Isolevuglandins Promote Mitochondrial Dysfunction and Electrophysiologic Abnormalities in Atrial Cardiomyocytes.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet the cellular and molecular mechanisms underlying the AF substrate remain unclear. Isolevuglandins (IsoLGs) are highly reactive lipid dicarbonyl products that mediate oxidative stress-related injury. In murine hypertension, the lipid dicarbonyl scavenger 2-hydroxybenzylamine (2-HOBA) reduced IsoLGs and AF susceptibility. We hypothesized that IsoLGs mediate detrimental pathophysiologic effects in atrial cardiomyocytes that promote the AF substrate. Using Seahorse XFp extracellular flux analysis and a luminescence assay, IsoLG exposure suppressed intracellular ATP production in atrial HL-1 cardiomyocytes. IsoLGs caused mitochondrial dysfunction, with reduced mitochondrial membrane potential, increased mitochondrial reactive oxygen species (ROS) with protein carbonylation, and mitochondrial DNA damage. Moreover, they generated cytosolic preamyloid oligomers previously shown to cause similar detrimental effects in atrial cells. In mouse atrial and HL-1 cells, patch clamp experiments demonstrated that IsoLGs rapidly altered action potentials (AP), implying a direct effect independent of oligomer formation by reducing the maximum Phase 0 upstroke slope and shortening AP duration due to ionic current modifications. IsoLG-mediated mitochondrial and electrophysiologic abnormalities were blunted or totally prevented by 2-HOBA. These findings identify IsoLGs as novel mediators of oxidative stress-dependent atrial pathophysiology and support the investigation of dicarbonyl scavengers as a novel therapeutic approach to prevent AF.

Small Molecule Activation of NAPE-PLD Enhances Efferocytosis by Macrophages.

N-Acyl-phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase that hydrolyzes N-acyl-phosphatidylethanolamines (NAPEs) to form N-acyl-ethanolamines (NAEs) and phosphatidic acid. Several lines of evidence suggest that reduced NAPE-PLD activity could contribute to cardiometabolic diseases. For instance, NAPEPLD expression is reduced in human coronary arteries with unstable atherosclerotic lesions, defective efferocytosis is implicated in the enlargement of necrotic cores of these lesions, and NAPE-PLD products such as palmitoylethanolamide and oleoylethanolamide have been shown to enhance efferocytosis. Thus, enzyme activation mediated by a small molecule may serve as a therapeutic treatment for cardiometabolic diseases. As a proof-of-concept study, we sought to identify small molecule activators of NAPE-PLD. High-throughput screening followed by hit validation and primary lead optimization studies identified a series of benzothiazole phenylsulfonyl-piperidine carboxamides that variably increased activity of both mouse and human NAPE-PLD. From this set of small molecules, two NAPE-PLD activators (VU534 and VU533) were shown to increase efferocytosis by bone-marrow derived macrophages isolated from wild-type mice, while efferocytosis was significantly reduced in Napepld-/- BMDM or after Nape-pld inhibition. Together, these studies demonstrate an essential role for NAPE-PLD in the regulation of efferocytosis and the potential value of NAPE-PLD activators as a strategy to treat cardiometabolic diseases.

HDL Function and Atherosclerosis: Reactive Dicarbonyls as Promising Targets of Therapy.

