8%, suggesting that IsoLG inhibited apoA-I from disassociating from HDL to interact with ABCA1. Intriguingly, IsoLG inhibited HDL’s protection against LPS-stimulated inflammatory response in macrophages at 0.03 eq as shown by IL-1β and TNFα mRNA expression comparable to LPS alone. At 0.1 eq IsoLG, HDL becomes pro-inflammatory, as indicated by a 927±309% increase in IL-1β mRNA expression (P<0.001). Unlike cholesterol efflux, these effects occurred independent of HDL apolipoprotein crosslinking. We report a novel pathway by which HDL becomes dysfunctional, by mechanisms involving IsoLG-mediated alterations of HDL proteins and structure. Future studies will pinpoint how IsoLG modifies HDL proteins (or lipids) to alter its function.