Mitochondrial CypD Acetylation Promotes Endothelial Dysfunction and Hypertension.

BACKGROUND: Nearly half of adults have hypertension, a major risk factor for cardiovascular disease. Mitochondrial hyperacetylation is linked to hypertension, but the role of acetylation of specific proteins is not clear. We hypothesized that acetylation of mitochondrial CypD (cyclophilin D) at K166 contributes to endothelial dysfunction and hypertension. METHODS: To test this hypothesis, we studied CypD acetylation in patients with essential hypertension, defined a pathogenic role of CypD acetylation in deacetylation mimetic CypD-K166R mutant mice and endothelial-specific GCN5L1 (general control of amino acid synthesis 5 like 1)-deficient mice using an Ang II (angiotensin II) model of hypertension. RESULTS: Arterioles from hypertensive patients had 280% higher CypD acetylation coupled with reduced Sirt3 (sirtuin 3) and increased GCN5L1 levels. GCN5L1 regulates mitochondrial protein acetylation and promotes CypD acetylation, which is counteracted by mitochondrial deacetylase Sirt3. In human aortic endothelial cells, GCN5L1 depletion prevents superoxide overproduction. Deacetylation mimetic CypD-K166R mice were protected from vascular oxidative stress, endothelial dysfunction, and Ang II-induced hypertension. Ang II-induced hypertension increased mitochondrial GCN5L1 and reduced Sirt3 levels resulting in a 250% increase in GCN5L1/Sirt3 ratio promoting CypD acetylation. Treatment with mitochondria-targeted scavenger of cytotoxic isolevuglandins (mito2HOBA) normalized GCN5L1/Sirt3 ratio, reduced CypD acetylation, and attenuated hypertension. The role of mitochondrial acetyltransferase GCN5L1 in the endothelial function was tested in endothelial-specific GCN5L1 knockout mice. Depletion of endothelial GCN5L1 prevented Ang II-induced mitochondrial oxidative stress, reduced the maladaptive switch of vascular metabolism to glycolysis, prevented inactivation of endothelial nitric oxide, preserved endothelial-dependent relaxation, and attenuated hypertension. CONCLUSIONS: These data support the pathogenic role of CypD acetylation in endothelial dysfunction and hypertension. We suggest that targeting cytotoxic mitochondrial isolevuglandins and GCN5L1 reduces CypD acetylation, which may be beneficial in cardiovascular disease.

Immunoproteasomal Processing of IsoLG-Adducted Proteins Is Essential for Hypertension.

BACKGROUND: Hypertension is characterized by CD8+ (cluster differentiation 8) T cell activation and infiltration into peripheral tissues. CD8+ T cell activation requires proteasomal processing of antigenic proteins. It has become clear that isoLG (isolevuglandin)-adduced peptides are antigenic in hypertension; however, IsoLGs inhibit the constitutive proteasome. We hypothesized that immunoproteasomal processing of isoLG-adducts is essential for CD8+ T cell activation and inflammation in hypertension. METHODS: IsoLG adduct processing was studied in murine dendritic cells (DCs), endothelial cells (ECs), and B8 fibroblasts. The role of the proteasome and the immunoproteasome in Ang II (angiotensin II)-induced hypertension was studied in C57BL/6 mice treated with bortezomib or the immunoproteasome inhibitor PR-957 and by studying mice lacking 3 critical immunoproteasome subunits (triple knockout mouse). We also examined hypertension in mice lacking the critical immunoproteasome subunit LMP7 (large multifunctional peptidase 7) specifically in either DCs or ECs. RESULTS: We found that oxidant stress increases the presence of isoLG adducts within MHC-I (class I major histocompatibility complex), and immunoproteasome overexpression augments this. Pharmacological or genetic inhibition of the immunoproteasome attenuated hypertension and tissue inflammation. Conditional deletion of LMP7 in either DCs or ECs attenuated hypertension and vascular inflammation. Finally, we defined the role of the innate immune receptors STING (stimulator of interferon genes) and TLR7/8 (toll-like receptor 7/8) as drivers of LMP7 expression in ECs. CONCLUSIONS: These studies define a previously unknown role of the immunoproteasome in DCs and ECs in CD8+ T cell activation. The immunoproteasome in DCs and ECs is critical for isoLG-adduct presentation to CD8+ T cells, and in the endothelium, this guides homing and infiltration of T cells to specific tissues.

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.

Isolevuglandin-Modified Cardiac Proteins Drive CD4+ T-Cell Activation in the Heart and Promote Cardiac Dysfunction.