Epidemiologic studies detected an inverse relationship between HDL (high-density lipoprotein) cholesterol (HDL-C) levels and atherosclerotic cardiovascular disease (ASCVD), identifying HDL-C as a major risk factor for ASCVD and suggesting atheroprotective functions of HDL. However, the role of HDL-C as a mediator of risk for ASCVD has been called into question by the failure of HDL-C-raising drugs to reduce cardiovascular events in clinical trials. Progress in understanding the heterogeneous nature of HDL particles in terms of their protein, lipid, and small RNA composition has contributed to the realization that HDL-C levels do not necessarily reflect HDL function. The most examined atheroprotective function of HDL is reverse cholesterol transport, whereby HDL removes cholesterol from plaque macrophage foam cells and delivers it to the liver for processing and excretion into bile. Indeed, in several studies, HDL has shown inverse associations between HDL cholesterol efflux capacity and ASCVD in humans. Inflammation plays a key role in the pathogenesis of atherosclerosis and vulnerable plaque formation, and a fundamental function of HDL is suppression of inflammatory signaling in macrophages and other cells. Oxidation is also a critical process to ASCVD in promoting atherogenic oxidative modifications of LDL (low-density lipoprotein) and cellular inflammation. HDL and its proteins including apoAI (apolipoprotein AI) and PON1 (paraoxonase 1) prevent cellular oxidative stress and LDL modifications. Importantly, HDL in humans with ASCVD is oxidatively modified rendering HDL dysfunctional and proinflammatory. Modification of HDL with reactive carbonyl species, such as malondialdehyde and isolevuglandins, dramatically impairs the antiatherogenic functions of HDL. Importantly, treatment of murine models of atherosclerosis with scavengers of reactive dicarbonyls improves HDL function and reduces systemic inflammation, atherosclerosis development, and features of plaque instability. Here, we discuss the HDL antiatherogenic functions in relation to oxidative modifications and the potential of reactive dicarbonyl scavengers as a therapeutic approach for ASCVD.

Small Molecule Activation of NAPE-PLD Enhances Efferocytosis by Macrophages.

N -acyl-phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase that hydrolyzes N -acyl-phosphatidylethanolamine (NAPEs) to form N -acyl-ethanolamides (NAEs) and phosphatidic acid. Several lines of evidence suggest that reduced NAPE-PLD activity could contribute to cardiometabolic diseases. For instance, NAPEPLD expression is reduced in human coronary arteries with unstable atherosclerotic lesions, defective efferocytosis is implicated in the enlargement of necrotic cores of these lesions, and NAPE-PLD products such as palmitoylethanolamide and oleoylethanolamide have been shown to enhance efferocytosis. Thus, enzyme activation mediated by a small molecule may serve as a therapeutic treatment for cardiometabolic diseases. As a proof-of-concept study, we sought to identify small molecule activators of NAPE-PLD. High-throughput screening followed by hit validation and primary lead optimization studies identified a series of benzothiazole phenylsulfonyl-piperidine carboxamides that variably increased activity of both mouse and human NAPE-PLD. From this set of small molecules, two NAPE-PLD activators (VU534 and VU533) were shown to increase efferocytosis by bone-marrow derived macrophages isolated from wild-type mice, while efferocytosis was significantly reduced in Napepld -/- BMDM or after Nape-pld inhibition. Together these studies demonstrate an essential role for NAPE-PLD in the regulation of efferocytosis and the potential value of NAPE-PLD activators as a strategy to treat cardiometabolic diseases.

Scavenging dicarbonyls with 5'-O-pentyl-pyridoxamine increases HDL net cholesterol efflux capacity and attenuates atherosclerosis and insulin resistance.

