Protective Effect of NO2-OA on Oxidative Stress, Gliosis, and Pro-Angiogenic Response in Müller Glial Cells.

Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α2-macroglobulin (α2M) and gliosis in Müller glial cells (MGCs, such as MIO-M1). It is well known that MGCs play important roles in healthy and sick retinas, including in PR. Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO2-OA) is beneficial against oxidative stress, gliosis, and the pro-angiogenic response in MGCs. Pure synthetic NO2-OA increased HO-1 expression in a time- and concentration-dependent manner, which was abrogated by the Nrf2 inhibitor trigonelline. In response to phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), NO2-OA prevented the ROS increase and reduced the gliosis induced by α2M. Finally, when hypoxic MGCs were incubated with NO2-OA, the increase in VEGF mRNA expression was not affected, but under hypoxia and inflammation (IL-1ß), NO2-OA significantly reduced VEGF mRNA levels. Furthermore, NO2-OA inhibited endothelial cell (BAEC) tubulogenesis. Our results highlight NO2-OA's protective effect on oxidative damage, gliosis; and the exacerbated pro-angiogenic response in MGCs.

Quantification of Reactive Oxygen Species Using 2',7'-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells.

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, ß-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since Müller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Assessment and characterization of tomato lipophilic electrophiles and their potential contribution to nutraceutical properties via SKN-1/Nrf2 signaling activation.

Phytochemical electrophiles are drawing significant attention due to their properties to modulate signaling pathways related to cellular homeostasis. The aim of this study was to develop new tools to examine the electrophilic activity in food and predict their beneficial effects on health. We developed a spectrophotometric assay based on the nitrobenzenethiol (NBT) reactivity, as a thiol-reactive nucleophile, to screen electrophiles in tomato fruits. The method is robust, simple, inexpensive, and could be applied to other types of food. We quantified the electrophile activity in a tomato collection and associated this activity with the pigment composition. Thus, we identified lycopene, ß- and γ-carotenes, 16 by-products of carotenoid oxidation and 18 unknown compounds as NBT-reactive by HPLC-MS/MS. The potential benefits of NBT-reactive compounds on health were evaluated in the in vivo model of C. elegans where they activated the SKN-1/Nrf2 pathway, evidencing the ability of electrophilic compounds to induce a biological response.

Nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating oxidized-LDL uptake and cholesterol efflux in RAW264.7 macrophages.

Macrophages play a pivotal role in the early stages of atherosclerosis development; they excessively accumulate cholesterol in the cytosol in response to modified Low Density Lipoprotein (mLDL). The mLDL are incorporated through scavenger receptors. CD36 is a high-affinity cell surface scavenger receptor that facilitates the binding and uptake of long-chain fatty acids and mLDL into the cell. Numerous structurally diverse ligands can initiate signaling responses through CD36 to regulate cell metabolism, migration, and angiogenesis. Nitro-fatty acids are endogenous electrophilic lipid mediators that react with and modulate the function of multiple enzymes and transcriptional regulatory proteins. These actions induce the expression of several anti-inflammatory and cytoprotective genes and limit pathologic responses in experimental models of atherosclerosis, cardiac ischemia/reperfusion, and inflammatory diseases. Pharmacological and genetic approaches were used to explore the actions of nitro-oleic acid (NO2-OA) on macrophage lipid metabolism. Pure synthetic NO2-OA dose-dependently increased CD36 expression in RAW264.7 macrophages and this up-regulation was abrogated in BMDM from Nrf2-KO mice. Ligand binding analysis revealed that NO2-OA specifically interacts with CD36, thus limiting the binding and uptake of mLDL. Docking analysis shows that NO2-OA establishes a low binding energy interaction with the alpha helix containing Lys164 in CD36. NO2-OA also restored autophagy flux in mLDL-loaded macrophages, thus reversing cholesterol deposition within the cell. In aggregate, these results indicate that NO2-OA reduces cholesterol uptake by binding to CD36 and increases cholesterol efflux by restoring autophagy.