BACKGROUND: Despite the well-established association between T-cell-mediated inflammation and nonischemic heart failure, the specific mechanisms triggering T-cell activation during the progression of heart failure and the antigens involved are poorly understood. We hypothesized that myocardial oxidative stress induces the formation of isolevuglandin (IsoLG)-modified proteins that function as cardiac neoantigens to elicit CD4+ T-cell receptor (TCR) activation and promote heart failure. METHODS: We used transverse aortic constriction in mice to trigger myocardial oxidative stress and T-cell infiltration. We profiled the TCR repertoire by mRNA sequencing of intramyocardial activated CD4+ T cells in Nur77GFP reporter mice, which transiently express GFP on TCR engagement. We assessed the role of antigen presentation and TCR specificity in the development of cardiac dysfunction using antigen presentation-deficient MhcII-/- mice and TCR transgenic OTII mice that lack specificity for endogenous antigens. We detected IsoLG protein adducts in failing human hearts. We also evaluated the role of reactive oxygen species and IsoLGs in eliciting T-cell immune responses in vivo by treating mice with the antioxidant TEMPOL and the IsoLG scavenger 2-hydroxybenzylamine during transverse aortic constriction, and ex vivo in mechanistic studies of CD4+ T-cell proliferation in response to IsoLG-modified cardiac proteins. RESULTS: We discovered that TCR antigen recognition increases in the left ventricle as cardiac dysfunction progresses and identified a limited repertoire of activated CD4+ T-cell clonotypes in the left ventricle. Antigen presentation of endogenous antigens was required to develop cardiac dysfunction because MhcII-/- mice reconstituted with CD4+ T cells and OTII mice immunized with their cognate antigen were protected from transverse aortic constriction-induced cardiac dysfunction despite the presence of left ventricle-infiltrated CD4+ T cells. Scavenging IsoLGs with 2-hydroxybenzylamine reduced TCR activation and prevented cardiac dysfunction. Mechanistically, cardiac pressure overload resulted in reactive oxygen species-dependent dendritic cell accumulation of IsoLG protein adducts, which induced robust CD4+ T-cell proliferation. CONCLUSIONS: Our study demonstrates an important role of reactive oxygen species-induced formation of IsoLG-modified cardiac neoantigens that lead to TCR-dependent CD4+ T-cell activation within the heart.

Dicarbonyl Electrophiles Mediate Inflammation-Induced Gastrointestinal Carcinogenesis.

BACKGROUND & AIMS: Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis. METHODS: The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori-induced carcinogenesis and in C57BL/6 mice treated with azoxymethane-dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori-infected INS-GAS mice was assessed by whole-exome sequencing. RESULTS: We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane-dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity. CONCLUSIONS: Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention.

Modified sites and functional consequences of 4-oxo-2-nonenal adducts in HDL that are elevated in familial hypercholesterolemia.

The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ∼46.5 to ∼18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia.

Simplified LC/MS assay for the measurement of isolevuglandin protein adducts in plasma and tissue samples.

Isolevuglandins (IsoLGs) are a family of highly reactive 4-ketoaldehydes formed by lipid peroxidation that modify the lysyl residues of cellular proteins. Modification of proteins by IsoLGs have been shown to contribute to disease processes such as the development of hypertension. Accurate quantitation of the extent of protein modification by IsoLGs is essential for understanding the mechanisms whereby these modifications contribute to disease and the efficacy of interventions designed to prevent this modification. The previously described LC/MS assay to quantitate IsoLG protein adducts was extremely labor-intensive and time consuming, and while it offered reasonably low intra-day variation for replicate samples, variation when replicate samples were processed on separate days was significant. These limitations significantly restricted utilization of this approach. We therefore performed a series of studies to optimize the assay. We now report a significantly simplified LC/MS assay for measurement of IsoLG protein adducts with increased sensitivity and lower intra-day and inter-day variability.

Pyrrole adducts in globin and plasma of workers exposed to hexane.

