Molecular mechanisms underlying NLRP3 inflammasome activation and IL-1ß production in air pollution fine particulate matter (PM2.5)-primed macrophages.

Exposure to air pollution fine particulate matter (PM2.5) aggravates respiratory and cardiovascular diseases. It has been proposed that PM2.5 uptake by alveolar macrophages promotes local inflammation that ignites a systemic response, but precise underlying mechanisms remain unclear. Here, we demonstrate that PM2.5 phagocytosis leads to NLRP3 inflammasome activation and subsequent release of the pro-inflammatory master cytokine IL-1ß. Inflammasome priming and assembly was time- and dose-dependent in inflammasome-reporter THP-1-ASC-GFP cells, and consistent across PM2.5 samples of variable chemical composition. While inflammasome activation was promoted by different PM2.5 surrogates, significant IL-1ß release could only be observed after stimulation with transition-metal rich Residual Oil Fly Ash (ROFA) particles. This effect was confirmed in primary human monocyte-derived macrophages and murine bone marrow-derived macrophages (BMDMs), and by confocal imaging of inflammasome-reporter ASC-Citrine BMDMs. IL-1ß release by ROFA was dependent on the NLRP3 inflammasome, as indicated by lack of IL-1ß production in ROFA-exposed NLRP3-deficient (Nlrp3-/-) BMDMs, and by specific NLRP3 inhibition with the pharmacological compound MCC950. In addition, while ROFA promoted the upregulation of pro-inflammatory gene expression and cytokines release, MCC950 reduced TNF-α, IL-6, and CCL2 production. Furthermore, inhibition of TNF-α with a neutralizing antibody decreased IL-1ß release in ROFA-exposed BMDMs. Using electron tomography, ROFA particles were observed inside intracellular vesicles and mitochondria, which showed signs of ultrastructural damage. Mechanistically, we identified lysosomal rupture, K+ efflux, and impaired mitochondrial function as important prerequisites for ROFA-mediated IL-1ß release. Interestingly, specific inhibition of superoxide anion production (O2•-) from mitochondrial respiratory Complex I, but not III, blunted IL-1ß release in ROFA-exposed BMDMs. Our findings unravel the mechanism by which PM2.5 promotes IL-1ß release in macrophages and provide a novel link between innate immune response and exposure to air pollution PM2.5.

Redox and inflammatory mechanisms linking air pollution particulate matter with cardiometabolic derangements.

Air pollution is the largest environmental risk factor for disease and premature death. Among the different components that are present in polluted air, fine particulate matter below 2.5 µm in diameter (PM2.5) has been identified as the main hazardous constituent. PM2.5 mainly arises from fossil fuel combustion during power generation, industrial processes, and transportation. Exposure to PM2.5 correlates with enhanced mortality risk from cardiovascular diseases (CVD), such as myocardial infarction and stroke. Over the last decade, it has been increasingly suggested that PM2.5 affects CVD already at the stage of risk factor development. Among the multiple biological mechanisms that have been described, the interplay between oxidative stress and inflammation has been consistently highlighted as one of the main drivers of pulmonary, systemic, and cardiovascular effects of PM2.5 exposure. In this context, PM2.5 uptake by tissue-resident immune cells in the lung promotes oxidative and inflammatory mediators release that alter tissue homeostasis at remote locations. This pathway is central for PM2.5 pathogenesis and might account for the accelerated development of risk factors for CVD, including obesity and diabetes. However, transmission and end-organ mechanisms that explain PM2.5-induced impaired function in metabolic active organs are not completely understood. In this review, the main features of PM2.5 physicochemical characteristics related to PM2.5 ability to induce oxidative stress and inflammation will be presented. Hallmark and recent epidemiological and interventional studies will be summarized and discussed in the context of current air quality guidelines and legislation, knowledge gaps, and inequities. Lastly, mechanistic studies at the intersection between redox metabolism, inflammation, and function will be discussed, with focus on heart and adipose tissue alterations. By offering an integrated analysis of PM2.5-induced effects on cardiometabolic derangements, this review aims to contribute to a better understanding of the pathogenesis and potential interventions of air pollution-related CVD.