LRP1-Mediated AggLDL Endocytosis Promotes Cholesteryl Ester Accumulation and Impairs Insulin Response in HL-1 Cells.

Abstract: The cardiovascular disease (CVD) frequently developed during metabolic syndrome and type-2 diabetes mellitus is associated with increased levels of aggregation-prone small LDL particles. Aggregated LDL (aggLDL) internalization is mediated by low-density lipoprotein receptor-related protein-1 (LRP1) promoting intracellular cholesteryl ester (CE) accumulation. Additionally, LRP1 plays a key function in the regulation of insulin receptor (IR) and glucose transporter type 4 (GLUT4) activities. Nevertheless, the link between LRP1, CE accumulation, and insulin response has not been previously studied in cardiomyocytes. We aimed to identify mechanisms through which aggLDL, by its interaction with LRP1, produce CE accumulation and affects the insulin-induced intracellular signaling and GLUT4 trafficking in HL-1 cells. We demonstrated that LRP1 mediates the endocytosis of aggLDL and promotes CE accumulation in these cells. Moreover, aggLDL reduced the molecular association between IR and LRP1 and impaired insulin-induced intracellular signaling activation. Finally, aggLDL affected GLUT4 translocation to the plasma membrane and the 2-NBDG uptake in insulin-stimulated cells. We conclude that LRP1 is a key regulator of the insulin response, which can be altered by CE accumulation through LRP1-mediated aggLDL endocytosis.

Nitro-fatty acid formation and metabolism.

Nitro-fatty acids (NO2-FA) are pleiotropic modulators of redox signaling pathways. Their effects on inflammatory signaling have been studied in great detail in cell, animal and clinical models primarily using exogenously administered nitro-oleic acid. While we know a considerable amount regarding NO2-FA signaling, endogenous formation and metabolism is relatively unexplored. This review will cover what is currently known regarding the proposed mechanisms of NO2-FA formation, dietary modulation of endogenous NO2-FA levels, pathways of NO2-FA metabolism and the detection of NO2-FA and corresponding metabolites.

New Insights into the Immunobiology of Mononuclear Phagocytic Cells and Their Relevance to the Pathogenesis of Cardiovascular Diseases.

Macrophages are the primary immune cells that reside within the myocardium, suggesting that these mononuclear phagocytes are essential in the orchestration of cardiac immunity and homeostasis. Independent of the nature of the injury, the heart triggers leukocyte activation and recruitment. However, inflammation is harmful to this vital terminally differentiated organ with extremely poor regenerative capacity. As such, cardiac tissue has evolved particular strategies to increase the stress tolerance and minimize the impact of inflammation. In this sense, growing evidences show that mononuclear phagocytic cells are particularly dynamic during cardiac inflammation or infection and would actively participate in tissue repair and functional recovery. They respond to soluble mediators such as metabolites or cytokines, which play central roles in the timing of the intrinsic cardiac stress response. During myocardial infarction two distinct phases of monocyte influx have been identified. Upon infarction, the heart modulates its chemokine expression profile that sequentially and actively recruits inflammatory monocytes, first, and healing monocytes, later. In the same way, a sudden switch from inflammatory macrophages (with microbicidal effectors) toward anti-inflammatory macrophages occurs within the myocardium very shortly after infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy. While in sterile injury, healing response is necessary to stop tissue damage; during an intracellular infection, the anti-inflammatory milieu in infected hearts would promote microbial persistence. The balance of mononuclear phagocytic cells seems to be also dynamic in atherosclerosis influencing plaque initiation and fate. This review summarizes the participation of mononuclear phagocyte system in cardiovascular diseases, keeping in mind that the immune system evolved to promote the reestablishment of tissue homeostasis following infection/injury, and that the effects of different mediators could modulate the magnitude and quality of the immune response. The knowledge of the effects triggered by diverse mediators would serve to identify new therapeutic targets in different cardiovascular pathologies.