OBJECTIVES: Urinary excretion of 2,5-hexanedione is currently used to estimate the exposure levels of hexane occurring to an individual during the previous work shift. However, because hexane exposures and urinary 2,5-hexanedione levels can vary considerably from day to day, and subchronic to chronic exposures to hexane are required to produce neuropathy, this biomarker may not accurately reflect the risk of an individual for developing hexane neuropathy. This investigation examines the potential of hexane-derived pyrrole adducts produced on globin and plasma proteins as markers for integrating cumulative exposures. Because the pyrrole markers incorporate bioactivation of hexane to 2,5-hexandione and the initial step of protein adduction involved in hexane-induced neuropathy, they potentially can serve as biomarkers of effect through reflecting pathogenetic events within the nervous system. Additionally, pyrrole formation is an irreversible reaction suggesting that hexane-derived protein pyrroles can be used to assess cumulative exposures to provide a better characterization of individual susceptibilities. METHODS: To examine the utility of the proposed markers, blood samples were obtained from eleven workers who used hexane for granulating metal powders in a slurry to produce metal machining die tools and four non-exposed volunteers. Globin and plasma were isolated, and the proteins were digested using pepsin, reacted with Ehrlich's reagent and the level of pyrrole adducts were determined by absorbance at 530 nm. To determine the dose-response curve and dynamic range of the assay, erythrocytes were incubated with a range of 2,5-hexanedione concentrations and the net absorbance at 530 nm of isolated globin was measured. RESULTS: Pyrrole was detected in both the globin and plasma samples of the workers exposed to hexane and the levels of pyrroles in plasma were positively correlated with the levels of pyrroles in globin for most of the workers. CONCLUSIONS: This investigation demonstrates that detectable levels of hexane-derived protein pyrrole adducts are produced on peripheral proteins following occupational exposures to hexane and supports the utility of measuring pyrroles for integrating cumulative exposures to hexane.

Reactive γ-ketoaldehydes promote protein misfolding and preamyloid oligomer formation in rapidly-activated atrial cells.

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid ß1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.

Modification of platelet proteins by malondialdehyde: prevention by dicarbonyl scavengers.

The thromboxane synthase converts prostaglandin H(2) to thromboxane A(2) and malondialdehyde (MDA) in approximately equimolar amounts. A reactive dicarbonyl, MDA forms covalent adducts of amino groups, including the ε-amine of lysine, but the importance of this reaction in platelets was unknown. Utilizing a novel LC/MS/MS method for analysis of one of the MDA adducts, the dilysyl-MDA cross-link, we demonstrated that dilysyl-MDA cross-links in human platelets are formed following platelet activation via the cyclooxygenase (COX)-1/thromboxane synthase pathway. Salicylamine and analogs of salicylamine were shown to react with MDA preferentially, thereby preventing formation of lysine adducts. Dilysyl-MDA cross-links were measured in two diseases known to be associated with increased platelet activation. Levels of platelet dilysyl-MDA cross-links were increased by 2-fold in metabolic syndrome relative to healthy subjects, and by 1.9-fold in sickle cell disease (SCD). In patients with SCD, the reduction of platelet dilysyl-MDA cross-links following administration of nonsteroidal anti-inflammatory drug provided evidence that MDA modifications of platelet proteins in this disease are derived from the COX pathway. In summary, MDA adducts of platelet proteins that cross-link lysines are formed on platelet activation and are increased in diseases associated with platelet activation. These protein modifications can be prevented by salicylamine-related scavengers.

Scavenging 4-Oxo-2-nonenal.

4-Oxo-2-nonenal (ONE), a product of cellular lipid oxidation, reacts nonspecifically with the lysine residues of proteins and is generated in increased amounts during degenerative diseases and cancer. We show that pyridoxamine, salicylamine, and related 2-aminomethylphenols react with ONE, to form pyrrolo[2,1-b][1,3]oxazines with the participation of both the amino and the phenolic groups. 2-Aminomethylphenols react with ONE as well as with the Michael adducts of ONE much more rapidly than lysine, suggesting their use for therapeutically scavenging ONE.

5'-O-Alkylpyridoxamines: Lipophilic Analogues of Pyridoxamine Are Potent Scavengers of 1,2-Dicarbonyls.

Pyridoxamine (PM) is a prospective drug for the treatment of diabetic complications. In order to make zwitterionic PM more lipophilic and improve its tissue distribution, PM derivatives containing medium length alkyl groups on the hydroxymethyl side chain were prepared. The synthesis of these alkylpyridoxamines (alkyl-PMs) starting from pyridoxine offers high yields and is amenable to bulk preparations. Interestingly, alkyl-PMs were found to react with methylglyoxal (MGO), a major toxic product of glucose metabolism and autoxidation, several orders of magnitude faster than PM. This suggests the formation of nonionic pyrido-1,3-oxazine as the key step in the reaction of PM with MGO. Since the primary target of MGO in proteins is the guanidine side chain of arginine, alkyl-PMs were shown to be more effective than PM in reducing the modification of N-α-benzoylarginine by MGO. Alkyl-PMs in the presence of MGO also protected the enzymatic activity of lysozyme that contains several arginine residues next to its active site. Alkyl-PMs can be expected to trap MGO and other toxic 1,2-carbonyl compounds more effectively than PM, especially in lipophilic tissue environments, thus protecting macromolecules from functional damage. This suggests potential therapeutic uses for alkyl-PMs in diabetes and other diseases characterized by the elevated levels of toxic dicarbonyl compounds.