Remote ischemic preconditioning prevents sarcolemmal-associated proteolysis by MMP-2 inhibition.

The death of myocytes occurs through different pathways, but the rupture of the plasma membrane is the key point in the transition from reversible to irreversible injury. In the myocytes, three major groups of structural proteins that link the extracellular and intracellular milieus and confer structural stability to the cell membrane: the dystrophin-associated protein complex, the vinculin-integrin link, and the spectrin-based submembranous cytoskeleton. The objective was to determine if remote ischemic preconditioning (rIPC) preserves membrane-associated cytoskeletal proteins (dystrophin and ß-dystroglycan) through the inhibition of metalloproteinase type 2 (MMP-2) activity. A second objective was to describe some of the intracellular signals of the rIPC, that modify mitochondrial function at the early reperfusion. Isolated rat hearts were subjected to 30 min of global ischemia and 120 min of reperfusion (I/R). rIPC was performed by 3 cycles of ischemia/reperfusion in the lower limb (rIPC). rIPC significantly decreased the infarct size, induced Akt/GSK-3 ß phosphorylation and inhibition of the MPTP opening. rIPC improved mitochondrial function, increasing membrane potential, ATP production and respiratory control. I/R increased ONOO- production, which activates MMP-2. This enzyme degrades ß-dystroglycan and dystrophin and collaborates to sarcolemmal disruption. rIPC attenuates the breakdown of ß-dystroglycan and dystrophin through the inhibition of MMP-2 activity. Furthermore, we confirm that rIPC activates different intracellular pathway that involves the an Akt/Gsk3ß and MPTP pore with preservation of mitochondrial function.

Serotonin transporter-deficient mice display enhanced adipose tissue inflammation after chronic high-fat diet feeding.

Introduction: Serotonin is involved in leukocyte recruitment during inflammation. Deficiency of the serotonin transporter (SERT) is associated with metabolic changes in humans and mice. A possible link and interaction between the inflammatory effects of serotonin and metabolic derangements in SERT-deficient mice has not been investigated so far. Methods: SERT-deficient (Sert -/-) and wild type (WT) mice were fed a high-fat diet, starting at 8 weeks of age. Metabolic phenotyping (metabolic caging, glucose and insulin tolerance testing, body and organ weight measurements, qPCR, histology) and assessment of adipose tissue inflammation (flow cytometry, histology, qPCR) were carried out at the end of the 19-week high-fat diet feeding period. In parallel, Sert -/- and WT mice received a control diet and were analyzed either at the time point equivalent to high-fat diet feeding or as early as 8-11 weeks of age for baseline characterization. Results: After 19 weeks of high-fat diet, Sert -/- and WT mice displayed similar whole-body and fat pad weights despite increased relative weight gain due to lower starting body weight in Sert -/-. In obese Sert -/- animals insulin resistance and liver steatosis were enhanced as compared to WT animals. Leukocyte accumulation and mRNA expression of cytokine signaling mediators were increased in epididymal adipose tissue of obese Sert -/- mice. These effects were associated with higher adipose tissue mRNA expression of the chemokine monocyte chemoattractant protein 1 and presence of monocytosis in blood with an increased proportion of pro-inflammatory Ly6C+ monocytes. By contrast, Sert -/- mice fed a control diet did not display adipose tissue inflammation. Discussion: Our observations suggest that SERT deficiency in mice is associated with inflammatory processes that manifest as increased adipose tissue inflammation upon chronic high-fat diet feeding due to enhanced leukocyte recruitment.

Circulating Autoantibodies Recognizing Immunodominant Epitopes From Human Apolipoprotein B Associate With Cardiometabolic Risk Factors, but Not With Atherosclerotic Disease.

