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AimsTrimethylamine N-oxide (TMAO), choline and betaine serum levels have been associated with metabolic diseases including type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). These associations could be mediated by insulin resistance. However, the relationships among these metabolites, insulin resistance and NAFLD have not been thoroughly investigated. Moreover, it has recently been suggested that TMAO could play a role in NAFLD by altering bile acid metabolism. We examined the association between circulating TMAO, choline and betaine levels and NAFLD in obese subjects.MethodsSerum TMAO, choline, betaine and bile acid levels were measured in 357 Mexican obese patients with different grades of NAFLD as determined by liver histology. Associations of NAFLD with TMAO, choline and betaine levels were tested. Moreover, association of TMAO levels with non-alcoholic steatohepatitis (NASH) was tested separately in patients with and without T2D.ResultsTMAO and choline levels were significantly associated with NAFLD histologic features and NASH risk. While increased serum TMAO levels were significantly associated with NASH in patients with T2D, in non-T2D subjects this association lost significance after adjusting for sex, BMI and HOMA2-IR. Moreover, circulating secondary bile acids were associated both with increased TMAO levels and NASH.ConclusionsIn obese patients, circulating TMAO levels were associated with NASH mainly in the presence of T2D. Functional studies are required to evaluate the role of insulin resistance and T2D in this association, both highly prevalent in NASH patients.

Homocysteine (Hcy)-thiolactone is toxic, induces epileptic seizures in rodents, and has been implicated in Alzheimer's disease. Paraoxonase 1 (Pon1), a component of high-density lipoprotein, hydrolyzes Hcy-thiolactone in vitro. Whether this reflects a physiological function and whether Pon1 can protect against Hcy-thiolactone toxicity was unknown. Here we show that Hcy-thiolactone was elevated in brains of Pon1-/- mice (1.5-fold, p = 0.047) and that Pon1-/- mice excrete more Hcy-thiolactone than wild type animals (2.4-fold, p = 0.047). The frequency of seizures induced by intraperitoneal injections of L-Hcy-thiolactone was significantly higher in Pon1-/- mice compared with wild type animals (52.8% versus 29.5%, p = 0.042); the latency of seizures was lower in Pon1-/- mice than in wild type animals (31.8 min versus 41.2 min, p = 0.019). Using the Pon1 null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself is neurotoxic in vivo. Our findings indicate that Pon1 protects mice against Hcy-thiolactone neurotoxicity by hydrolyzing it in the brain, and suggest a mechanism by which Pon1 can protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease. [ABSTRACT FROM AUTHOR]

We recently showed that NOTUM, a liver-secreted Wnt inhibitor, can acutely promote browning of white adipose. We now report studies of chronic overexpression of NOTUM in liver indicating that it protects against diet-induced obesity and improves glucose homeostasis in mice. Adeno-associated virus (AAV) vectors were used to overexpress GFP or mouse Notum in the livers of male C57BL/6J mice and the mice were fed an obesifying diet. After 14 weeks of high fat, high sucrose diet feeding, the AAV-Notum mice exhibited decreased obesity and improved glucose tolerance compared to the AAV-GFP mice. Gene expression and immunoblotting analysis of the inguinal fat and brown fat revealed increased expression of beige/brown adipocyte markers in the AAV-Notum group, suggesting enhanced thermogenic capacity by NOTUM. A β3 adrenergic receptor agonist-stimulated lipolysis test suggested increased lipolysis capacity by NOTUM. The levels of collagen and C-C motif chemokine ligand 2 (CCL2) in the epididymal white adipose tissue of the AAV-Notum mice were significantly reduced, suggesting decreased fibrosis and inflammation, respectively. RNA sequencing analysis of inguinal white adipose of 4-week chow diet-fed mice revealed a highly significant enrichment of extracellular matrix (ECM) functional cluster among the down-regulated genes in the AAV-Notum group, suggesting a potential mechanism contributing to improved glucose homeostasis. Our in vitro studies demonstrated that recombinant human NOTUM protein blocked the inhibitory effects of WNT3A on brown adipocyte differentiation. Furthermore, NOTUM attenuated WNT3A's effects on upregulation of TGF-β signaling and its downstream targets. Overall, our data suggest that NOTUM modulates adipose tissue function by promoting thermogenic capacity and inhibiting fibrosis through inhibition of Wnt signaling., (© 2021. The Author(s).)

This study demonstrates, for the first time, that renal tubular excretion of trimethylamine N -oxide (TMAO) is inhibited by concomitant loop diuretic administration. The observed marked accumulation in the renal parenchyma, and to lesser extent, plasma, implies differential distributions of TMAO across various tissues and/or systems as a consequence of efflux channel control. A better understanding of TMAO renal clearance and its potential interactions with current and future therapies in patients with heart failure are warranted., Competing Interests: Dr. Li was supported as a 2016-2017 Fellow of the Sarnoff Cardiovascular Research Foundation. Drs. Hazen and Tang were supported by grants from the National Institutes of Health (NIH) and the Office of Dietary Supplements (R01DK106000, R01HL103866, and R01HL126827). Dr. Hazen was supported by a grant from the National Institutes of Health (1P01HL-147823). Dr. Wang was supported by a grant from the National Institutes of Health (R01HL130819). Dr. Lusis was supported by a grant from the National Institutes of Health (R01HL144651). Drs. Hazen and Lusis were supported in part by grants from the Leducq Fondation (17CVD01). Drs. Wang and Hazen are co-inventors on pending and issued patents held by the Cleveland Clinic related to cardiovascular diagnostics and therapeutics, and have received royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland Heart Lab, a fully owned subsidiary of Quest Diagnostics, and Procter and Gamble. Dr. Hazen has been a paid consultant for Procter and Gamble; and has received research funding from Pfizer Inc., and Roche Diagnostics. Dr. Tang has been a consultant for Sequana Medical Inc and Owkin Inc., unrelated to the contents of this paper. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2021 The Authors.)

Patients with chronic kidney disease (CKD) have elevated circulating levels of trimethylamine N-oxide (TMAO), a metabolite derived from gut microbes and associated with cardiovascular diseases. High circulating levels of TMAO and its dietary precursor, choline, predict increased risk for development of CKD in apparently healthy subjects, and studies in mice fed TMAO or choline suggest that TMAO can contribute to kidney impairment and renal fibrosis. Here we examined the interactions between TMAO, kidney disease, and cardiovascular disease in mouse models. We observed that while female hyperlipidemic apoE KO mice fed a 0.2% adenine diet for 14 weeks developed CKD with elevated plasma levels of TMAO, provision of a non-lethal inhibitor of gut microbial trimethylamine (TMA) production, iodomethylcholine (IMC), significantly reduced multiple markers of renal injury (plasma creatinine, cystatin C, FGF23, and TMAO), reduced histopathologic evidence of fibrosis, and markedly attenuated development of microalbuminuria. In addition, while the adenine-induced CKD model significantly increased heart weight, a surrogate marker for myocardial hypertrophy, this was largely prevented by IMC supplementation. Surprisingly, adenine feeding did not increase atherosclerosis and significantly decreased the expression of inflammatory genes in the aorta compared to the control groups, effects unrelated to TMAO levels. Our data demonstrate that inhibition of TMAO production attenuated CKD development and cardiac hypertrophy in mice, suggesting that TMAO reduction may be a novel strategy in treating CKD and its cardiovascular disease complications.