Levuglandin forms adducts with histone h4 in a cyclooxygenase-2-dependent manner, altering its interaction with DNA.

Inflammation and subsequent cyclooxygenase-2 (COX-2) activity has long been linked with the development of cancer, although little is known about any epigenetic effects of COX-2. A product of COX-2 activation, levuglandin (LG) quickly forms covalent bonds with nearby primary amines, such as those in lysine, which leads to LG-protein adducts. Here, we demonstrate that COX-2 activity causes LG-histone adducts in cultured cells and liver tissue, detectable through LC-MS, with the highest incidence in histone H4. Adduction is blocked by a γ-ketoaldehyde scavenger, which has no effect on COX-2 activity as measured by PGE2 production. Formation of the LG-histone adduct is associated with an increased histone solubility in NaCl, indicating destabilization of the nucleosome structure; this is also reversed with scavenger treatment. These data demonstrate that COX-2 activity can cause histone adduction and loosening of the nucleosome complex, which could lead to altered transcription and contribute to carcinogenesis.

Isolevuglandin-modified phosphatidylethanolamine is metabolized by NAPE-hydrolyzing phospholipase D.

Lipid aldehydes including isolevuglandins (IsoLGs) and 4-hydroxynonenal modify phosphatidylethanolamine (PE) to form proinflammatory and cytotoxic adducts. Therefore, cells may have evolved mechanisms to degrade and prevent accumulation of these potentially harmful compounds. To test if cells could degrade isolevuglandin-modified phosphatidylethanolamine (IsoLG-PE), we generated IsoLG-PE in human embryonic kidney 293 (HEK293) cells and human umbilical cord endothelial cells and measured its stability over time. We found that IsoLG-PE levels decreased more than 75% after 6 h, suggesting that IsoLG-PE was indeed degraded. Because N-acyl phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) has been described as a key enzyme in the hydrolysis of N-acyl phosphatidylethanoamine (NAPE) and both NAPE and IsoLG-PE have large aliphatic headgroups, we considered the possibility that this enzyme might also hydrolyze IsoLG-PE. We found that knockdown of NAPE-PLD expression using small interfering RNA (siRNA) significantly increased the persistence of IsoLG-PE in HEK293 cells. IsoLG-PE competed with NAPE for hydrolysis by recombinant mouse NAPE-PLD, with the catalytic efficiency (V(max)/K(m)) for hydrolysis of IsoLG-PE being 30% of that for hydrolysis of NAPE. LC-MS/MS analysis confirmed that recombinant NAPE-PLD hydrolyzed IsoLG-PE to IsoLG-ethanolamine. These results demonstrate that NAPE-PLD contributes to the degradation of IsoLG-PE and suggest that a major physiological role of NAPE-PLD may be to degrade aldehyde-modified PE, thereby preventing the accumulation of these harmful compounds.

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.

Immunoproteasomal Processing of Isolevuglandin Adducts in Hypertension.

Isolevuglandins (isoLGs) are lipid aldehydes that form in the presence of reactive oxygen species (ROS) and drive immune activation. We found that isoLG-adducts are presented within the context of major histocompatibility complexes (MHC-I) by an immunoproteasome dependent mechanism. Pharmacologic inhibition of LMP7, the chymotrypsin subunit of the immunoproteasome, attenuates hypertension and tissue inflammation in the angiotensin II (Ang II) model of hypertension. Genetic loss of function of all immunoproteasome subunits or conditional deletion of LMP7 in dendritic cell (DCs) or endothelial cells (ECs) attenuated hypertension, reduced aortic T cell infiltration, and reduced isoLG-adduct MHC-I interaction. Furthermore, isoLG adducts structurally resemble double-stranded DNA and contribute to the activation of STING in ECs. These studies define a critical role of the immunoproteasome in the processing and presentation of isoLG-adducts. Moreover they define a role of LMP7 as a regulator of T cell activation and tissue infiltration in hypertension.

Phosphatidylethanolamines modified by γ-ketoaldehyde (γKA) induce endoplasmic reticulum stress and endothelial activation.