Peanut Seed Cultivars with Contrasting Resistance to Aspergillus parasiticus Colonization Display Differential Temporal Response of Protease Inhibitors.

Significant efforts are being made to minimize aflatoxin contamination in peanut seeds and one possible strategy is to understand and exploit the mechanisms of plant defense against fungal infection. In this study we have identified and characterized, at biochemical and molecular levels, plant protease inhibitors (PPIs) produced in peanut seeds of the resistant PI 337394 and the susceptible Forman cultivar during Aspergillus parasiticus colonization. With chromatographic methods and 2D-electrophoresis-mass spectrometry we have isolated and identified four variants of Bowman-Birk trypsin inhibitor (BBTI) and a novel Kunitz-type protease inhibitor (KPI) produced in response to A. parasiticus colonization. KPI was detected only in the resistant cultivar, while BBTI was produced in the resistant cultivar in a higher concentration than susceptible cultivar and with different isoforms. The kinetic expression of KPI and BBTI genes along with trypsin inhibitory activity was analyzed in both cultivars during infection. In the susceptible cultivar an early PPI activity response was associated with BBTI occurrence. Meanwhile, in the resistant cultivar a later response with a larger increase in PPI activity was associated with BBTI and KPI occurrence. The biological significance of PPI in seed defense against fungal infection was analyzed and linked to inhibitory properties on enzymes released by the fungus during infection, and to the antifungal effect of KPI.

Fatty acid nitroalkenes induce resistance to ischemic cardiac injury by modulating mitochondrial respiration at complex II.

Nitro-fatty acids (NO2-FA) are metabolic and inflammatory-derived electrophiles that mediate pleiotropic signaling actions. It was hypothesized that NO2-FA would impact mitochondrial redox reactions to induce tissue-protective metabolic shifts in cells. Nitro-oleic acid (OA-NO2) reversibly inhibited complex II-linked respiration in isolated rat heart mitochondria in a pH-dependent manner and suppressed superoxide formation. Nitroalkylation of Fp subunit was determined by BME capture and the site of modification by OA-NO2 defined by mass spectrometric analysis. These effects translated into reduced basal and maximal respiration and favored glycolytic metabolism in H9C2 cardiomyoblasts as assessed by extracellular H(+) and O2 flux analysis. The perfusion of NO2-FA induced acute cardioprotection in an isolated perfused heart ischemia/reperfusion (IR) model as evidenced by significantly higher rate-pressure products. Together these findings indicate that NO2-FA can promote cardioprotection by inducing a shift from respiration to glycolysis and suppressing reactive species formation in the post-ischemic interval.

Fatty acid nitroalkenes ameliorate glucose intolerance and pulmonary hypertension in high-fat diet-induced obesity.

AIMS: Obesity is a risk factor for diabetes and cardiovascular diseases, with the incidence of these disorders becoming epidemic. Pathogenic responses to obesity have been ascribed to adipose tissue (AT) dysfunction that promotes bioactive mediator secretion from visceral AT and the initiation of pro-inflammatory events that induce oxidative stress and tissue dysfunction. Current understanding supports that suppressing pro-inflammatory and oxidative events promotes improved metabolic and cardiovascular function. In this regard, electrophilic nitro-fatty acids display pleiotropic anti-inflammatory signalling actions. METHODS AND RESULTS: It was hypothesized that high-fat diet (HFD)-induced inflammatory and metabolic responses, manifested by loss of glucose tolerance and vascular dysfunction, would be attenuated by systemic administration of nitrooctadecenoic acid (OA-NO2). Male C57BL/6j mice subjected to a HFD for 20 weeks displayed increased adiposity, fasting glucose, and insulin levels, which led to glucose intolerance and pulmonary hypertension, characterized by increased right ventricular (RV) end-systolic pressure (RVESP) and pulmonary vascular resistance (PVR). This was associated with increased lung xanthine oxidoreductase (XO) activity, macrophage infiltration, and enhanced expression of pro-inflammatory cytokines. Left ventricular (LV) end-diastolic pressure remained unaltered, indicating that the HFD produces pulmonary vascular remodelling, rather than LV dysfunction and pulmonary venous hypertension. Administration of OA-NO2 for the final 6.5 weeks of HFD improved glucose tolerance and significantly attenuated HFD-induced RVESP, PVR, RV hypertrophy, lung XO activity, oxidative stress, and pro-inflammatory pulmonary cytokine levels. CONCLUSIONS: These observations support that the pleiotropic signalling actions of electrophilic fatty acids represent a therapeutic strategy for limiting the complex pathogenic responses instigated by obesity.