Paraoxonase 1(PON1) is an HDL-associated protein, which metabolizes inflammatory, oxidized lipids associated with atherosclerotic plaque development. Because oxidized lipid mediators have also been implicated in the pathogenesis of rheumatoid arthritis (RA), we evaluated the role of PON1 in murine inflammatory arthritis. K/BxN serum transfer (STIA) or collagen antibody transfer (CAIA) was used for arthritis induction in B6 mice homozygous for the PON1 human transgene [PON1Tg], PON1 knock-out mice [PON1KO], and wild type littermate control mice [WT]. Experiments were also performed in K/BxN mice with chronic arthritis, and in RA patients and healthy controls. Arthritis activity in K/BxN mice was associated with a marked dyslipidemia, lower PON1 activity and higher bioactive lipid mediators (BLM), as well as a dysregulated hepatic lipid gene expression profile. Higher serum PON1 activity correlated with lower BLM and lower arthritis activity in both K/BxN mice and RA patients. Overexpression of the human PON1 transgene was associated with reduced inflammatory arthritis, which correlated strongly with higher circulating PON1 activity, upregulation of the hepatic glutathione pathway, and reduction of circulating BLM. These results implicate PON1 as a potential novel therapeutic target for joint disease in RA with potential for vascular benefit, which warrants further investigation.

Objective: Air pollution is associated with increased cardiovascular morbidity and mortality, as well as dyslipidemia and metabolic syndrome. Our goal was to dissect the mechanisms involved. Approach and Results: We assessed the effects of exposure to air pollution on lipid metabolism in mice through assessment of plasma lipids and lipoproteins, oxidized fatty acids 9-HODE (9-hydroxyoctadecadienoic) and 13-HODE (13-hydroxyoctadecadienoic), lipid, and carbohydrate metabolism. Findings were corroborated, and mechanisms were further assessed in HepG2 hepatocytes in culture. ApoE knockout mice exposed to inhaled diesel exhaust (DE, 6 h/d, 5 days/wk for 16 weeks) exhibited elevated plasma cholesterol and triglyceride levels, increased hepatic triglyceride content, and higher hepatic levels of 9-HODE and 13-HODE, as compared to control mice exposed to filtered air. A direct effect of DE exposure on hepatocytes was demonstrated by treatment of HepG2 cells with a methanol extract of DE particles followed by loading with oleic acid. As observed in vivo, this led to increased triglyceride content and significant downregulation of ACAD9 mRNA expression. Treatment of HepG2 cells with DE particles and oleic acid did not alter de novo lipogenesis but inhibited total, mitochondrial, and ATP-linked oxygen consumption rate, indicative of mitochondrial dysfunction. Treatment of isolated mitochondria, prepared from mouse liver, with DE particles and oleic acid also inhibited mitochondrial complex activity and β-oxidation., Conclusions: DE exposure leads to dyslipidemia and liver steatosis in ApoE knockout mice, likely due to mitochondrial dysfunction and decreased lipid catabolism.

Objective- FMO (flavin-containing monooxygenase) 3 converts bacterial-derived trimethylamine to trimethylamine N-oxide (TMAO), an independent risk factor for cardiovascular disease. We generated FMO3 knockout (FMO3KO) mouse to study its effects on plasma TMAO, lipids, glucose/insulin metabolism, thrombosis, and atherosclerosis. Approach and Results- Previous studies with an antisense oligonucleotide (ASO) knockdown strategy targeting FMO3 in LDLRKO (low-density lipoprotein receptor knockout) mice resulted in major reductions in TMAO levels and atherosclerosis, but also showed effects on plasma lipids, insulin, and glucose. Although FMO3KO mice generated via CRISPR/Cas9 technology bred onto the LDLRKO background did exhibit similar effects on TMAO levels, the effects on lipid metabolism were not as pronounced as with the ASO knockdown model. These differences could result from either off-target effects of the ASO or from a developmental adaptation to the FMO3 deficiency. To distinguish these possibilities, we treated wild-type and FMO3KO mice with control or FMO3 ASOs. FMO3-ASO treatment led to the same extent of lipid-lowering effects in the FMO3KO mice as the wild-type mice, indicating off-target effects. The levels of TMAO in LDLRKO mice fed an atherogenic diet are very low in both wild-type and FMO3KO mice, and no significant effect was observed on atherosclerosis. When FMO3KO and wild-type mice were maintained on a 0.5% choline diet, FMO3KO showed a marked reduction in both TMAO and in vivo thrombosis potential. Conclusions- FMO3KO markedly reduces systemic TMAO levels and thrombosis potential. However, the previously observed large effects of an FMO3 ASO on plasma lipid levels appear to be due partly to off-target effects.

(1) Background: Paraoxonase 2 (PON2) is a ubiquitously expressed protein localized to endoplasmic reticulum and mitochondria. Previous studies have shown that PON2 exhibits anti-oxidant and anti-inflammatory functions, and PON2-deficient (PON2-def) mice are more susceptible to atherosclerosis. Furthermore, PON2 deficiency leads to impaired mitochondrial function. (2) Methods: In this study, we examined the susceptibility of PON2-def mice to diet-induced obesity. (3) Results: After feeding of an obesifying diet, the PON2-def mice exhibited significantly increased body weight due to increased fat mass weight as compared to the wild-type (WT) mice. The increased adiposity was due, in part, to increased adipocyte hypertrophy. PON2-def mice had increased fasting insulin levels and impaired glucose tolerance after diet-induced obesity. PON2-def mice had decreased oxygen consumption and energy expenditure. Furthermore, the oxygen consumption rate of subcutaneous fat pads from PON2-def mice was lower compared to WT mice. Gene expression analysis of the subcutaneous fat pads revealed decreased expression levels of markers for beige adipocytes in PON2-def mice. (4) Conclusions: We concluded that altered systemic energy balance, perhaps due to decreased beige adipocytes and mitochondrial dysfunction in white adipose tissue of PON2-def mice, leads to increased obesity in these mice.

The mammalian paraoxonases (PONs 1, 2 and 3) are a family of esterases that are highly conserved within and between species. They exhibit antioxidant and anti-inflammatory activities. However, their physiological function(s) and native substrates are uncertain. Previous structure-activity relationship studies demonstrate that PONs have a high specificity for lipophilic lactones, suggesting that such compounds may be representative of native substrates. This report describes the ability of PONs to hydrolyze two bioactive δ-lactones derived from arachidonic acid, 5,6-dihydroxy-eicosatrienoic acid lactone (5,6-DHTL) and cyclo-epoxycyclopentenone (cyclo-EC). Both lactones were very efficiently hydrolyzed by purified PON3. PON1 efficiently hydrolyzed 5,6-DHTL, but with a specific activity about 15-fold lower than PON3. 5,6-DHTL was a poor substrate for PON2. Cyclo-EC was a poor substrate for PON1 and not hydrolyzed by PON2. Studies with the PON inhibitor EDTA and a serine esterase inhibitor indicated that the PONs are the main contributors to hydrolysis of the lactones in human and mouse liver homogenates. Studies with homogenates from PON3 knockout mouse livers indicated that >80% of the 5,6-DHTL and cyclo-EC lactonase activities were attributed to PON3. The findings provide further insight into the structural requirements for PONs substrates and support the hypothesis that PONs, particularly PON1 and PON3, evolved to hydrolyze and regulate a class of lactone lipid mediators derived from polyunsaturated fatty acids., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Essentials Microbe-dependent production of trimethylamine N-oxide (TMAO) contributes to thrombosis risk. The impact of host flavin monooxygenase 3 (FMO3) modulation on platelet function is unknown. Genetic manipulation of FMO3 in mice alters systemic TMAO levels and thrombosis potential. Genetic manipulation of FMO3 is associated with alteration of gut microbial community structure., Summary: Background Gut microbes play a critical role in the production of trimethylamine N-oxide (TMAO), an atherogenic metabolite that impacts platelet responsiveness and thrombosis potential. Involving both microbe and host enzymatic machinery, TMAO generation utilizes a metaorganismal pathway, beginning with ingestion of trimethylamine (TMA)-containing dietary nutrients such as choline, phosphatidylcholine and carnitine, which are abundant in a Western diet. Gut microbial TMA lyases use these nutrients as substrates to produce TMA, which upon delivery to the liver via the portal circulation, is converted into TMAO by host hepatic flavin monooxygenases (FMOs). Gut microbial production of TMA is rate limiting in the metaorganismal TMAO pathway because hepatic FMO activity is typically in excess. Objectives FMO3 is the major FMO responsible for host generation of TMAO; however, a role for FMO3 in altering platelet responsiveness and thrombosis potential in vivo has not yet been explored. Methods The impact of FMO3 suppression (antisense oligonucleotide-targeting) and overexpression (as transgene) on plasma TMAO levels, platelet responsiveness and thrombosis potential was examined using a murine FeCl 3 -induced carotid artery injury model. Cecal microbial composition was examined using 16S analyses. Results Modulation of FMO3 directly impacts systemic TMAO levels, platelet responsiveness and rate of thrombus formation in vivo. Microbial composition analyses reveal taxa whose proportions are associated with both plasma TMAO levels and in vivo thrombosis potential. Conclusions The present studies demonstrate that host hepatic FMO3, the terminal step in the metaorganismal TMAO pathway, participates in diet-dependent and gut microbiota-dependent changes in both platelet responsiveness and thrombosis potential in vivo., (© 2018 International Society on Thrombosis and Haemostasis.)

