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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 α (TNFα) and interleukin-1β (IL-1β) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ∼46.5 to ∼18.4% (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 injury due to elevated levels of reactive oxygen species is implicated in cardiovascular diseases, Alzheimer's disease, lung and liver diseases, and many cancers. Antioxidant therapies have generally been ineffective at treating these diseases, potentially due to ineffective doses but also due to interference with critical host defense and signaling processes. Therefore, alternative strategies to prevent oxidative injury are needed. Elevated levels of reactive oxygen species induce lipid peroxidation, generating reactive lipid dicarbonyls. These lipid oxidation products may be the most salient mediators of oxidative injury, as they cause cellular and organ dysfunction by adducting to proteins, lipids, and DNA. Small-molecule compounds have been developed in the past decade to selectively and effectively scavenge these reactive lipid dicarbonyls. This review outlines evidence supporting the role of lipid dicarbonyls in disease pathogenesis, as well as preclinical data supporting the efficacy of novel dicarbonyl scavengers in treating or preventing disease.

Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized lipids/lipoproteins, little is known about how kidney injury impacts the intestinal lymphatic network, or lipoproteins transported therein. To study this, we used puromycin aminoglycoside-treated rats and NEP25 transgenic mice to show that proteinuric injury expanded the intestinal lymphatic network, activated lymphatic endothelial cells and increased mesenteric lymph flow. The lymph was found to contain increased levels of cytokines, immune cells, and isolevuglandin (a highly reactive dicarbonyl) and to have a greater output of apolipoprotein AI. Plasma levels of cytokines and isolevuglandin were not changed. However, isolevuglandin was also increased in the ileum of proteinuric animals, and intestinal epithelial cells exposed to myeloperoxidase produced more isolevuglandin. Apolipoprotein AI modified by isolevuglandin directly increased lymphatic vessel contractions, activated lymphatic endothelial cells, and enhanced the secretion of the lymphangiogenic promoter vascular endothelial growth factor-C by macrophages. Inhibition of isolevuglandin synthesis by a carbonyl scavenger reduced intestinal isolevuglandin adduct level and lymphangiogenesis. Thus, our data reveal a novel mediator, isolevuglandin modified apolipoprotein AI, and uncover intestinal lymphatic network structure and activity as a new pathway in the crosstalk between kidney and intestine that may contribute to the adverse impact of kidney disease on other organs.

Gut microbes play vital roles in host health and disease. A number of commensal bacteria have been used as vectors for genetic engineering to create living therapeutics. This review highlights recent advances in engineering gut bacteria for the treatment of chronic diseases such as metabolic diseases, cancer, inflammatory bowel diseases, and autoimmune disorders. KEY POINTS: • Bacterial homing to tumors has been exploited to deliver therapeutics in mice models. • Engineered bacteria show promise in mouse models of metabolic diseases. • Few engineered bacterial treatments have advanced to clinical studies.

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 (n = 10, 233.4 ± 158.3 ng/mg) were found to be significantly higher than in healthy controls (n = 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 [(3)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.

We report a case of disappearing high-density lipoprotein (HDL) syndrome caused by oxidative modification of HDL and by autoantibodies against modified HDL, with subsequent diagnosis of myeloma. An elderly Caucasian man had normal lipid levels with HDL cholesterol (HDL-C) levels in the upper 70 mg/dL range from 1999 to 2003. In 2003, his HDL-C levels began to progressively fall, and by 2011, they were undetectable (5 mg/dL) when measured with a Beckman Synchron LX auto analyzer. Analyses of the plasma sample from 2011 using ultracentrifugation (Vertical Auto Profile), nuclear magnetic resonance, and Ace EXCEL auto analyzer have shown that HDL-C levels were easily detectable (47-54 mg/dL), although reduced compared with his pre-2003 values. Analyses of his plasma sample from 2011 also showed the presence of lipid-adducted apolipoprotein A1 (apoA1) and high titer of antibodies against the adducted apoA1. Interestingly, a negative correlation between HDL-C levels and the titer of antibodies against apoA1 adducts was found in the control cohort. Finally, we show that in the mouse system, an antibody against apoA1 increases the clearance of HDL from plasma. This case of smoldering myeloma preceded by acquired, severe HDL-C deficiency, likely because of oxidative modifications of the HDL protein leading to the formation of autoantibodies, interference with clinical measurement of HDL-C, and increased plasma clearance of HDL, adds to the list of diagnostic considerations for unexplained HDL-C decreases over time.

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 LPS-induced 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

Obesity increases the risk for cardiometabolic diseases. N-acyl phosphatidylethanolamines (NAPEs) are precursors of N-acylethanolamides, which are endogenous lipid satiety factors. Incorporating engineered bacteria expressing NAPEs into the gut microbiota retards development of diet induced obesity in wild-type mice. Because NAPEs can also exert anti-inflammatory effects, we hypothesized that administering NAPE-expressing bacteria to low-density lipoprotein receptor (Ldlr)−/− mice fed a Western diet would improve various indices of cardiometabolic disease manifested by these mice. NAPE-expressing E. coli Nissle 1917 (pNAPE-EcN), control Nissle 1917 (pEcN), or vehicle (veh) were given via drinking water to Ldlr−/− mice for 12 weeks. Compared to pEcN or veh treatment, pNAPE-EcN significantly reduced body weight and adiposity, hepatic triglycerides, fatty acid synthesis genes, and increased expression of fatty acid oxidation genes. pNAPE-EcN also significantly reduced markers for hepatic inflammation and early signs of fibrotic development. Serum cholesterol was reduced with pNAPE-EcN, but atherosclerotic lesion size showed only a non-significant trend for reduction. However, pNAPE-EcN treatment reduced lesion necrosis by 69% indicating an effect on preventing macrophage inflammatory death. Our results suggest that incorporation of NAPE expressing bacteria into the gut microbiota can potentially serve as an adjuvant therapy to retard development of cardiometabolic disease.

Isolevuglandins are 4-ketoaldehydes formed by peroxidation of arachidonic acid. Isolevuglandins react rapidly with primary amines including the lysyl residues of proteins to form irreversible covalent modifications. This review highlights evidence for the potential role of isolevuglandin modification in the disease processes, especially atherosclerosis, and some of the tools including small molecule dicarbonyl scavengers utilized to assess their contributions to disease.

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 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.

Products of lipid peroxidation include a number of reactive lipid aldehydes such as malondialdehyde, 4-hydroxy-nonenal, 4-oxo-nonenal, and isolevuglandins (IsoLGs). Although these all contribute to disease processes, the most reactive are the IsoLGs, which rapidly adduct to lysine and other cellular primary amines, leading to changes in protein function, cross-linking and immunogenicity. Their rapid reactivity means that only IsoLG adducts, and not the unreacted aldehyde, can be readily measured. This high reactivity also makes it challenging for standard cellular defense mechanisms such as aldehyde reductases and oxidases to dispose of them before they react with proteins and other cellular amines. This led us to seek small molecule primary amines that might trap and inactivate IsoLGs before they could modify cellular proteins or other endogenous cellular amines such as phosphatidylethanolamines to cause disease. Our studies identified 2-aminomethylphenols including 2-hydroxybenzylamine as IsoLG scavengers. Subsequent studies showed that they also trap other lipid dicarbonyls that react with primary amines such as 4-oxo-nonenal and malondialdehyde, but not hydroxyalkenals like 4-hydroxy-nonenal that preferentially react with soft nucleophiles. This review describes the use of these 2-aminomethylphenols as dicarbonyl scavengers to assess the contribution of IsoLGs and other amine-reactive lipid dicarbonyls to disease and as therapeutic agents.