Biomimetic nitration of conjugated linoleic acid: formation and characterization of naturally occurring conjugated nitrodienes.

Nitro-conjugated linoleic acids (NO2-cLA), endogenous nitrodiene lipids which act as inflammatory signaling mediators, were isolated and single isomers purified from the biomimetic acidic nitration products of conjugated linoleic acid (CLA). Structures were elucidated by means of detailed NMR and HPLC-MS/MS spectroscopic analysis and the relative double bond configurations assigned. Additional synthetic methods produced useful quantities and similar isomeric distributions of these unusual and reactive compounds for biological studies and isotopic standards, and the potential conversion of nitro-linoleic to nitro-conjugated linoleic acids was explored via a facile base-catalyzed isomerization. This represents one of the few descriptions of naturally occurring conjugated nitro dienes (in particular, 1-nitro 1,3-diene), an unusual and highly reactive motif with few biological examples extant.

Modulation of nitro-fatty acid signaling: prostaglandin reductase-1 is a nitroalkene reductase.

Inflammation, characterized by the activation of both resident and infiltrated immune cells, is accompanied by increased production of oxidizing and nitrating species. Nitrogen dioxide, the proximal nitrating species formed under these conditions, reacts with unsaturated fatty acids to yield nitroalkene derivatives. These electrophilic products modulate protein function via post-translational modification of susceptible nucleophilic amino acids. Nitroalkenes react with Keap1 to instigate Nrf2 signaling, activate heat shock response gene expression, and inhibit NF-κB-mediated signaling, inducing net anti-inflammatory and tissue-protective metabolic responses. We report the purification and characterization of a NADPH-dependent liver enzyme that reduces the nitroalkene moiety of nitro-oleic acid, yielding the inactive product nitro-stearic acid. Prostaglandin reductase-1 (PtGR-1) was identified as a nitroalkene reductase by protein purification and proteomic studies. Kinetic measurements, inhibition studies, immunological and molecular biology approaches as well as clinical analyses confirmed this identification. Overexpression of PtGR-1 in HEK293T cells promoted nitroalkene metabolism to inactive nitroalkanes, an effect that abrogated the Nrf2-dependent induction of heme oxygenase-1 expression by nitro-oleic acid. These results situate PtGR-1 as a critical modulator of both the steady state levels and signaling activities of fatty acid nitroalkenes in vivo.

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine.

The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca-9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 ± 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl ß-oxidation metabolites. High resolution mass determinations and coelution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with mercury chloride. This reaction increases the level of free nitroalkene fraction >10-fold and displays a K(D) of 7.5 × 10(-6) M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive noninvasive index of metabolic and inflammatory status.

Nitrated fatty acids: synthesis and measurement.

Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. The discovery of peroxynitrite and peroxidase-induced nitration of biomolecules led to the initial reports of endogenous nitrated fatty acids. These species increase during ischemia/reperfusion, but concentrations are often at or near the limits of detection. Here, we describe multiple methods for nitrated fatty acid synthesis and sample extraction from complex biological matrices and a rigorous method of qualitative and quantitative detection of nitrated fatty acids by liquid chromatography-mass spectrometry. In addition, optimized instrument conditions and caveats regarding data interpretation are discussed.