Background: Mitochondrial oxidation is a major source of reactive oxygen species (ROS) and mitochondrial dysfunction plays a central role in development of heart failure (HF). Paraoxonase 2 deficient (PON2-def) mitochondria are impaired in function. In this study, we tested whether PON2-def aggravates HF progression., Methods and Results: Using qPCR, immunoblotting and lactonase activity assay, we demonstrate that PON2 activity was significantly decreased in failing hearts despite increased PON2 expression. To determine the cardiac-specific function of PON2, we performed heart transplantations in which PON2-def and wild type (WT) donor hearts were implanted into WT recipient mice. Beating scores of the donor hearts, assessed at 4 weeks post-transplantation, were significantly decreased in PON2-def hearts when compared to WT donor hearts. By using a transverse aortic constriction (TAC) model, we found PON2 deficiency significantly exacerbated left ventricular remodeling and cardiac fibrosis post-TAC. We further demonstrated PON2 deficiency significantly enhanced ROS generation in heart tissues post-TAC. ROS generation was measured through dihydroethidium (DHE) using high-pressure liquid chromatography (HPLC) with a fluorescent detector. By using neonatal cardiomyocytes treated with CoCl 2 to mimic hypoxia, we found PON2 deficiency dramatically increased ROS generation in the cardiomyocytes upon CoCl 2 treatment. In response to a short CoCl 2 exposure, cell viability and succinate dehydrogenase (SDH) activity assessed by MTT assay were significantly diminished in PON2-def cardiomyocytes compared to those in WT cardiomyocytes. PON2-def cardiomyocytes also had lower baseline SDH activity. By using adult mouse cardiomyocytes and mitochondrial ToxGlo assay, we found impaired cellular ATP generation in PON2-def cells compared to that in WT cells, suggesting that PON2 is necessary for proper mitochondrial function., Conclusion: Our study suggests a cardioprotective role for PON2 in both experimental and human heart failure, which may be associated with the ability of PON2 to improve mitochondrial function and diminish ROS generation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Inter-tissue communication via secreted proteins has been established as a vital mechanism for proper physiologic homeostasis. Here, we report a bioinformatics framework using a mouse reference population, the Hybrid Mouse Diversity Panel (HMDP), which integrates global multi-tissue expression data and publicly available resources to identify and functionally annotate novel circuits of tissue-tissue communication. We validate this method by showing that we can identify known as well as novel endocrine factors responsible for communication between tissues. We further show the utility of this approach by identification and mechanistic characterization of two new endocrine factors. Adipose-derived Lipocalin-5 is shown to enhance skeletal muscle mitochondrial function, and liver-secreted Notum promotes browning of white adipose tissue, also known as "beiging." We demonstrate the general applicability of the method by providing in vivo evidence for three additional novel molecules mediating tissue-tissue interactions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The choline-derived metabolite trimethylamine N-oxide (TMAO) has been demonstrated to contribute to atherosclerosis and is associated with coronary artery disease risk., Methods and Results: We explored the impact of TMAO on endothelial and smooth muscle cell function in vivo, focusing on disease-relevant outcomes for atherogenesis. Initially, we observed that aortas of LDLR(-/-) mice fed a choline diet showed elevated inflammatory gene expression compared with controls. Acute TMAO injection at physiological levels was sufficient to induce the same inflammatory markers and activate the well-known mitogen-activated protein kinase, extracellular signal-related kinase, and nuclear factor-κB signaling cascade. These observations were recapitulated in primary human aortic endothelial cells and vascular smooth muscle cells. We also found that TMAO promotes recruitment of activated leukocytes to endothelial cells. Through pharmacological inhibition, we further showed that activation of nuclear factor-κB signaling was necessary for TMAO to induce inflammatory gene expression in both of these relevant cell types as well as endothelial cell adhesion of leukocytes., Conclusions: Our results suggest a likely contributory mechanism for TMAO-dependent enhancement in atherosclerosis and cardiovascular risks., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

We report the engineering and characterization of paraoxonase-3 knockout mice (Pon3KO). The mice were generally healthy but exhibited quantitative alterations in bile acid metabolism and a 37% increased body weight compared to the wild-type mice on a high fat diet. PON3 was enriched in the mitochondria-associated membrane fraction of hepatocytes. PON3 deficiency resulted in impaired mitochondrial respiration, increased mitochondrial superoxide levels, and increased hepatic expression of inflammatory genes. PON3 deficiency did not influence atherosclerosis development on an apolipoprotein E null hyperlipidemic background, but it did lead to a significant 60% increase in atherosclerotic lesion size in Pon3KO mice on the C57BL/6J background when fed a cholate-cholesterol diet. On the diet, the Pon3KO had significantly increased plasma intermediate-density lipoprotein/LDL cholesterol and bile acid levels. They also exhibited significantly elevated levels of hepatotoxicity markers in circulation, a 58% increase in gallstone weight, a 40% increase in hepatic cholesterol level, and increased mortality. Furthermore, Pon3KO mice exhibited decreased hepatic bile acid synthesis and decreased bile acid levels in the small intestine compared with wild-type mice. Our study suggests a role for PON3 in the metabolism of lipid and bile acid as well as protection against atherosclerosis, gallstone disease, and obesity., (© FASEB.)

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein-associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other's function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.

We examined the effects of a natural secondary bile acid, hyodeoxycholic acid (HDCA), on lipid metabolism and atherosclerosis in LDL receptor-null (LDLRKO) mice. Female LDLRKO mice were maintained on a Western diet for 8 wk and then divided into 2 groups that received chow, or chow + 1.25% HDCA, diets for 15 wk. We observed that mice fed the HDCA diet were leaner and exhibited a 37% (P<0.05) decrease in fasting plasma glucose level. HDCA supplementation significantly decreased atherosclerotic lesion size at the aortic root region, the entire aorta, and the innominate artery by 44% (P<0.0001), 48% (P<0.01), and 94% (P<0.01), respectively, as compared with the chow group. Plasma VLDL/IDL/LDL cholesterol levels were significantly decreased, by 61% (P<0.05), in the HDCA group as compared with the chow diet group. HDCA supplementation decreased intestinal cholesterol absorption by 76% (P<0.0001) as compared with the chow group. Furthermore, HDL isolated from the HDCA group exhibited significantly increased ability to mediate cholesterol efflux ex vivo as compared with HDL of the chow diet group. In addition, HDCA significantly increased the expression of genes involved in cholesterol efflux, such as Abca1, Abcg1, and Apoe, in a macrophage cell line. Thus, HDCA is a candidate for antiatherosclerotic drug therapy.