Rationale: Atherosclerosis is a chronic inflammatory disease of large arteries that involves an autoimmune response with autoreactive T cells and auto-antibodies recognizing Apolipoprotein B (ApoB), the core protein of low-density lipoprotein (LDL). Here, we aimed to establish a clinical association between circulating human ApoB auto-antibodies with atherosclerosis and its clinical risk factors using a novel assay to detect auto-antibodies against a pool of highly immunogenic ApoB-peptides. Methods and Results: To detect polyclonal IgM- and IgG-antibodies recognizing ApoB, we developed a chemiluminescent sandwich ELISA with 30 ApoB peptides selected by an in silico assay for a high binding affinity to MHC-II, which cover more than 80% of known MHC-II variants in a Caucasian population. This pre-selection of immunogenic self-peptides accounted for the high variability of human MHC-II, which is fundamental to allow T cell dependent generation of IgG antibodies. We quantified levels of ApoB-autoantibodies in a clinical cohort of 307 patients that underwent coronary angiography. Plasma anti-ApoB IgG and IgM concentrations showed no differences across healthy individuals (n = 67), patients with coronary artery disease (n = 179), and patients with an acute coronary syndrome (n = 61). However, plasma levels of anti-ApoB IgG, which are considered pro-inflammatory, were significantly increased in patients with obesity (p = 0.044) and arterial hypertension (p < 0.0001). In addition, patients diagnosed with the metabolic syndrome showed significantly elevated Anti-ApoB IgG (p = 0.002). Even when normalized for total plasma IgG, anti-ApoB IgG remained highly upregulated in hypertensive patients (p < 0.0001). We observed no association with triglycerides, total cholesterol, VLDL, or LDL plasma levels. However, total and normalized anti-ApoB IgG levels negatively correlated with HDL. In contrast, total and normalized anti-ApoB IgM, that have been suggested as anti-inflammatory, were significantly lower in diabetic patients (p = 0.012) and in patients with the metabolic syndrome (p = 0.005). Conclusion: Using a novel ELISA method to detect auto-antibodies against ApoB in humans, we show that anti-ApoB IgG associate with cardiovascular risk factors but not with the clinical appearance of atherosclerosis, suggesting that humoral immune responses against ApoB are shaped by cardiovascular risk factors but not disease status itself. This novel tool will be helpful to develop immune-based risk stratification for clinical atherosclerosis in the future.

The Role of Tumor Necrosis Factor Associated Factors (TRAFs) in Vascular Inflammation and Atherosclerosis.

TNF receptor associated factors (TRAFs) represent a family of cytoplasmic signaling adaptor proteins that regulate, bundle, and transduce inflammatory signals downstream of TNF- (TNF-Rs), interleukin (IL)-1-, Toll-like- (TLRs), and IL-17 receptors. TRAFs play a pivotal role in regulating cell survival and immune cell function and are fundamental regulators of acute and chronic inflammation. Lately, the inhibition of inflammation by anti-cytokine therapy has emerged as novel treatment strategy in patients with atherosclerosis. Likewise, growing evidence from preclinical experiments proposes TRAFs as potent modulators of inflammation in atherosclerosis and vascular inflammation. Yet, TRAFs show a highly complex interplay between different TRAF-family members with partially opposing and overlapping functions that are determined by the level of cellular expression, concomitant signaling events, and the context of the disease. Therefore, inhibition of specific TRAFs may be beneficial in one condition and harmful in others. Here, we carefully discuss the cellular expression and signaling events of TRAFs and evaluate their role in vascular inflammation and atherosclerosis. We also highlight metabolic effects of TRAFs and discuss the development of TRAF-based therapeutics in the future.

Chronic exposure to polluted urban air aggravates myocardial infarction by impaired cardiac mitochondrial function and dynamics.

Air pollution exposure positively correlates with increased cardiovascular morbidity and mortality rates, mainly due to myocardial infarction (MI). Herein, we aimed to study the metabolic mechanisms underlying this association, focusing on the evaluation of cardiac mitochondrial function and dynamics, together with its impact over MI progression. An initial time course study was performed in BALB/c mice breathing filtered air (FA) or urban air (UA) in whole-body exposure chambers located in Buenos Aires City downtown for up to 16 weeks (n = 8 per group and time point). After 12 weeks, lung inflammatory cell recruitment was evident in UA-exposed mice. Interestingly, impaired redox metabolism, characterized by decreased lung SOD activity and increased GSSG levels and NOX activity, precede local inflammation in this group. At this selected time point, additional mice were exposed to FA or UA (n = 12 per group) and alveolar macrophage PM uptake and nitric oxide (NO) production was observed in UA-exposed mice, together with increased pro-inflammatory cytokine levels (TNF-α and IL-6) in BAL and plasma. Consequently, impaired heart tissue oxygen metabolism and altered mitochondrial ultrastructure and function were observed in UA-exposed mice after 12 weeks, characterized by decreased active state respiration and ATP production rates, and enhanced mitochondrial H2O2 production. Moreover, disturbed cardiac mitochondrial dynamics was detected in this group. This scenario led to a significant increase in the area of infarcted tissue following myocardial ischemia reperfusion injury in vivo, from 43 ± 3% of the area at risk in mice breathing FA to 66 ± 4% in UA-exposed mice (n = 6 per group, p < 0.01), together with a sustained increase in LVEDP during myocardial reperfusion. Taken together, our data unravel cardiac mitochondrial mechanisms that contribute to the understanding of the adverse health effects of urban air pollution exposure, and ultimately highlight the importance of considering environmental factors in the development of cardiovascular diseases.

Modulating Autoimmunity against LDL: Development of a Vaccine against Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the arterial wall that leads to the build-up of occluding atherosclerotic plaques. Its clinical sequelae, myocardial infarction and stroke, represent the most frequent causes of death worldwide. Atherosclerosis is a multifactorial pathology that involves traditional risk factors and chronic low-grade inflammation in the atherosclerotic plaque and systemically. This process is accompanied by a strong autoimmune response that involves autoreactive T cells in lymph nodes and atherosclerotic plaques, as well as autoantibodies that recognize low-density lipoprotein (LDL) and its main protein component apolipoprotein B (ApoB). In the past 60 years, numerous preclinical observations have suggested that immunomodulatory vaccination with LDL, ApoB, or its peptides has the potential to specifically dampen autoimmunity, enhance tolerance to atherosclerosis-specific antigens, and protect from experimental atherosclerosis in mouse models. Here, we summarize and discuss mechanisms, challenges, and therapeutic opportunities of immunomodulatory vaccination and other strategies to enhance protective immunity in atherosclerosis.

Oxidative metabolism in the cardiorespiratory system after an acute exposure to nickel-doped nanoparticles in mice.

There is an increasing concern over the harmful effects that metallic nanoparticles (NP) may produce on human health. Due to their redox properties, nickel (Ni) and Ni-containing NP are particularly relevant. Hence, the aim of this study was to establish the toxicological mechanisms in the cardiorespiratory oxidative metabolism initiated by an acute exposure to Ni-doped-NP. Mice were intranasally instilled with silica NP containing Ni (II) (Ni-NP) (1 mg Ni (II)/kg body weight) or empty NP as control, and 1 h after exposure lung, plasma, and heart samples were obtained to assess the redox metabolism. Results showed that, NP were mainly retained in the lungs triggering a significantly increased tissue O2 consumption rate, leading to Ni-NP-increased reactive oxygen species production by NOX activity, and mitochondrial H2O2 production rate. In addition, an oxidant redox status due to an altered antioxidant system showed by lung GSH/GSSG ratio decreased, and SOD activity increased, resulting in an increased phospholipid oxidation. Activation of circulating polymorphonuclear leukocytes, along with GSH/GSSG ratio decreased, and phospholipid oxidation were found in the Ni-NP-group plasma samples. Consequently, in distant organs such as heart, Ni-NP inhalation alters the tissue redox status. Our results showed that the O2 metabolism analysis is a critical area of study following Ni-NP inhalation. Therefore, this work provides novel data linking the redox metabolisms alterations elicited by exposure to Ni (II) adsorbed to NP and cardiorespiratory toxicity.

Genetic Deficiency of TRAF5 Promotes Adipose Tissue Inflammation and Aggravates Diet-Induced Obesity in Mice.