OBJECTIVE: Oxidative stress contributes to the development of insulin resistance (IR) and atherosclerosis. Peroxidation of lipids produces reactive dicarbonyls such as Isolevuglandins (IsoLG) and malondialdehyde (MDA) that covalently bind plasma/cellular proteins, phospholipids, and DNA leading to altered function and toxicity. We examined whether scavenging reactive dicarbonyls with 5'-O-pentyl-pyridoxamine (PPM) protects against the development of IR and atherosclerosis in Ldlr-/- mice. METHODS: Male or female Ldlr-/- mice were fed a western diet (WD) for 16 weeks and treated with PPM versus vehicle alone. Plaque extent, dicarbonyl-lysyl adducts, efferocytosis, apoptosis, macrophage inflammation, and necrotic area were measured. Plasma MDA-LDL adducts and the in vivo and in vitro effects of PPM on the ability of HDL to reduce macrophage cholesterol were measured. Blood Ly6Chi monocytes and ex vivo 5-ethynyl-2'-deoxyuridine (EdU) incorporation into bone marrow CD11b+ monocytes and CD34+ hematopoietic stem and progenitor cells (HSPC) were also examined. IR was examined by measuring fasting glucose/insulin levels and tolerance to insulin/glucose challenge. RESULTS: PPM reduced the proximal aortic atherosclerosis by 48% and by 46% in female and male Ldlr-/- mice, respectively. PPM also decreased IR and hepatic fat and inflammation in male Ldlr-/- mice. Importantly, PPM decreased plasma MDA-LDL adducts and prevented the accumulation of plaque MDA- and IsoLG-lysyl adducts in Ldlr-/- mice. In addition, PPM increased the net cholesterol efflux capacity of HDL from Ldlr-/- mice and prevented both the in vitro impairment of HDL net cholesterol efflux capacity and apoAI crosslinking by MPO generated hypochlorous acid. Moreover, PPM decreased features of plaque instability including decreased proinflammatory M1-like macrophages, IL-1ß expression, myeloperoxidase, apoptosis, and necrotic core. In contrast, PPM increased M2-like macrophages, Tregs, fibrous cap thickness, and efferocytosis. Furthermore, PPM reduced inflammatory monocytosis as evidenced by decreased blood Ly6Chi monocytes and proliferation of bone marrow monocytes and HSPC from Ldlr-/- mice. CONCLUSIONS: PPM has pleotropic atheroprotective effects in a murine model of familial hypercholesterolemia, supporting the therapeutic potential of reactive dicarbonyl scavenging in the treatment of IR and atherosclerotic cardiovascular disease.

Oxidative modification of HDL by lipid aldehydes impacts HDL function.

Reduced levels of high-density lipoprotein (HDL) cholesterol correlate with increased risk for atherosclerotic cardiovascular diseases and HDL performs functions including reverse cholesterol transport, inhibition of lipid peroxidation, and suppression of inflammation, that would appear critical for cardioprotection. However, several large clinical trials utilizing pharmacologic interventions that elevated HDL cholesterol levels failed to provide cardioprotection to at-risk individuals. The reasons for these unexpected results have only recently begun to be elucidated. HDL cholesterol levels and HDL function can be significantly discordant, so that elevating HDL cholesterol levels may not necessarily lead to increased functional capacity, particularly under conditions that cause HDL to become oxidatively modified, resulting in HDL dysfunction. Here we review evidence that oxidative modifications of HDL, including by reactive lipid aldehydes generated by lipid peroxidation, reduce HDL functionality and that dicarbonyl scavengers that protect HDL against lipid aldehyde modification are beneficial in pre-clinical models of atherosclerotic cardiovascular disease.

Dicarbonyl-modified lipoproteins contribute to proteinuric kidney injury.

Lipoprotein modification by reactive dicarbonyls, including isolevuglandin (IsoLG), produces dysfunctional particles. Kidneys participate in lipoprotein metabolism, including tubular uptake. However, the process beyond the proximal tubule is unclear, as is the effect of kidney injury on this pathway. We found that patients and animals with proteinuric injury have increased urinary apolipoprotein AI (apoAI), IsoLG, and IsoLG adduct enrichment of the urinary apoAI fraction compared with other proteins. Proteinuric mice, induced by podocyte-specific injury, showed more tubular absorption of IsoLG-apoAI and increased expression of lipoprotein transporters in proximal tubular cells compared with uninjured animals. Renal lymph reflects composition of the interstitial compartment and showed increased apoAI and IsoLG in proteinuric animals, supporting a tubular cell-interstitium-lymph pathway for renal handling of lipoproteins. IsoLG-modified apoAI was not only a marker of renal injury but also directly damaged renal cells. IsoLG-apoAI increased inflammatory cytokines in cultured tubular epithelial cells (TECs), activated lymphatic endothelial cells (LECs), and caused greater contractility of renal lymphatic vessels than unmodified apoAI. In vivo, inhibition of IsoLG by a dicarbonyl scavenger reduced both albuminuria and urinary apoAI and decreased TEC and LEC injury, lymphangiogenesis, and interstitial fibrosis. Our results indicate that IsoLG-modified apoAI is, to our knowledge, a novel pathogenic mediator and therapeutic target in kidney disease.