Oxidative stress is a determinant of liver steatosis and the progression to more severe forms of disease. The present study investigated the effect of paraoxonase-1 (PON1) deficiency on histological alterations and hepatic metabolism in mice fed a high-fat high-cholesterol diet. We performed nontargeted metabolomics on liver tissues from 8 male PON1-deficient mice and 8 wild-type animals fed a high-fat, high-cholesterol diet for 22 weeks. We also measured 8-oxo-20-deoxyguanosine, reduced and oxidized glutathione, malondialdehyde, 8-isoprostanes and protein carbonyl concentrations. Results indicated lipid droplets in 14.5% of the hepatocytes of wild-type mice and in 83.3% of the PON1-deficient animals (P < 0.001). The metabolomic assay included 322 biochemical compounds, 169 of which were significantly decreased and 16 increased in PON1-deficient mice. There were significant increases in lipid peroxide concentrations and oxidative stress markers. We also found decreased glycolysis and the Krebs cycle. The urea cycle was decreased, and the pyrimidine cycle had a significant increase in orotate. The pathways of triglyceride and phospholipid synthesis were significantly increased. We conclude that PON1 deficiency is associated with oxidative stress and metabolic alterations leading to steatosis in the livers of mice receiving a high-fat high-cholesterol diet.

Hyperlipidemia blunts anabolic effects of intermittent parathyroid hormone (PTH) on cortical bone, and the responsiveness to PTH are restored in part by oral administration of the antioxidant ApoA-I mimetic peptide, D-4F. To evaluate the mechanism of this rescue, hyperlipidemic mice overexpressing the high-density lipoprotein-associated antioxidant enzyme, paraoxonase 1 (Ldlr(-/-)PON1(tg)) were generated, and daily PTH injections were administered to Ldlr(-/-)PON1(tg) and to littermate Ldlr(-/-) mice. Expression of bone regulatory genes was determined by realtime RT-qPCR, and cortical bone parameters of the femoral bones by micro-computed tomographic analyses. PTH-treated Ldlr(-/-)PON1(tg) mice had significantly greater expression of PTH receptor (PTH1R), activating transcription factor-4 (ATF4), and osteoprotegerin (OPG) in femoral cortical bone, as well as significantly greater cortical bone mineral content, thickness, and area in femoral diaphyses compared with untreated Ldlr(-/-)PON1(tg) mice. In contrast, in control mice (Ldlr(-/-)) without PON1 overexpression, PTH treatment did not induce these markers. Calvarial bone of PTH-treated Ldlr(-/-)PON1(tg) mice also had significantly greater expression of osteoblastic differentiation marker genes as well as BMP-2-target and Wnt-target genes. Untreated Ldlr(-/-)PON1(tg) mice had significantly greater expression of PTHR1 than untreated Ldlr(-/-) mice, whereas sclerostin expression was reduced. In femoral cortical bones, expression levels of transcription factors, FoxO1 and ATF4, were also elevated in the untreated, control Ldlr(-/-)PON1(tg) mice, suggesting enhancement of cellular protection against oxidants. These findings suggest that PON1 restores responsiveness to PTH through effects on oxidant stress, PTH receptor expression, and/or Wnt signaling., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Circulating trimethylamine-N-oxide (TMAO) levels are strongly associated with atherosclerosis. We now examine genetic, dietary, and hormonal factors regulating TMAO levels. We demonstrate that two flavin mono-oxygenase family members, FMO1 and FMO3, oxidize trimethylamine (TMA), derived from gut flora metabolism of choline, to TMAO. Further, we show that FMO3 exhibits 10-fold higher specific activity than FMO1. FMO3 overexpression in mice significantly increases plasma TMAO levels while silencing FMO3 decreases TMAO levels. In both humans and mice, hepatic FMO3 expression is reduced in males compared to females. In mice, this reduction in FMO3 expression is due primarily to downregulation by androgens. FMO3 expression is induced by dietary bile acids by a mechanism that involves the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor. Analysis of natural genetic variation among inbred strains of mice indicates that FMO3 and TMAO are significantly correlated, and TMAO levels explain 11% of the variation in atherosclerosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We studied the influence of PON1 on metabolic alterations induced by oxidized LDL when incubated with endothelial cells. HUVEC cells were incubated with native LDL, oxidized LDL, oxidized LDL plus HDL from wild type mice, and oxidized LDL plus HDL from PON1-deficient mice. Results showed alterations in carbohydrate and phospholipid metabolism and increased apoptosis in cells incubated with oxidized LDL. These changes were partially prevented by wild type mouse HDL, but the effects were less effective with HDL from PON1-deficient mice. Our results suggest that PON1 may play a significant role in endothelial cell survival by protecting cells from alterations in the respiratory chain induced by oxidized LDL. These results extend current knowledge on the protective role of HDL and PON1 against oxidation and apoptosis in endothelial cells.

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.

Objective: Chronic infection has long been postulated as a stimulus for atherogenesis. Pseudomonas aeruginosa infection has been associated with increased atherosclerosis in rats, and these bacteria produce a quorum-sensing molecule 3-oxo-dodecynoyl-homoserine lactone (3OC12-HSL) that is critical for colonization and virulence. Paraoxonase 2 (PON2) hydrolyzes 3OC12-HSL and also protects against the effects of oxidized phospholipids thought to contribute to atherosclerosis. We now report the response of human aortic endothelial cells (HAECs) to 3OC12-HSL and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) in relation to PON2 expression., Methods and Results: Using expression profiling and network modeling, we identified the unfolded protein response (UPR), cell cycle genes, and the mitogen-activated protein kinase signaling pathway to be heavily involved in the HAEC response to 3OC12-HSL. The network also showed striking similarities to a network created based on HAEC response to Ox-PAPC, a major component of minimally modified low-density lipoprotein. HAECs in which PON2 was silenced by small interfering RNA showed increased proinflammatory response and UPR when treated with 3OC12-HSL or Ox-PAPC., Conclusion: 3OC12-HSL and Ox-PAPC influence similar inflammatory and UPR pathways. Quorum sensing molecules, such as 3OC12-HSL, contribute to the proatherogenic effects of chronic infection. The antiatherogenic effects of PON2 include destruction of quorum sensing molecules.

Therapies that raise levels of HDL, which is thought to exert atheroprotective effects via effects on endothelium, are being examined for the treatment or prevention of coronary artery disease (CAD). However, the endothelial effects of HDL are highly heterogeneous, and the impact of HDL of patients with CAD on the activation of endothelial eNOS and eNOS-dependent pathways is unknown. Here we have demonstrated that, in contrast to HDL from healthy subjects, HDL from patients with stable CAD or an acute coronary syndrome (HDLCAD) does not have endothelial antiinflammatory effects and does not stimulate endothelial repair because it fails to induce endothelial NO production. Mechanistically, this was because HDLCAD activated endothelial lectin-like oxidized LDL receptor 1 (LOX-1), triggering endothelial PKCβII activation, which in turn inhibited eNOS-activating pathways and eNOS-dependent NO production. We then identified reduced HDL-associated paraoxonase 1 (PON1) activity as one molecular mechanism leading to the generation of HDL with endothelial PKCβII-activating properties, at least in part due to increased formation of malondialdehyde in HDL. Taken together, our data indicate that in patients with CAD, HDL gains endothelial LOX-1- and thereby PKCβII-activating properties due to reduced HDL-associated PON1 activity, and that this leads to inhibition of eNOS-activation and the subsequent loss of the endothelial antiinflammatory and endothelial repair-stimulating effects of HDL.