Objective: The accumulation of inflammatory leukocytes is a prerequisite of adipose tissue inflammation during cardiometabolic disease. We previously reported that a genetic deficiency of the intracellular signaling adaptor TRAF5 (TNF [tumor necrosis factor] receptor-associated factor 5) accelerates atherosclerosis in mice by increasing inflammatory cell recruitment. Here, we tested the hypothesis that an impairment of TRAF5 signaling modulates adipose tissue inflammation and its metabolic complications in a model of diet-induced obesity in mice. Approach and Results: To induce diet-induced obesity and adipose tissue inflammation, wild-type or Traf5-/- mice consumed a high-fat diet for 18 weeks. Traf5-/- mice showed an increased weight gain, impaired insulin tolerance, and increased fasting blood glucose. Weight of livers and peripheral fat pads was increased in Traf5-/- mice, whereas lean tissue weight and growth were not affected. Flow cytometry of the stromal vascular fraction of visceral adipose tissue from Traf5-/- mice revealed an increase in cytotoxic T cells, CD11c+ macrophages, and increased gene expression of proinflammatory cytokines and chemokines. At the level of cell types, expression of TNF[alpha], MIP (macrophage inflammatory protein)-1[alpha], MCP (monocyte chemoattractant protein)-1, and RANTES (regulated on activation, normal T-cell expressed and secreted) was significantly upregulated in Traf5-deficient adipocytes but not in Traf5-deficient leukocytes from visceral adipose tissue. Finally, Traf5 expression was lower in adipocytes from obese patients and mice and recovered in adipose tissue of obese patients one year after bariatric surgery. Conclusions: We show that a genetic deficiency of TRAF5 in mice aggravates diet-induced obesity and its metabolic derangements by a proinflammatory response in adipocytes. Our data indicate that TRAF5 may promote anti-inflammatory and obesity-preventing signaling events in adipose tissue.

Energy management and mitochondrial dynamics in cerebral cortex during endotoxemia.

Mitochondria play an essential role in inflammatory processes such as sepsis or endotoxemia, contributing to organ-cellular redox metabolism, emerging as the energy hub of the cell, and as an important center of action of second messengers. In this work, we aimed to elucidate the energy state, redox balance, and mitochondrial remodeling status in cerebral cortex in an experimental model of endotoxemia. Female Sprague-Dawley rats were subjected to a single dose of LPS (ip 8 mg kg-1 body weight) for 6 h. State 3 O2 consumption was observed increased, ATP production and P/O ratio were observed decreased, probably indicating an inefficient oxidative phosphorylation process. O2- production and both systemic and tissue NO markers were observed increased in treated animals. The existence of nitrated proteins suggests an alteration in the local redox balance and possible harmful effects over energetic processes. Increases in PGC-1α and mtTFA expression, and in OPA-1 expression, suggest an increase in de novo formation of mitochondria and fusion of pre-existing mitochondria. The observed elongation of mitochondria correlates with the occurrence of mild mitochondrial dysfunction and increased levels of systemic NO. Our work presents novel results that contribute to unravel the mechanism by which the triad endotoxemia-redox homeostasis-energy management interact in the cerebral cortex, leading to propose a relevant mechanism for future developing therapeutics with the aim of preserving this organ from inflammatory and oxidative damage.

Urban air pollution induces alterations in redox metabolism and mitochondrial dysfunction in mice brain cortex.

Previous reports indicate that the central nervous system (CNS) is a target of air pollution, causing tissue damage and functional alterations. Oxidative stress and neuroinflammation have been pointed out as possible mechanisms mediating these effects. The aim of this work was to study the chronic effects of urban air pollution on mice brain cortex, focusing on oxidative stress markers, and mitochondrial function. Male 8-week-old BALB/c mice were exposed to filtered air (FA, control) or urban air (UA) inside whole-body exposure chambers, located in a highly polluted area of Buenos Aires city, for up to 4 weeks. Glutathione levels, assessed as GSH/GSSG ratio, were decreased after 1 and 2 weeks of exposure to UA (45% and 25% respectively vs. FA; p < 0.05). A 38% increase in lipid peroxidation was found after 1 week of UA exposure (p < 0.05). Regarding protein oxidation, carbonyl content was significantly increased at week 2 in UA-exposed mice, compared to FA-group, and an even higher increment was found after 4 weeks of exposure (week 2: 40% p < 0.05, week 4: 54% p < 0.001). NADPH oxidase (NOX) and glutathione peroxidase (GPx) activities were augmented at all the studied time points, while superoxide dismutase (Cu,Zn-SOD cytosolic isoform) and glutathione reductase (GR) activities were increased only after 4 weeks of UA exposure (p < 0.05). The increased NOX activity was accompanied with higher expression levels of NOX2 regulatory subunit p47phox, and NOX4 (p < 0.05). Also, UA mice showed impaired mitochondrial function due to a 50% reduction in O2 consumption in active state respiration (p < 0.05), a 29% decrease in mitochondrial inner membrane potential (p < 0.05), a 65% decrease in ATP production rate (p < 0.01) and a 30% increase in H2O2 production (p < 0.01). Moreover, respiratory complexes I-III and II-III activities were decreased in UA group (30% and 36% respectively vs. FA; p < 0.05). UA exposed mice showed alterations in mitochondrial function, increased oxidant production evidenced by NOX activation, macromolecules damage and the onset of the enzymatic antioxidant system. These data indicate that oxidative stress and impaired mitochondrial function may play a key role in CNS damage mechanisms triggered by air pollution.

Inflammatory Cell Recruitment in Cardiovascular Disease.

Atherosclerosis, the main underlying pathology for myocardial infarction and stroke, is a chronic inflammatory disease of middle-sized to large arteries that is initiated and maintained by leukocytes infiltrating into the subendothelial space. It is now clear that the accumulation of pro-inflammatory leukocytes drives progression of atherosclerosis, its clinical complications, and directly modulates tissue-healing in the infarcted heart after myocardial infarction. This inflammatory response is orchestrated by multiple soluble mediators that enhance inflammation systemically and locally, as well as by a multitude of partially tissue-specific molecules that regulate homing, adhesion, and transmigration of leukocytes. While numerous experimental studies in the mouse have refined our understanding of leukocyte accumulation from a conceptual perspective, only a few anti-leukocyte therapies have been directly validated in humans. Lack of tissue-tropism of targeted factors required for leukocyte accumulation and unspecific inhibition strategies remain the major challenges to ultimately translate therapies that modulate leukocytes accumulation into clinical practice. Here, we carefully describe receptor and ligand pairs that guide leukocyte accumulation into the atherosclerotic plaque and the infarcted myocardium, and comment on potential future medical therapies.

ApoB-Specific CD4+ T Cells in Mouse and Human Atherosclerosis.

Atherosclerosis is a chronic inflammatory condition of the arterial wall that leads to the formation of vessel-occluding plaques within the subintimal space of middle-sized and larger arteries. While traditionally understood as a myeloid-driven lipid-storage disease, growing evidence suggests that the accumulation of low-density lipoprotein cholesterol (LDL-C) ignites an autoimmune response with CD4+ T-helper (TH) cells that recognize self-peptides from Apolipoprotein B (ApoB), the core protein of LDL-C. These autoreactive CD4+ T cells home to the atherosclerotic plaque, clonally expand, instruct other cells in the plaque, and induce clinical plaque instability. Recent developments in detecting antigen-specific cells at the single cell level have demonstrated that ApoB-reactive CD4+ T cells exist in humans and mice. Their phenotypes and functions deviate from classical immunological concepts of distinct and terminally differentiated TH immunity. Instead, ApoB-specific CD4+ T cells have a highly plastic phenotype, can acquire several, partially opposing and mixed transcriptional programs simultaneously, and transit from one TH subset into another over time. In this review, we highlight adaptive immune mechanisms in atherosclerosis with a focus on CD4+ T cells, introduce novel technologies to detect ApoB-specific CD4+ T cells at the single cell level, and discuss the potential impact of ApoB-driven autoimmunity in atherosclerosis.

Deficiency of Endothelial CD40 Induces a Stable Plaque Phenotype and Limits Inflammatory Cell Recruitment to Atherosclerotic Lesions in Mice.

OBJECTIVES: The co-stimulatory CD40L-CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis. METHODS AND RESULTS: Atherosclerotic plaques of apolipoprotein E-deficient (Apoe -/- ) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCreERT2-driven) deficiency of CD40 in Apoe -/- mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs. CONCLUSION: Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.

Mitochondrial function is impaired in the primary visual cortex in an experimental glaucoma model.