DC ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.

BACKGROUND: Salt sensitivity of blood pressure is an independent predictor of cardiovascular morbidity and mortality. The exact mechanism by which salt intake increases blood pressure and cardiovascular risk is unknown. We previously found that sodium entry into antigen-presenting cells (APCs) via the amiloride-sensitive epithelial sodium channel EnaC (epithelial sodium channel) leads to the formation of IsoLGs (isolevuglandins) and release of proinflammatory cytokines to activate T cells and modulate salt-sensitive hypertension. In the current study, we hypothesized that ENaC-dependent entry of sodium into APCs activates the NLRP3 (NOD [nucleotide-binding and oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome via IsoLG formation leading to salt-sensitive hypertension. METHODS: We performed RNA sequencing on human monocytes treated with elevated sodium in vitro and Cellular Indexing of Transcriptomes and Epitopes by Sequencing analysis of peripheral blood mononuclear cells from participants rigorously phenotyped for salt sensitivity of blood pressure using an established inpatient protocol. To determine mechanisms, we analyzed inflammasome activation in mouse models of deoxycorticosterone acetate salt-induced hypertension as well as salt-sensitive mice with ENaC inhibition or expression, IsoLG scavenging, and adoptive transfer of wild-type dendritic cells into NLRP3 deficient mice. RESULTS: We found that high levels of salt exposure upregulates the NLRP3 inflammasome, pyroptotic and apoptotic caspases, and IL (interleukin)-1ß transcription in human monocytes. Cellular Indexing of Transcriptomes and Epitopes by Sequencing revealed that components of the NLRP3 inflammasome and activation marker IL-1ß dynamically vary with changes in salt loading/depletion. Mechanistically, we found that sodium-induced activation of the NLRP3 inflammasome is ENaC and IsoLG dependent. NLRP3 deficient mice develop a blunted hypertensive response to elevated sodium, and this is restored by the adoptive transfer of NLRP3 replete APCs. CONCLUSIONS: These findings reveal a mechanistic link between ENaC, inflammation, and salt-sensitive hypertension involving NLRP3 inflammasome activation in APCs. APC activation via the NLRP3 inflammasome can serve as a potential diagnostic biomarker for salt sensitivity of blood pressure.

Isolevuglandins disrupt PU.1-mediated C1q expression and promote autoimmunity and hypertension in systemic lupus erythematosus.

We describe a mechanism responsible for systemic lupus erythematosus (SLE). In humans with SLE and in 2 SLE murine models, there was marked enrichment of isolevuglandin-adducted proteins (isoLG adducts) in monocytes and dendritic cells. We found that antibodies formed against isoLG adducts in both SLE-prone mice and humans with SLE. In addition, isoLG ligation of the transcription factor PU.1 at a critical DNA binding site markedly reduced transcription of all C1q subunits. Treatment of SLE-prone mice with the specific isoLG scavenger 2-hydroxybenzylamine (2-HOBA) ameliorated parameters of autoimmunity, including plasma cell expansion, circulating IgG levels, and anti-dsDNA antibody titers. 2-HOBA also lowered blood pressure, attenuated renal injury, and reduced inflammatory gene expression uniquely in C1q-expressing dendritic cells. Thus, isoLG adducts play an essential role in the genesis and maintenance of systemic autoimmunity and hypertension in SLE.

Elucidation of physico-chemical principles of high-density lipoprotein-small RNA binding interactions.