Conjugated linoleic acid is a preferential substrate for fatty acid nitration.

The oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 10(5) greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO(2)-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.

The cytoplasmic tyrosine kinase Arg regulates gastrulation via control of actin organization.

Coordinated cell movements are crucial for vertebrate gastrulation and are controlled by multiple signals. Although many factors are shown to mediate non-canonical Wnt pathways to regulate cell polarity and intercalation during gastrulation, signaling molecules acting in other pathways are less investigated and the connections between various signals and cytoskeleton are not well understood. In this study, we show that the cytoplasmic tyrosine kinase Arg modulates gastrulation movements through control of actin remodeling. Arg is expressed in the dorsal mesoderm at the onset of gastrulation, and both gain- and loss-of-function of Arg disrupted axial development in Xenopus embryos. Arg controlled migration of anterior mesendoderm, influenced cell decision on individual versus collective migration, and modulated spreading and protrusive activities of anterior mesendodermal cells. Arg also regulated convergent extension of the trunk mesoderm by influencing cell intercalation behaviors. Arg modulated actin organization to control dynamic F-actin distribution at the cell-cell contact or in membrane protrusions. The functions of Arg required an intact tyrosine kinase domain but not the actin-binding motifs in its carboxyl terminus. Arg acted downstream of receptor tyrosine kinases to regulate phosphorylation of endogenous CrkII and paxillin, adaptor proteins involved in activation of Rho family GTPases and actin reorganization. Our data demonstrate that Arg is a crucial cytoplasmic signaling molecule that controls dynamic actin remodeling and mesodermal cell behaviors during Xenopus gastrulation.

Gas-phase fragmentation analysis of nitro-fatty acids.

Nitro-fatty acids are electrophilic signaling mediators formed in increased amounts during inflammation by nitric oxide and nitrite-dependent redox reactions. A more rigorous characterization of endogenously-generated species requires additional understanding of their gas-phase induced fragmentation. Thus, collision induced dissociation (CID) of nitroalkane and nitroalkene groups in fatty acids were studied in the negative ion mode to provide mass spectrometric tools for their structural characterization. Fragmentation of nitroalkanes occurred mainly through loss of the NO(2)(-) anion or neutral loss of HNO(2). The CID of nitroalkenes proceeds via a more complex cyclization, followed by fragmentation to nitrile and aldehyde products. Gas-phase fragmentation of nitroalkene functional groups with additional γ or δ unsaturation occurred through a multiple step cyclization reaction process, leading to 5 and 6 member ring heterocyclic products and carbon chain fragmentation. Cyclization products were not obtained during nitroalkane fragmentation, highlighting the role of double bond π electrons during NO(2)(-) rearrangements, stabilization and heterocycle formation. The proposed structures, mechanisms and products of fragmentation are supported by analysis of (13)C and (15)N labeled parent molecules, 6 different nitroalkene positional isomers, 6 nitroalkane positional isomers, accurate mass determinations at high resolution and quantum mechanics calculations. Multiple key diagnostic ion fragments were obtained through this analysis, allowing for the precise placement of double bonds and sites of fatty acid nitration, thus supporting an ability to predict nitro positions in biological samples.

Electrophilic fatty acids regulate matrix metalloproteinase activity and expression.