Glaucoma is a neurodegenerative disease that affects eye structures and brain areas related to the visual system. Oxidative stress plays a key role in the development and progression of the disease. The aims of the present study were to evaluate the mitochondrial function and its participation in the brain redox metabolism in an experimental glaucoma model. 3-month-old female Wistar rats were subjected to cauterization of two episcleral veins of the left eye to elevate the intraocular pressure. Seven days after surgery, animals were sacrificed, the brain was carefully removed and the primary visual cortex was dissected. Mitochondrial bioenergetics and ROS production, and the antioxidant enzyme defenses from both mitochondrial and cytosolic fractions were evaluated. When compared to control, glaucoma decreased mitochondrial ATP production (23%, p < 0.05), with an increase in superoxide and hydrogen peroxide production (30%, p < 0.01 and 28%, p < 0.05, respectively), whereas no changes were observed in membrane potential and oxygen consumption rate. In addition, the glaucoma group displayed a decrease in complex II activity (34%, p < 0.01). Moreover, NOX4 expression was increased in glaucoma compared to the control group (27%, p < 0.05). Regarding the activity of enzymes associated with the regulation of the redox status, glaucoma showed an increase in mitochondrial SOD activity (34%, p < 0.05), mostly due to an increase in Mn-SOD (50%, p < 0.05). A decrease in mitochondrial GST activity was observed (11%, p < 0.05). GR and TrxR activity were decreased in both mitochondrial (16%, p < 0.05 and 20%, p < 0.05 respectively) and cytosolic (21%, p < 0.01 and 50%, p < 0.01 respectively) fractions in the glaucoma group. Additionally, glaucoma showed an increase in cytoplasmatic GPx (50%, p < 0.01). In this scenario, redox imbalance took place resulting in damage to mitochondrial lipids (39%, p < 0.01) and proteins (70%, p < 0.05). These results suggest that glaucoma leads to mitochondrial function impairment in brain visual targets, that is accompanied by an alteration in both mitochondrial and cytoplasmatic enzymatic defenses. As a consequence of redox imbalance, oxidative damage to macromolecules takes place and can further affect vital cellular functions. Understanding the role of the mitochondria in the development and progression of the disease could bring up new neuroprotective therapies.

Alterations in oxygen metabolism are associated to lung toxicity triggered by silver nanoparticles exposure.

Along with the AgNP applications development, the concern about their possible toxicity has increasingly gained attention. As the respiratory system is one of the main exposure routes, the aim of this study was to evaluate the harmful effects developed in the lung after an acute AgNP exposure. In vivo studies using Balb/c mice intranasally instilled with 0.1 mg AgNP/kg b.w, were performed. 99mTc-AgNP showed the lung as the main organ of deposition, where, in turn, AgNP may exert barrier injury observed by increased protein content and total cell count in BAL samples. In vivo acute exposure showed altered lung tissue O2 consumption due to increased mitochondrial active respiration and NOX activity. Both O2 consumption processes release ROS triggering the antioxidant system as observed by the increased SOD, catalase and GPx activities and a decreased GSH/GSSG ratio. In addition, increased protein oxidation was observed after AgNP exposure. In A549 cells, exposure to 2.5 µg/mL AgNP during 1 h resulted in augment NOX activity, decreased mitochondrial ATP associated respiration and higher H2O2 production rate. Lung 3D tissue model showed AgNP-initiated barrier alterations as TEER values decreased and morphological alterations. Taken together, these results show that AgNP exposure alters O2 metabolism leading to alterations in oxygen metabolism lung toxicity. AgNP-triggered oxidative damage may be responsible for the impaired lung function observed due to alveolar epithelial injury.

Role of Mitochondria in the Redox Signaling Network and Its Outcomes in High Impact Inflammatory Syndromes.