PON3 is a member of the paraoxonase gene family that includes PON1 and PON2. For example, PON3 and PON1 share approximately 60% identity at the amino acid level. Recent studies have demonstrated that PON3 is present in human and rabbit HDL but not in mouse HDL. Mouse PON3 appears to be cell-associated and is expressed in a wide range of tissues such as liver, adipose, macrophage, and the artery wall. In vitro studies have shown that PON3 can prevent LDL oxidation and destroy bacterial quorum-sensing molecules. Previous studies also showed that human PON3 transgenic mice were protected from obesity and atherosclerosis in both the C57BL/6 J wild-type and LDLR knockout genetic background. Administration of adenovirus expressing the human PON3 gene into apoE -/- mice also decreased atherosclerotic lesion formation. In order to further understand the functions of PON3 in physiology and disease, we performed in situ hybridization analysis to examine Pon3 gene expression patterns in newborn and adult mice, in various tissues, including atherosclerotic lesions of apoE -/- mice. Our results show relatively high levels of Pon3 mRNA labeling in the adrenal gland, submaxillary gland, lung, liver, adipose, pancreas, large intestine, and other tissues of newborn mice. In the adult mouse, Pon3 mRNA levels were much lower in the corresponding tissues as mentioned above for the newborn mouse. Sections of the aortic root from the hearts of both wild-type and apoE -/- mice displayed moderate levels of Pon3 mRNA labeling. Pon3 mRNA was also detected in the atherosclerotic lesion areas at the aortic root of apoE -/- hearts. Our data revealed that mouse Pon3 is expressed in a wide range of tissues, and that its expression is temporally controlled.

Paraoxonases (PONs) are a family of lactonases with promiscuous enzyme activity that has been implicated in multiple diseases. PON2 is intracellularly located, is the most ubiquitously expressed PON, and has the highest lactonase activity of the PON family members. Whereas some single-nucleotide polymorphisms (SNPs) in PON1 have resulted in altered enzymatic activity in serum, to date the functional consequences of SNPs on PON2 function remain unknown. We hypothesized that a common PON2 SNP would result in impaired lactonase activity. Substitution of cysteine for serine at codon 311 in recombinant PON2 resulted in normal protein production and localization but altered glycosylation and decreased lactonase activity. Moreover, we screened 200 human lung samples for the PON2 Cys(311) variant and found that in vivo this mutation impaired lactonase activity. These data suggest that impaired lactonase activity may play a role in innate immunity, atherosclerosis, and other diseases associated with the PON2 311 SNP.

Purpose of Review: The paraoxonase (PON) gene family includes three members, PON1, PON2, and PON3. In-vitro and mouse studies have demonstrated that all three PONs are atheroprotective. Some, but not all, human epidemiologic studies have observed associations between PON gene polymorphisms and risk of cardiovascular disease (CVD). In this review, we summarize studies published within the last year, elucidating involvement of PON1 and PON2 in oxidative stress, CVD, and innate immune responses., Recent Findings: In a prospective study, the PON1 192QQ genotype and low PON1 activity were associated with increased systemic oxidative stress and increased risk for CVD. PON1 expression protected against Pseudomonas aeruginosa lethality in Drosophila, suggesting that PON1 can interfere with quorum sensing in vivo. PON2 attenuated macrophage triglyceride accumulation via inhibition of diacylglycerol acyltransferase 1. Overexpression of PON2 protected against endoplasmic reticulum stress-induced apoptosis when the stress was induced by interference with protein modification but not when endoplasmic reticulum stress was induced by Ca2+ deregulation., Summary: Both mouse and human studies have demonstrated the antioxidative and atheroprotective effects of PON1. The mechanisms by which PON2 exerts its atheroprotective effects are emerging. Large-scale epidemiologic studies are needed to further examine the relationship between PON2 genetic polymorphisms and risk for CVD. Elucidation of the physiologic substrates of the PON proteins is of particular importance to further advance this field.

Genetic disorders of homocysteine (Hcy) or folate metabolism or high-methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy-thiolactone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine beta-synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (N-Hcy-protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how N-Hcy-protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma N-Hcy-protein was elevated 10-fold in mice fed a high-methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton-coupled folate transporter (Pcft) gene resulted in a 10- to 30-fold increase in plasma or serum N-Hcy-protein levels. Liver N-Hcy-protein was elevated 3.4-fold in severely and 11-fold in extremely hyperhomocysteinemic Cbs-deficient mice, 3.6-fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr-deficient animals, suggesting that mice have a capacity to prevent accumulation of N-Hcy-protein in their organs. These findings provide evidence that N-Hcy-protein is an important metabolite associated with Hcy pathophysiology in the mouse.

Oxidative stress generated from endogenous and exogenous sources causes oxidative DNA damage. The most frequent mutagenic base lesion 7,8-dihydro-8-oxoguanine and the resulting mismatched adenine are removed by OGG1 and MYH in mammals. Deficiencies in human MYH or mouse MYH and OGG1 result in tumor predisposition but the underlying molecular mechanism is not fully understood. To facilitate the study of the roles of MYH and OGG1 in the protection against oxidative stress, we generated mouse embryonic fibroblast cell lines deficient in these genes. Myh and Ogg1 double knockout cells were more sensitive than wild type to oxidants (hydrogen peroxide and t-butyl hydroperoxide), but not to cis-platinum or gamma-irradiations. The low dosage oxidative stress resulted in more reduction of S phase and increase of G(2)/M phase in Myh(-/-)Ogg1(-/-) cells than in wild-type cells, but a similar level of cell death in both cells. The oxidants also induced more multinucleated cells in Myh(-/-)Ogg1(-/-) cells than in wild-type, accompanied by centrosome amplification and multipolar spindle formation. Thus, under oxidative stress, Myh and Ogg1 are likely required for normal cell-cycle progression and nuclear division, suggesting multiple roles of Myh and Ogg1 in the maintenance of genome stability and tumor prevention.

Cadmium, a ubiquitous environmental contaminant, damages several major organs in humans and other mammals. The molecular mechanisms for damage are not known. At high doses (5 mg/kg cadmium chloride or higher), testicular damage in mice, rats, and other rodents includes interstitial edema, hemorrhage, and changes in the seminiferous tubules affecting spermatogenesis. Necrosis is evident by 48 h. The goal of this study was to fine map and identify the cdm gene, a gene that when mutated prevents cadmium-induced testicular toxicity in mouse strains with a mutation in this gene. A serine-threonine phosphatase, calcineurin (CN), subunit A, alpha isoform (Ppp3ca), was one of the seven candidates in the cdm region that was narrowed from 5.6 to 2.0 Mb on mouse chromosome 3. An inhibitor of CN, the immunosuppressant, FK506, prevented cadmium-induced testicular damage in five pathological categories, including vascular endothelial and seminiferous epithelial endpoints. Inductively coupled plasma-mass spectrometry revealed that FK506 protected without lowering the amount of cadmium in the testes. Ppp3ca(-/-) mice were investigated but were found to exhibit endogenous testicular abnormalities, making them an inappropriate model for determining whether the inactivation of the Ppp3ca gene would afford protection from cadmium-induced testicular toxicity. The protection afforded by FK506, found by the current study, indicated that CN is likely to be important in the mechanism of cadmium toxicity in the testis and possibly other organs.