Nitro-fatty acids (NO(2)-FA) are electrophilic signaling mediators formed by reactions of nitric oxide and nitrite. NO(2)-FA exert anti-inflammatory signaling actions through post-translational protein modifications. We report that nitro-oleic acid (OA-NO(2)) stimulates proMMP-7 and proMMP-9 proteolytic activity via adduction of the conserved cysteine switch domain thiolate. Biotin-labeled OA-NO(2) showed this adduction occurs preferentially with latent forms of MMP, confirming a role for thiol alkylation by OA-NO(2) in MMP activation. In addition to regulating pro-MMP activation, MMP expression was modulated by OA-NO(2) via activation of peroxisome proliferator-activated receptor-γ. MMP-9 transcription was decreased in phorbol 12-myristate 13-acetate-stimulated THP-1 macrophages to an extent similar to that induced by the peroxisome proliferator-activated receptor-γ agonist Rosiglitazone. This was affirmed using a murine model of atherosclerosis, ApoE(-/-) mice, where in vivo OA-NO(2) administration suppressed MMP expression in atherosclerotic lesions. These findings reveal that electrophilic fatty acid derivatives can serve as effectors during inflammation, first by activating pro-MMP proteolytic activity via alkylation of the cysteine switch domain, and then by transcriptionally inhibiting MMP expression, thereby limiting the further progression of inflammatory processes.

Electrophilic nitro-fatty acids activate NRF2 by a KEAP1 cysteine 151-independent mechanism.

Nitro-fatty acids (NO(2)-FAs) are electrophilic signaling mediators formed in vivo via nitric oxide (NO)- and nitrite (NO(2)(-))-dependent reactions. Nitro-fatty acids modulate signaling cascades via reversible covalent post-translational modification of nucleophilic amino acids in regulatory proteins and enzymes, thus altering downstream signaling events, such as Keap1-Nrf2-antioxidant response element (ARE)-regulated gene expression. In this study, we investigate the molecular mechanisms by which 9- and 10-nitro-octadec-9-enoic acid (OA-NO(2)) activate the transcription factor Nrf2, focusing on the post-translational modifications of cysteines in the Nrf2 inhibitor Keap1 by nitroalkylation and its downstream responses. Of the two regioisomers, 9-nitro-octadec-9-enoic acid was a more potent ARE inducer than 10-nitro-octadec-9-enoic acid. The most OA-NO(2)-reactive Cys residues in Keap1 were Cys(38), Cys(226), Cys(257), Cys(273), Cys(288), and Cys(489). Of these, Cys(273) and Cys(288) accounted for ∼50% of OA-NO(2) reactions in a cellular milieu. Notably, Cys(151) was among the least OA-NO(2)-reactive of the Keap1 Cys residues, with mutation of Cys(151) having no effect on net OA-NO(2) reaction with Keap1 or on ARE activation. Unlike many other Nrf2-activating electrophiles, OA-NO(2) enhanced rather than diminished the binding between Keap1 and the Cul3 subunit of the E3 ligase for Nrf2. OA-NO(2) can therefore be categorized as a Cys(151)-independent Nrf2 activator, which in turn can influence the pattern of gene expression and therapeutic actions of nitroalkenes.

Activated α(2) macroglobulin induces matrix metalloproteinase 9 expression by low-density lipoprotein receptor-related protein 1 through MAPK-ERK1/2 and NF-κB activation in macrophage-derived cell lines.

Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP-9) expression and protein secretion through the activation of MAPK-ERK and NF-κB signaling pathways. Previously, we demonstrated that activated α(2)-macroglulin (α(2)M*) through the interaction with its receptor low-density lipoprotein receptor-related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK-ERK1/2. In the present work, we examined whether α(2)M*/LRP1interaction could induce the MMP-9 production in J774 and Raw264.7 macrophage-derived cell lines. It was shown that α(2)M* promoted MMP-9 expression and protein secretion by LRP1 in both macrophage-derived cell lines, which was mediated by the activation of MAPK-ERK1/2 and NF-κB. Both intracellular signaling pathways activated by α(2)M* were effectively blocked by calphostin-C, suggesting involvement of PKC. In addition, we demonstrate that α(2)M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA-AM, the α(2)M*-induced MAPK-ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF-κB, it was shown that the α(2)M*-induced MMP-9 protein secretion was inhibited, indicating that the MMP production promoted by the α(2)M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression.