Cardiovascular disease risk depends on high-density lipoprotein (HDL) function, not HDL-cholesterol. Isolevuglandins (IsoLGs) are lipid dicarbonyls that react with lysine residues of proteins and phosphatidylethanolamine. IsoLG adducts are elevated in atherosclerosis. The consequences of IsoLG modification of HDL have not been studied. We hypothesized that IsoLG modification of apoA-I deleteriously alters HDL function. We determined the effect of IsoLG on HDL structure-function and whether pentylpyridoxamine (PPM), a dicarbonyl scavenger, can preserve HDL function. IsoLG adducts in HDL derived from patients with familial hypercholesterolemia ( = 10, 233.4 ± 158.3 ng/mg) were found to be significantly higher than in healthy controls ( = 7, 90.1 ± 33.4 pg/mg protein). Further, HDL exposed to myeloperoxidase had elevated IsoLG-lysine adducts (5.7 ng/mg protein) compared with unexposed HDL (0.5 ng/mg protein). Preincubation with PPM reduced IsoLG-lysine adducts by 67%, whereas its inactive analogue pentylpyridoxine did not. The addition of IsoLG produced apoA-I and apoA-II cross-links beginning at 0.3 molar eq of IsoLG/mol of apoA-I (0.3 eq), whereas succinylaldehyde and 4-hydroxynonenal required 10 and 30 eq. IsoLG increased HDL size, generating a subpopulation of 16-23 nm. 1 eq of IsoLG decreased HDL-mediated [H]cholesterol efflux from macrophages via ABCA1, which corresponded to a decrease in HDL-apoA-I exchange from 47.4% to only 24.8%. This suggests that IsoLG inhibits apoA-I from disassociating from HDL to interact with ABCA1. The addition of 0.3 eq of IsoLG ablated HDL's ability to inhibit LPS-stimulated cytokine expression by macrophages and increased IL-1β expression by 3.5-fold. The structural-functional effects were partially rescued with PPM scavenging.

The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ؎ 33.8 pmol/ mg) than healthy controls (15.3 ؎ 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor ␣ (TNF␣) and interleukin-1␤ (IL-1␤) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ϳ46.5 to ϳ18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia. Oxidative stress and increased net production of free radicals represent an important pathogenic mechanism in atherosclerosis. Free radicals react with unsaturated fatty acids in a chain reaction to yield lipid peroxides and secondary aldehyde products. These lipid aldehydes are highly reactive and selectively modify proteins or lipids to cause cellular and tissue damage. An important role for reactive aldehydes in the pathogenesis of atherosclerosis is suggested by increased aldehyde-protein adducts in plasma (1, 2), aortic atherosclerotic lesions (3-6), and lipoproteins. Adduction to apolipoprotein B in low-density lipoprotein (LDL) 2 enhances their recognition and uptake by macrophages (7, 8) which converts macrophages to lipid-laden foam cells (9-11). HDL normally protects LDL from aldehyde adduction by serving as a "sink" for lipid peroxides and their reactive by-products (12), but when HDL becomes modified, it results in numerous dysfunctions of HDL (13, 14). HNE is one of the most investigated aldehydic end products of oxidative breakdown of membrane n-6 polyunsaturated fatty acids. Its adduction to LDL accelerates its uptake by macrophages (15), and its adduction to HDL causes crosslinks of apoA-I and inhibits its ability to activate lecithin-cholesterol acyltransferase (LCAT) (14). However, the role of it...

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.

The lipid aldehyde 4-oxo-2-nonenal (ONE) derived from peroxidation of n-6 polyunsaturated fatty acids and generated in parallel to 4-hydroxynonenal (HNE) is a highly reactive protein crosslinker. Crosslinking of proteins in high-density lipoprotein (HDL) by lipid peroxidation products causes HDL dysfunction and contributes to atherogenesis. While HNE is relatively well studied, the relevance of ONE in atherosclerosis and in modifying HDL has not been examined. In the present study, we found a significant increase in ONE-ketoamide (lysine) adducts in HDL derived from patients with familial hypercholesterolemia (FH) (1620 ± 985.4 pmol/mg) compared to healthy controls (664 ± 219.5 pmol/mg). ONE crosslinked apoA-I on HDL at a concentration of >3 mol ONE per 10 mol apoA-I (0.3 eq), which is 100-fold lower than HNE but comparable to the potent protein crosslinker, isolevuglandin. ONE-modified HDL partially inhibited the ability of HDL to protect against LPSinduced TNFα and IL-1β mRNA expression in murine macrophages. At 3 eq., ONE dramatically decreased the ability of apoA-I to exchange from HDL, from ~46.5% to only ~18.4% (P<0.001). Surprisingly, ONE-modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC/MS/MS analysis showed modification of Lys12, Lys23, Lys96, and Lys226 of apoA-I by ONEketoamide adducts. Compared to other dicarbonyl scavengers, pentylpyridoxamine (PPM) was most efficacious at blocking ONE-induced protein crosslinking in HDL. Our studies show that ONE HDL adducts are elevated in FH who have severe hypercholesterolemia and atherosclerosis and causes HDL dysfunction. We demonstrate the use of

Background: Lipid peroxidation products such as hydroxy-2-nonenal (HNE) are elevated in atherosclerosis and cause HDL dysfunction. Generated in parallel to HNE is 4-oxo-2-nonenal (ONE), a less studied lipid dicarbonyl possibly due to its far greater reactivity. The consequences of ONE modification of HDL have not been studied. We have recently found that scavengers of lipid dicarbonyls such as salicylamine (SAM) and pentylpyridoxamine (PPM) prevent HDL dysfunction induced by malondialdehyde and isolevuglandin (IsoLG). In this study, we examine the impact of ONE adduct formation on HDL structure-function, and the scavenging abilities of various small molecules in preventing ONE modification. Methods and Results: By Western blot analysis, ONE crosslinked apoA-I on HDL at a concentration of 3 ONE molecules for every 10 apoA-I proteins (0.3 molar equivalence, eq.), which is 100 fold lower than HNE but comparable to IsoLG. ONE-mediated crosslinking of HDL proteins preferentially produced a 39 kDa band on SDS-PAGE, likely an apoA-I/apoA-II heterodimer. ONE-modified HDL partially inhibited the ability of HDL to protect against the inflammatory response of macrophages (as shown in TNFα and IL-1β mRNA expression), but did not render HDL pro-inflammatory. At 3 eq., ONE dramatically decreased the ability of apoA-I to exchange among HDLs, from ~46.5% to only ~18.4% (P<0.001). HDL-mediated macrophage cholesterol efflux was decreased by ~70.6% (P<0.005) and 56.1% (P<0.001) by HDL modified by 0.3 and 3 eq. ONE, respectively. Investigation of various scavenger analogues in protecting HDL from ONE modification showed that while SAM and its fluoro- and chloro- analogues partially prevented ONE-mediated HDL protein crosslinking, PPM nearly completely blocked crosslinking. PPM pretreatment of HDL prior to 3 eq. ONE modification was also able to restore HDL-mediated macrophage cholesterol efflux, from 56.1~ to ~83.0% (P<0.01) while the inactive analogue pentylpyridoxine had no effect. Conclusions: Our study is the first to show that ONE causes HDL dysfunction, and demonstrates that not all modified HDLs result in the same “dysfunction”. We also demonstrate the use of PPM in preferentially scavenging ONE in biological systems.