Heme oxygenase (HO-1) is the rate-limiting enzyme in the catabolism of heme, which leads to the generation of biliverdin, iron, and carbon monoxide. It has been shown to have important antioxidant and antiinflammatory properties that result in a vascular antiatherogenic effect. To determine whether HO-1 expression in macrophages constitutes a significant component of the protective role in atherosclerosis, we evaluated the effect of decreased or absent HO-1 expression in peritoneal macrophages on oxidative stress and inflammation in vitro, and the effect of complete deficiency of HO-1 expression in macrophages in atherosclerotic lesion formation in vivo. We found that compared with HO-1(+/+) controls, peritoneal macrophages from HO-1(-/-) and HO-1(+/-) mice exhibited (1) increased reactive oxygen species (ROS) generation, (2) increased proinflammatory cytokines such as monocyte chemotactic protein 1 (MCP-1) and interleukin 6 (IL-6), and (3) increased foam cell formation when treated with oxLDL, attributable in part to increased expression of scavenger receptor A (SR-A). Bone marrow transplantation experiments performed in lethally irradiated LDL-R null female mice, reconstituted with bone marrow from HO-1(-/-) versus HO-1(+/+) mice, revealed that HO-1(-/-) reconstituted animals exhibited atherosclerotic lesions with a greater macrophage content as evaluated by immunohistochemistry and planimetric assessment. We conclude that HO-1 expression in macrophages constitutes an important component of the antiatherogenic effect by increasing antioxidant protection and decreasing the inflammatory component of atherosclerotic lesions.

Genetic disorders of homocysteine (Hcy) metabolism or a high-methionine diet lead to elevations of plasma Hcy levels. In humans, severe genetic hyperhomocysteinemia results in premature death from vascular complications whereas dietary hyperhomocysteinemia is often used to induce atherosclerosis in animal models. Hcy is mistakenly selected in place of methionine by methionyl-tRNA synthetase during protein biosynthesis, which results in the formation of Hcy-thiolactone and initiates a pathophysiological pathway that has been implicated in human vascular disease. However, whether genetic deficiencies in Hcy metabolism or a high-methionine diet affect Hcy-thiolactone levels in mammals has been unknown. Here we show that plasma Hcy-thiolactone is elevated 59-fold and 72-fold in human patients with hyperhomocysteinemia secondary to mutations in methylenetetrahydrofolate reductase and cystathionine beta-synthase genes, respectively. We also show that mice, like humans, eliminate Hcy-thiolactone by urinary excretion; in contrast to humans, however, mice also eliminate significant amounts of plasma total Hcy (approximately 38%) by urinary excretion. In mice, hyperhomocysteinemia secondary to a high-methionine diet leads to 3.7-fold and 25-fold increases in plasma and urinary Hcy-thiolactone levels, respectively. Thus, we conclude that hyperhomocysteinemia leads to significant increases in the atherogenic metabolite Hcy-thiolactone in humans and mice.

Paraoxonase 3 (PON3) is a member of the PON family, which includes PON1, PON2, and PON3. Recently, PON3 was shown to prevent the oxidation of low-density lipoprotein in vitro. To test the role of PON3 in atherosclerosis and related traits, 2 independent lines of human PON3 transgenic (Tg) mice on the C57BL/6J (B6) background were constructed. Human PON3 mRNA was detected in various tissues, including liver, lung, kidney, brain, adipose, and aorta, of both lines of Tg mice. The human PON3 mRNA levels in the livers of PON3 Tg mice were 4- to 7-fold higher as compared with the endogenous mouse Pon3 mRNA levels. Human PON3 protein and activity were detected in the livers of Tg mice as well. No significant differences in plasma total, high-density lipoprotein, and very-low-density lipoprotein/low-density lipoprotein cholesterol and triglyceride and glucose levels were observed between the PON3 Tg and non-Tg mice. Interestingly, atherosclerotic lesion areas were significantly smaller in both lines of male PON3 Tg mice as compared with the male non-Tg littermates on B6 background fed an atherogenic diet. When bred onto the low-density lipoprotein receptor knockout mouse background, the male PON3 Tg mice also exhibited decreased atherosclerotic lesion areas and decreased expression of monocyte chemoattractant protein-1 in the aorta as compared with the male non-Tg littermates. In addition, decreased adiposity and lower circulating leptin levels were observed in both lines of male PON3 Tg mice as compared with the male non-Tg mice. In an F2 cross, adipose Pon3 mRNA levels inversely correlated with adiposity and related traits. Our study demonstrates that elevated PON3 expression significantly decreases atherosclerotic lesion formation and adiposity in male mice. PON3 may play an important role in protection against obesity and atherosclerosis.

Pseudomonas aeruginosa is an important cause of nosocomial infections and is frequently present in the airways of cystic fibrosis patients. Quorum sensing mediates P. aeruginosa's virulence and biofilm formation through density-dependent interbacterial signaling with autoinducers. N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is the major autoinducer in P. aeruginosa. We have previously shown that human airway epithelia and paraoxonases (PONs) degrade 3OC12-HSL. This study investigated the role of PON1, PON2, and PON3 in airway epithelial cell inactivation of 3OC12-HSL. All three PONs were present in murine tracheal epithelial cells, with PON2 and PON3 expressed at the highest levels. Lysates of tracheal epithelial cells from PON2, but not PON1 or PON3, knockout mice had impaired 3OC12-HSL inactivation compared with wild-type mice. In contrast, PON1-, PON2-, or PON3-targeted deletions did not affect 3OC12-HSL degradation by intact epithelia. Overexpression of PON2 enhanced 3OC12-HSL degradation by human airway epithelial cell lysates but not by intact epithelia. Finally, using a quorum-sensing reporter strain of P. aeruginosa, we found that quorum sensing was enhanced in PON2-deficient airway epithelia. In summary, these results show that loss of PON2 impairs 3OC12-HSL degradation by airway epithelial cells and suggests that diffusion of 3OC12-HSL into the airway cells can be the rate-limiting step for degradation of the molecule.

Paraoxonases (PONs) are a family of proteins that may play a significant role in providing relief from both toxic environmental chemicals as well as physiological oxidative stress. Although the physiological roles of the PON family of proteins, PON1, PON2, and PON3, remain unknown, epidemiological, biochemical, and mouse genetic studies of PON1 suggest an anti-atherogenic function for paraoxonases. To determine whether PON2 plays a role in the development of atherosclerosis in vivo, we generated PON2-deficient mice. When challenged with a high fat, high cholesterol diet for 15 weeks, serum levels of high density lipoprotein cholesterol, triglycerides, and glucose were not significantly different between wild-type and PON2-deficient mice. In contrast, serum levels of very low density lipoprotein (VLDL)/low density lipoprotein (LDL) cholesterol were significantly lower (-32%) in PON2-deficient mice compared with wild-type mice. However, despite lower levels of VLDL/LDL cholesterol, mice deficient in PON2 developed significantly larger (2.7-fold) atherosclerotic lesions compared with their wild-type counterparts. Enhanced inflammatory properties of LDL, attenuated anti-atherogenic capacity of high density lipoprotein, and a heightened state of oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages appear to be the main mechanisms behind the larger atherosclerotic lesions in PON2-deficient mice. These results demonstrate that PON2 plays a protective role in atherosclerosis.