Extracellular small RNAs (sRNAs) are abundant in many biofluids, but little is known about their mechanisms of transport and stability in RNase-rich environments. We previously reported that high-density lipoproteins (HDLs) in mice were enriched with multiple classes of sRNAs derived from the endogenous transcriptome, but also from exogenous organisms. Here, we show that human HDL transports tRNA-derived sRNAs (tDRs) from host and nonhost species, the profiles of which were found to be altered in human atherosclerosis. We hypothesized that HDL binds to tDRs through apolipoprotein A-I (apoA-I) and that these interactions are conferred by RNA-specific features. We tested this using microscale thermophoresis and electrophoretic mobility shift assays and found that HDL binds to tDRs and other single-stranded sRNAs with strong affinity but did not bind to double-stranded RNA or DNA. Furthermore, we show that natural and synthetic RNA modifications influenced tDR binding to HDL. We demonstrate that reconstituted HDL bound to tDRs only in the presence of apoA-I, and purified apoA-I alone were able to bind sRNA. Conversely, phosphatidylcholine vesicles did not bind tDRs. In summary, we conclude that HDL binds to single-stranded sRNAs likely through nonionic interactions with apoA-I. These results highlight binding properties that likely enable extracellular RNA communication and provide a foundation for future studies to manipulate HDL-sRNA interactions for therapeutic approaches to prevent or treat disease.

Selective measurement of NAPE-PLD activity via a PLA1/2-resistant fluorogenic N-acyl-phosphatidylethanolamine analog.

N-acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase enzyme that converts NAPEs to bioactive N-acyl-ethanolamides. Altered NAPE-PLD activity may contribute to pathogenesis of obesity, diabetes, atherosclerosis, and neurological diseases. Selective measurement of NAPE-PLD activity is challenging, however, because of alternative phospholipase pathways for NAPE hydrolysis. Previous methods to measure NAPE-PLD activity involved addition of exogenous NAPE followed by TLC or LC/MS/MS, which are time and resource intensive. Recently, NAPE-PLD activity in cells has been assayed using the fluorogenic NAPE analogs PED-A1 and PED6, but these substrates also detect the activity of serine hydrolase-type lipases PLA1 and PLA2. To create a fluorescence assay that selectively measured cellular NAPE-PLD activity, we synthesized an analog of PED-A1 (flame-NAPE) where the sn-1 ester bond was replaced with an N-methyl amide to create resistance to PLA1 hydrolysis. Recombinant NAPE-PLD produced fluorescence when incubated with either PED-A1 or flame-NAPE, whereas PLA1 only produced fluorescence when incubated with PED-A1. Furthermore, fluorescence in HepG2 cells using PED-A1 could be partially blocked by either biothionol (a selective NAPE-PLD inhibitor) or tetrahydrolipstatin (an inhibitor of a broad spectrum of serine hydrolase-type lipases). In contrast, fluorescence assayed in HepG2 cells using flame-NAPE could only be blocked by biothionol. In multiple cell types, the phospholipase activity detected using flame-NAPE was significantly more sensitive to biothionol inhibition than that detected using PED-A1. Thus, using flame-NAPE to measure phospholipase activity provides a rapid and selective method to measure NAPE-PLD activity in cells and tissues.

Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence.

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.

Myeloperoxidase-induced modification of HDL by isolevuglandins inhibits paraoxonase-1 activity.

Reduced activity of paraoxonase 1 (PON1), a high-density lipoprotein (HDL)-associated enzyme, has been implicated in the development of atherosclerosis. Post-translational modifications of PON1 may represent important mechanisms leading to reduced PON1 activity. Under atherosclerotic conditions, myeloperoxidase (MPO) is known to associate with HDL. MPO generates the oxidants hypochlorous acid and nitrogen dioxide, which can lead to post-translational modification of PON1, including tyrosine modifications that inhibit PON1 activity. Nitrogen dioxide also drives lipid peroxidation, leading to the formation of reactive lipid dicarbonyls such as malondialdehyde and isolevuglandins, which modify HDL and could inhibit PON1 activity. Because isolevuglandins are more reactive than malondialdehyde, we used in vitro models containing HDL, PON1, and MPO to test the hypothesis that IsoLG formation by MPO and its subsequent modification of HDL contributes to MPO-mediated reductions in PON1 activity. Incubation of MPO with HDL led to modification of HDL proteins, including PON1, by IsoLG. Incubation of HDL with IsoLG reduced PON1 lactonase and antiperoxidation activities. IsoLG modification of recombinant PON1 markedly inhibited its activity, while irreversible IsoLG modification of HDL before adding recombinant PON1 only slightly inhibited the ability of HDL to enhance the catalytic activity of recombinant PON1. Together, these studies support the notion that association of MPO with HDL leads to lower PON1 activity in part via IsoLG-mediated modification of PON1, so that IsoLG modification of PON1 could contribute to increased risk for atherosclerosis, and blocking this modification might prove beneficial to reduce atherosclerosis.