Peroxidation of plasma lipoproteins has been implicated in the endothelial cell activation and monocyte adhesion that initiate atherosclerosis, but the exact mechanisms underlying this activation remain unclear. Lipid peroxidation generates lipid aldehydes, including the γ-ketoaldehydes (γKA), also termed isoketals or isolevuglandins, that readily modify the amine headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde modification of PE could mediate some of the proinflammatory effects of lipid peroxidation. We found that PE modified by γKA (γKA-PE) induced THP-1 monocyte adhesion to human umbilical cord endothelial cells. γKA-PE also induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in human umbilical cord endothelial cells. To determine the structural requirements for γKA-PE activity, we tested several related compounds. PE modified by 4-oxo-pentanal induced THP-1 adhesion, but N-glutaroyl-PE and C(18:0)N-acyl-PE did not, suggesting that an N-pyrrole moiety was essential for cellular activity. As the N-pyrrole headgroup might distort the membrane, we tested the effect of the pyrrole-PEs on membrane parameters. γKA-PE and 4-oxo-pentanal significantly reduced the temperature for the liquid crystalline to hexagonal phase transition in artificial bilayers, suggesting that these pyrrole-PE markedly altered membrane curvature. Additionally, fluorescently labeled γKA-PE rapidly internalized to the endoplasmic reticulum (ER); γKA-PE induced C/EBP homologous protein CHOP and BiP expression and p38 MAPK activity, and inhibitors of ER stress reduced γKA-PE-induced C/EBP homologous protein CHOP and BiP expression as well as EC activation, consistent with γKA-PE inducing ER stress responses that have been previously linked to inflammatory chemokine expression. Thus, γKA-PE is a potential mediator of the inflammation induced by lipid peroxidation.

IsoLGs (Isolevuglandins) Drive Neutrophil Migration in Hypertension and Are Essential for the Formation of Neutrophil Extracellular Traps.

BACKGROUND: IsoLGs (isolevuglandins) are electrophilic products of lipid peroxidation formed in the presence of reactive oxygen species. IsoLGs contribute to hypertension by an unknown mechanism. Studies have shown that reactive oxygen species production drives the formation of neutrophil extracellular traps (NETs) and that NETs accumulate within the aorta and kidneys of patients with hypertension. The purpose of this study was to determine the role of isoLGs in neutrophil migration and NET formation (NETosis) in hypertension. METHODS: Mice were treated with Ang II (angiotensin II) and the specific isoLG scavenger 2-hydroxybenzylamine and examined for tissue neutrophil and NET accumulation by single-cell sequencing and flow cytometry. Isolated human neutrophils were studied to determine the role of isoLGs in NETosis and neutrophil chromatin expansion by immunofluorescence and live cell confocal microscopy. RESULTS: Single-cell sequencing performed on sham, Ang II, and Ang II+2-hydroxybenzylamine treated mice revealed neutrophils as a primary target of 2-hydroxybenzylamine. Peripheral neutrophil migration, aortic NET accumulation, and renal NET accumulation is blocked with 2-hydroxybenzylamine treatment. In isolated human neutrophils, isoLGs accumulate during NETosis and scavenging of isoLGs prevents NETosis. IsoLGs drive neutrophil chromatin expansion during NETosis and disrupt nucleosome structure. CONCLUSIONS: These observations identified a critical role of isoLGs in neutrophil migration and NETosis in hypertension and provide a potential therapy for NET-associated diseases including hypertension and associated end organ damage.

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.

Isoketals form cytotoxic phosphatidylethanolamine adducts in cells.

Levuglandins and their stereo- and regio-isomers (termed isolevuglandins or isoketals) are gamma-ketoaldehydes (IsoK) that rapidly react with lysines to form stable protein adducts. IsoK protein adduct levels increase in several pathological conditions including cardiovascular disease. IsoKs can induce ion channel dysfunction and cell death, potentially by adducting to cellular proteins. However, IsoKs also adduct to phosphatidylethanolamine (PE) in vitro, and whether PE adducts form in cells or contribute to the effects of IsoKs is unknown. When radiolabeled IsoK was added to HEK293 cells, 40% of the radiolabel extracted into the chloroform lower phase suggesting the possible formation of PE adducts. We therefore developed methods to measure IsoK-PE adducts in cells. IsoK-PE was quantified by LC/MS/MS after hydrolysis to IsoK-ethanolamine by Streptomyces chromofuscus phospholipase D. In HEK293 and human umbilical vein endothelial cells (HUVEC), IsoK dose-dependently increased PE adduct concentrations to a greater extent than protein adduct. To test the biological significance of IsoK-PE formation, we treated HUVEC with IsoK-PE. IsoK-PE dose dependently induced cytotoxicity (LC(50) 2.2 muM). These results indicate that cellular PE is a significant target of IsoKs, and that formation of PE adducts may mediate some of the biological effects of IsoKs relevant to disease.