The inducible nitric oxide synthase (iNOS) is abundantly expressed by smooth muscle cells and macrophages in atherosclerotic lesions. Apolipoprotein E-deficient (apoE(-/-)) mice develop early and advanced atherosclerotic lesions. The role of iNOS in both early and advanced atherosclerotic formation was determined in apoE(-/-) mice. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 12 weeks of age on chow diet, iNOS(-/-)/apoE(-/-) mice developed comparable sizes of early atherosclerotic lesions in the aortic root as did iNOS(+/+)/apoE(-/-) mice (30,993+/-4746 vs. 26,648+/-6815 microm(2)/section; P=0.608). After being fed the Western diet for 12 weeks, iNOS(-/-)/apoE(-/-) mice developed significantly smaller advanced lesions than iNOS(+/+)/apoE(-/-) mice (458,734+/-14,942 vs. 519,570+/-22,098 microm(2)/section; P=0.029). This reduction in lesion formation could not be explained by differences in plasma lipid levels. To examine whether iNOS contributed to LDL oxidation, smooth muscle cells were isolated from the aorta, activated with TNF-alpha, and then incubated with native LDL in the absence or presence of N-Omega-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by smooth muscle cells from iNOS(+/+)/apoE(-/-) mice (P=0.048), but it had no effect on LDL oxidation by cells from iNOS(-/-)/apoE(-/-) mice. iNOS(-/-)/apoE(-/-) mice had a significantly lower plasma lipoperoxide level on the Western diet (2.74+/-0.23 vs. 3.89+/-0.41 microM MDA; P=0.021) but not on chow diet (1.02+/-0.07 vs. 1.51+/-0.29 microM MDA; P=0.11). Thus, the absence of iNOS-mediated LDL oxidation may contribute to the reduction in advanced lesion formation of iNOS(-/-)/apoE(-/-) mice.

Paraoxonase-1 (PON1), an enzyme that metabolizes organophosphate insecticides, is secreted by the liver and transported in the blood complexed to HDL. In humans and mice, low plasma levels of PON1 have also been linked to the development of atherosclerosis. We previously reported that hepatic Pon1 expression was decreased when C57BL/6J mice were fed a high-fat, high-cholesterol diet supplemented with cholic acid (CA). In the current study, we used wild-type and farnesoid X receptor (FXR) null mice to demonstrate that this repression is dependent upon CA and FXR. PON1 mRNA levels were also repressed when HepG2 cells, derived from a human hepatoma, were incubated with natural or highly specific synthetic FXR agonists. In contrast, fibroblast growth factor-19 (FGF-19) mRNA levels were greatly induced by these same FXR agonists. Furthermore, treatment of HepG2 cells with recombinant human FGF-19 significantly decreased PON1 mRNA levels. Finally, deletion studies revealed that the proximal -230 to -96 bp region of the PON1 promoter contains regulatory element(s) necessary for promoter activity and bile acid repression. These data demonstrate that human PON1 expression is repressed by bile acids through the actions of FXR and FGF-19.

The pathogenic bacterium Pseudomonas aeruginosa uses acyl-HSL quorum-sensing signals to regulate genes controlling virulence and biofilm formation. We found that paraoxonase 1 (PON1), a mammalian lactonase with an unknown natural substrate, hydrolyzed the P. aeruginosa acyl-HSL 3OC12-HSL. In in vitro assays, mouse serum-PON1 was required and sufficient to degrade 3OC12-HSL. Furthermore, PON2 and PON3 also degraded 3OC12-HSL effectively. Serum-PON1 prevented P. aeruginosa quorum-sensing and biofilm formation in vitro by inactivating the quorum-sensing signal. Although 3OC12-HSL production by P. aeruginosa was important for virulence in a mouse sepsis model, Pon1-knock-out mice were paradoxically protected. These mice showed increased levels of PON2 and PON3 mRNA in epithelial tissues suggesting a possible compensatory mechanism. Thus, paraoxonase interruption of bacterial communication represents a novel mechanism to modulate quorum-sensing by bacteria. The consequences for host immunity are yet to be determined.

Objectives: The Q192R polymorphism of paraoxonase (PON1) has been shown to affect hydrolysis of organophosphorus compounds. The Q192 and R192 alloforms exhibit equivalent catalytic efficiencies of hydrolysis for diazoxon, the oxon form of the pesticide (DZ). However, the R192 alloform has a higher catalytic efficiency of hydrolysis than does the Q192 alloform for chlorpyrifos oxon (CPO), the oxon form of the pesticide chlorpyrifos (CPS). The current study examined the relevance of these observations for in-vivo exposures to chlorpyrifos and chlorpyrifos oxon., Methods: Using a transgenic mouse model we examined the relevance of the Q192R polymorphism for exposure to CPS and CPO in vivo. Transgenic mice were generated that expressed either human PON1Q192 or PON1R192 at equivalent levels, in the absence of endogenous mouse PON1. Dose-response and time course experiments were performed on adult mice exposed dermally to CPS or CPO. Morbidity and acetylcholinesterase (AChE) activity in the brain and diaphragm were determined in the first 24 h following exposure., Results: Mice expressing PON1Q192 were significantly more sensitive to CPO, and to a lesser extent CPS, than were mice expressing PON1R192. The time course of inhibition following exposure to 1.2 mg/kg CPO revealed maximum inhibition of brain AChE at 6-12 h, with PON1R192, PON1Q192, and PON1 mice exhibiting 40, 70 and 85% inhibition, respectively, relative to control mice. The effect of PON1 removal on the dose-response curve for CPS exposure was remarkably consistent with a PBPK/PD model of CPS exposure., Conclusion: These results indicate that individuals expressing only the PON1Q192 allele would be more sensitive to the adverse effects of CPO or CPS exposure, especially if they are expressing a low level of plasma PON1Q192.

Individual differences in detoxication capacities for specific organophosphorous (OP) compounds are due largely to differences in catalytic efficiency or abundance of the HDL-associated enzyme, paraoxonase (PON1). First, we provide evidence that children less than 2 years of age represent a particularly susceptible population for OP exposure due to low abundance of PON1 and variable onset of plasma PON1 activity. Second, we describe studies examining the neurotoxic effects of chronic, low-level OP pesticide exposure in mice. PON1 knockout (PON1(-/-)) and wild-type mice were exposed chronically (PN4 to PN21) to low levels of chlorpyrifos oxon (CPO). Endpoints included cholinesterase activity, histopathology, gene expression, and behavior. Even at PN4, when PON1 levels were low in wild-type mice, PON1(-/-) mice were more sensitive to inhibition of brain cholinesterase by CPO. At PN22, and persisting as long as 4 months, chronic developmental exposure to 0.18 mg/kg/d or 0.25 mg/kg/d CPO resulted in perinuclear vacuolization of cells in a discrete area of the neocortex and irregular distribution of neurons in the cortical plate, with an increase in the number of affected cells at 0.25mg/kg/d. Third, we describe a transgenic mouse model in which human transgenes encoding either hPON1Q192 or hPON1R192 were expressed at equal levels in place of mouse PON1. The developmental onset of expression followed the mouse time course and was identical for the two transgenes, allowing these mice to be used to assess the importance of the Q192R polymorphism during development. Adult mice expressing hPON1R192 were significantly more resistant than hPON1Q192 mice to CPO toxicity. Our studies indicate that children less than 2 years old, especially those homozygous for PON1Q192, would be predicted to be particularly susceptible to CPO toxicity.