Inflammation is associated with the release of soluble mediators that drive cellular activation and migration of inflammatory leukocytes to the site of injury, together with endothelial expression of adhesion molecules, and increased vascular permeability. It is a stepwise tightly regulated process that has been evolved to cope with a wide range of different inflammatory stimuli. However, under certain physiopathological conditions, the inflammatory response overwhelms local regulatory mechanisms and leads to systemic inflammation that, in turn, might affect metabolism in distant tissues and organs. In this sense, as mitochondria are able to perceive signals of inflammation is one of the first organelles to be affected by a dysregulation in the systemic inflammatory response, it has been associated with the progression of the physiopathological mechanisms. Mitochondria are also an important source of ROS (reactive oxygen species) within most mammalian cells and are therefore highly involved in oxidative stress. ROS production might contribute to mitochondrial damage in a range of pathologies and is also important in a complex redox signaling network from the organelle to the rest of the cell. Therefore, a role for ROS generated by mitochondria in regulating inflammatory signaling was postulated and mitochondria have been implicated in multiple aspects of the inflammatory response. An inflammatory condition that affects mitochondrial function in different organs is the exposure to air particulate matter (PM). Both after acute and chronic pollutants exposure, PM uptake by alveolar macrophages have been described to induce local cell activation and recruitment, cytokine release, and pulmonary inflammation. Afterwards, inflammatory mediators have been shown to be able to reach the bloodstream and induce a systemic response that affects metabolism in distant organs different from the lung. In this proinflammatory environment, impaired mitochondrial function that leads to bioenergetic dysfunction and enhanced production of oxidants have been shown to affect tissue homeostasis and organ function. In the present review, we aim to discuss the latest insights into the cellular and molecular mechanisms that link systemic inflammation and mitochondrial dysfunction in different organs, taking the exposure to air pollutants as a case model.

NADPH oxidase and mitochondria are relevant sources of superoxide anion in the oxinflammatory response of macrophages exposed to airborne particulate matter.

Exposure to ambient air particulate matter (PM) is associated with increased cardiorespiratory morbidity and mortality. In this context, alveolar macrophages exhibit proinflammatory and oxidative responses as a result of the clearance of particles, thus contributing to lung injury. However, the mechanisms linking these pathways are not completely clarified. Therefore, the oxinflammation phenomenon was studied in RAW 264.7 macrophages exposed to Residual Oil Fly Ash (ROFA), a PM surrogate rich in transition metals. While cell viability was not compromised under the experimental conditions, a proinflammatory phenotype was observed in cells incubated with ROFA 100 µg/mL, characterized by increased levels of TNF-α and NO production, together with PM uptake. This inflammatory response seems to precede alterations in redox metabolism, characterized by augmented levels of H2O2, diminished GSH/GSSG ratio, and increased SOD activity. This scenario resulted in increased oxidative damage to phospholipids. Moreover, alterations in mitochondrial respiration were observed following ROFA incubation, such as diminished coupling efficiency and spare respiratory capacity, together with augmented proton leak. These findings were accompanied by a decrease in mitochondrial membrane potential. Finally, NADPH oxidase (NOX) and mitochondria were identified as the main sources of superoxide anion () in our model. These results indicate that PM exposure induces direct activation of macrophages, leading to inflammation and increased reactive oxygen species production through NOX and mitochondria, which impairs antioxidant defense and may cause mitochondrial dysfunction.

Antioxidant Activity of Flavonoid Rich Fraction of Ligaria cuneifolia (Loranthaceae).

Ligaria cuneifolia (Ruiz & Pav.) Tiegh. (Loranthaceae), the 'Argentine mistletoe', is a hemiparasite species largely used in folk medicine. The aim of this study was to evaluate the antioxidant activity using in vitro, ex vivo, and in vivo methods. A screening of phenolics was performed by UV spectroscopy on different fractions. The antioxidant capacity was evaluated in vitro by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH. ) assay on a crude extract (CE), ethyl acetate fraction (EAF), and aqueous fraction (AF). The results suggest that EAF concentrates the antioxidant capacity and was selected for further analysis. Capillary electrophoresis was employed to monitor the individual antioxidant capacity and the potential contributors to this effect. Ex vivo assays showed an efficient inhibition of tert-butyl hydroperoxide-induced rat liver phospholipid oxidation, as well as rat brain autoxidation, and H2 O2 -induced DNA damage in blood monocytes. In vivo, the topical application of EAF significantly decreased skin chemiluminescence in a mice model.