Kidney injury-mediated disruption of intestinal lymphatics involves dicarbonyl-modified lipoproteins.

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.

Sodium activates human monocytes via the NADPH oxidase and isolevuglandin formation.

AIMS: Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro. METHODS AND RESULTS: To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments. Exposure of human monocytes to elevated Na+ex vivo caused a co-ordinated response involving isolevuglandin (IsoLG)-adduct formation, acquisition of a dendritic cell (DC)-like morphology, expression of activation markers CD83 and CD16, and increased production of pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-6, and IL-1ß. High salt also caused a marked change in monocyte gene expression as detected by RNA sequencing and enhanced monocyte migration to the chemokine CC motif chemokine ligand 5. NADPH-oxidase inhibition attenuated monocyte activation and IsoLG-adduct formation. The increase in IsoLG-adducts correlated with risk factors including body mass index, pulse pressure. Monocytes exposed to high salt stimulated IL-17A production from autologous CD4+ and CD8+ T cells. In addition, to evaluate the effect of salt in vivo, monocytes and T cells isolated from humans were adoptively transferred to immunodeficient NSG mice. Salt feeding of humanized mice caused monocyte-dependent activation of human T cells reflected by proliferation and accumulation of T cells in the bone marrow. Moreover, we performed a cross-sectional study in 70 prehypertensive subjects. Blood was collected for flow cytometric analysis and 23Na magnetic resonance imaging was performed for tissue sodium measurements. Monocytes from humans with high skin Na+ exhibited increased IsoLG-adduct accumulation and CD83 expression. CONCLUSION: Human monocytes exhibit co-ordinated increases in parameters of activation, conversion to a DC-like phenotype and ability to activate T cells upon both in vitro and in vivo sodium exposure. The ability of monocytes to be activated by sodium is related to in vivo cardiovascular disease risk factors. We therefore propose that in addition to the kidney and vasculature, immune cells like monocytes convey salt-induced cardiovascular risk in humans.

Scavenging Reactive Lipids to Prevent Oxidative Injury.

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.

Scavenging of reactive dicarbonyls with 2-hydroxybenzylamine reduces atherosclerosis in hypercholesterolemic Ldlr-/- mice.

Lipid peroxidation generates reactive dicarbonyls including isolevuglandins (IsoLGs) and malondialdehyde (MDA) that covalently modify proteins. Humans with familial hypercholesterolemia (FH) have increased lipoprotein dicarbonyl adducts and dysfunctional HDL. We investigate the impact of the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in Ldlr-/- mice, a model of FH. Compared to hypercholesterolemic Ldlr-/- mice treated with vehicle or 4-HOBA, a nonreactive analogue, 2-HOBA decreases atherosclerosis by 60% in en face aortas, without changing plasma cholesterol. Ldlr-/- mice treated with 2-HOBA have reduced MDA-LDL and MDA-HDL levels, and their HDL display increased capacity to reduce macrophage cholesterol. Importantly, 2-HOBA reduces the MDA- and IsoLG-lysyl content in atherosclerotic aortas versus 4-HOBA. Furthermore, 2-HOBA reduces inflammation and plaque apoptotic cells and promotes efferocytosis and features of stable plaques. Dicarbonyl scavenging with 2-HOBA has multiple atheroprotective effects in a murine FH model, supporting its potential as a therapeutic approach for atherosclerotic cardiovascular disease.

Engineering the gut microbiota to treat chronic diseases.

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.