Objective: High density lipoprotein (HDL)-associated paraoxonase 1 (PON1), hydrolyzes oxidized lipids in oxidized low density lipoprotein (LDL) and thus protects against atherosclerosis development. Increased susceptibility to atherosclerosis observed in PON1 knockout (PON1(0)) mice was associated with increased LDL lipid peroxidation as well as increased macrophage oxidative stress. Thus, the aim of the present study is to characterize the direct effect of PON1 on oxidative status processes in macrophages., Methods and Results: We used in vitro and in vivo models of PON1 expression in macrophages, as PON1 is not synthesized by these cells. Peritoneal macrophages (MPM) harvested from PON1(0) mice were transfected with human (hPON1). These cells exhibited reduced total peroxide levels by 47% and decreased capacity to release superoxide anions by 69%, associated with a small but significant increment of the reduced form of glutathione (GSH), a major cellular anti-oxidant, compared to control cells. MPM were also harvested from PON1 transgenic (PON1Tg) mice. Unexpectedly, these cells expressed hPON1 (mRNA and activity). Compared to MPM derived from control C57BL/6J mice, PON1Tg mouse MPM exhibited 35% decreased cellular total peroxide levels, decreased capacity to produce superoxide anions and 47% decreased capacity to oxidize LDL. PON1Tg mouse MPM were also characterized by 51% increased levels of GSH, compared to control MPM. Similarly, MPM harvested from PON1Tg on the genetic background of the atherosclerotic apolipoprotein E knockout (PON1Tg/E(0)) mice also exhibited decreased oxidative stress, compared to E(0) mouse MPM. Aortas obtained from these mice were characterized by decreased lipid peroxide levels, decreased capacity to oxidize LDL, and also increased GSH levels, compared to aortas obtained from E(0) mice. The decreased macrophage and aortic oxidative stress in PON1Tg/E(0) mice was associated with 2.7-fold decreased atherosclerotic lesion size in comparison to E(0) mice., Conclusions: PON1 directly reduced macrophage and aortic oxidative status, which was associated with decreased superoxide anion production and increased glutathione content. These phenomena could be responsible for the observed attenuated atherosclerosis development in PON1Tg mice in comparison to control mice.

Epidemiologic, genetic, and biochemical studies support an antiatherogenic role for paraoxonase (PON) 1. While the precise mechanism by which PON1 protects against the development of atherosclerosis is unclear, in vitro studies and the results from PON1 knockout and transgenic mice suggest that this protective effect may be attributed to PON1's ability to attenuate the oxidative modification of lipoprotein particles. The two other members of the PON gene family, namely, PON2 and PON3, have also been reported to possess antioxidant properties and may exhibit antiatherogenic capacities as well. Previous studies have demonstrated that PON1 expression is downregulated by oxidative stress. In contrast, more recent studies have shown that PON2 expression is upregulated in response to oxidative stress-inducing agents, while PON3 expression remains unchanged. While the physiological function of these proteins is unknown, studies currently underway using PON2 and PON3 knockout and transgenic mice should enable us to tease out the apparently redundant functions of these three proteins and yield clues as to their physiological function as well as their role in atherogenesis.

Heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme catabolism, exhibits potent antioxidant and anti-inflammatory properties. We developed HO-1 transgenic (Tg) mice using a rat HO-1 genomic transgene under the control of the endogenous promoter. Transgene expression was demonstrated by RT-PCR in all studied tissues, and a modest HO-1 overexpression was documented by Western, ELISA, and enzyme activity assays. To assess the effect of local vs systemic HO-1 in the acute rejection response, we used Tg mice as organ donors or recipients of MHC-incompatible heart grafts. In the local HO-1 overexpression model, Tg allografts survived 10.5 +/- 0.7 days (n = 10), compared with 6.5 +/- 0.4 days (n = 6) for wild-type donor controls (p = 0.0001). In the systemic HO-1 overexpression model, Tg recipients maintained allografts for 26.8 +/- 3.4 days (n = 10), compared with 6.3 +/- 0.1 days (n = 12) in wild-type controls (p = 0.00009). Inhibition of HO activity by treatment with tin protoporphyrin blunted survival advantage in Tg mice and resulted in acute graft rejection (n = 3). Increased carboxyhemoglobin levels were consistently noted in Tg mice. Comparisons of grafts at day 4 indicated that HO-1 overexpression was inversely associated with vasculitis/inflammatory cell infiltrate in both models. Hearts transplanted into Tg recipients showed decreased CD4(+) lymphocyte infiltration and diminished immune activation, as judged by CD25 expression. Thus, although local and systemic HO-1 overexpression improved allograft outcomes, systemic HO-1 led to a more robust protection and resulted in a significant blunting of host immune activation. This Tg mouse provides a valuable tool to study mechanisms by which HO-1 exerts beneficial effects in organ transplantation.

Background: Paraoxonase (PON1), a HDL-associated enzyme, protects against toxicity from specific organophosphorus compounds and oxidized lipids. Common polymorphisms in the PON1 gene have been identified and characterized in the coding region, 5' regulatory region and 3' UTR. The Q192R coding region polymorphism determines substrate-dependent differences in catalytic efficiency of hydrolysis. The -108CT polymorphism in the 5' regulatory region has a significant effect on PON1 expression, with the -108C allele expressing on average twice the level of plasma PON1 as the -108T allele. In addition to the effects of regulatory and coding region polymorphisms on PON1 levels and activity, plasma PON1 levels are also developmentally regulated. Since PON1 levels are important in determining resistance to specific organophosphorus compounds, the time course of appearance of PON1 in newborns is of great interest., Results: We report here that PON1 levels plateau between 6 to 15 months of age, and that variability in the age at which PON1 levels plateau is quite variable among individuals. In mice and rats, plasma PON1 activity reaches a plateau at 3 weeks of age. In mice that lack endogenous PON1, human transgenes encoding either PON1(Q192) or PON1(R192) under the control of the human PON1 regulatory sequences exhibited a similar time course of expression as that seen in wild-type mice, indicating conservation of the developmental regulatory elements between mouse and human PON1.

Human serum paraoxonase (PON1), an HDL-associated esterase, protects lipoproteins against oxidation, probably by hydrolyzing specific lipid peroxides. As arterial macrophages play a key role in oxidative stress in early atherogenesis, the aim of the present study was to examine the effect of PON1 on macrophage oxidative stress. For this purpose we used mouse arterial and peritoneal macrophages (MPM) that were harvested from two populations of PON1 knockout (KO) mice: one on the genetic background of C57BL/6J (PON1(0)) and the other one on the genetic background of apolipoproteinE KO (PON1(0)/E(0)). Serum and LDL, but not HDL, lipids peroxidation was increased in PON1(0), compared to C57BL/6J mice, by 84% and by 220%, respectively. Increased oxidative stress was shown in peritoneal and in arterial macrophages derived from either PON1(0) or PON1(0)/E(0) mice, compared to their appropriate controls. Macrophage oxidative stress was expressed by increased lipid peroxides content in MPM from PON1(0) and from PON1(0)/E(0) mice by 48% and by 80%, respectively, and by decreased reduced glutathione (GSH) content, compared to the appropriate controls. Furthermore, increased capacity of MPM from PON1(0) and PON1(0)/E(0) mice to oxidize LDL (by 40% and by 19%, respectively) and to release superoxide anions was observed. In accordance with these results, PON1(0) mice MPM exhibited 130% increased translocation of the cytosolic p47phox component of NADPH-oxidase to the macrophage plasma membrane, suggesting increased activation of macrophage NADPH-oxidase in PON1(0) mice, compared to control mice MPM. The increase in oxidative stress in PON1-deficient mice was observed despite the presence of the two other members of the PON gene family. PON2 and PON3 activities and mRNA expression were both found to be present in PON1-deficient mice MPM. Upon incubation of PON1(0)/E(0) derived macrophages with human PON1 (7.5 arylesterase units/ml), cellular peroxides content was decreased by 18%, macrophage superoxide anion release was decreased by 33%, and macrophage-mediated oxidation of LDL was reduced by 22%. Finally, a 42% increase in the atherosclerotic lesion area was observed in PON1(0)/E(0) mice, in comparison to E(0) mice under regular chow diet. We thus concluded that PON1 can directly reduce oxidative stress in macrophages and in serum, and that PON1-deficiency results in increased oxidative stress not only in serum, but also in macrophages, a phenomenon that can contribute to the accelerated atherosclerosis shown in PON1-deficient mice.