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Redox and inflammatory mechanisms linking urban air pollution PM2.5 exposure and cardiometabolic derangements
Dr. Timoteo Marchini | Cardiology and Angiology, Medical Center, University of Freiburg, Freiburg im Breisgau, Germany | Germany
Air pollution fine particulate matter (PM2.5) is a risk factor for the development of cardiometabolic disorders, by yet unclear mechanisms. In this study, we aimed to evaluate the impact of PM2.5 exposure, with a special focus on unravelling redox, inflammatory, and metabolic pathways. First, male 8-week-old C57BL/6 mice received 1 mg/kg body weight of a PM2.5 surrogate (ROFA, Residual Oil Fly Ash) or PBS (control) by intranasal instillation. A biphasic lung inflammatory cell recruitment was observed in ROFA-exposed mice with neutrophils peaking at 6 h post-exposure and macrophages peaking at 72 h, together with increased pro-inflammatory gene expression and cytokine levels (TNF-α, IL-6, CCL2). Bulk mRNA sequencing of sorted alveolar macrophages revealed a pro-inflammatory gene expression signature and altered pathways for redox and lipid metabolism. Upregulated differentially expressed genes were validated by a customised cytokine bead assay in BAL and plasma, which showed a sustained increase for up to 72 h in ROFA-exposed mice. In parallel, decreased metabolic gene expression (Ucp1, Elovl3, Adrb3) in brown adipose tissue suggests reduced lipolysis and thermogenesis, despite ongoing white adipose tissue inflammation. To further explore this observation, another set of mice were exposed to ROFA or PBS and monitored in metabolic cages for 48 h. Despite enhanced physical activity and lowered caloric intake, ROFA-exposed mice showed significantly reduced heat production. Lastly, consequences of PM2.5 inhalation were evaluated in a real-life mice model of exposure to polluted urban air for 16 weeks. Increased weight gain, impaired glucose homeostasis, and adipose tissue inflammation were observed in mice breathing urban air (27±8 µg PM2.5/m3) versus filtered air (2±1 µg PM2.5/m3), together with altered metabolic gene expression in adipose tissue. Our findings indicate that air pollution PM2.5 exposure blunts metabolic pathways in adipose tissue and promotes obesity, likely due to pulmonary and systemic inflammation.
SFRR-E YIA L 2:
Methodological, statistical and interpretive considerations in personalised redox biology
Dr. Nikos Margaritelis | Aristotle University of Thessaloniki (AUTh), Thessaloniki, Greece | Greece
Emerging evidence suggests that the presence of oxidative stress per se does not rationalize the use of antioxidants indiscriminately, emphasizing the need to identify responsive phenotypes for personalized interventions. This partially originates from the fact that all people inherit and acquire different or even unique biological and behavioural characteristics. As a result, the impact of any, for example nutritional or pharmacological, redox treatment that aims to regulate our physiology may be differentiated, resulting in beneficial, harmful or neutral outcomes. On this basis, the issue of individual responsiveness attracted the interest of researchers across diverse scientific fields, while ‘personalized’ - also known as ‘precision’ or ‘subject-tailored’ - treatments became the ultimate translational goal. Unfortunately, the ‘personalized treatment’ still remains more of a buzzword than a substantive and applicable concept, at least in redox biology. A potential explanation for this issue is the lack of specificity in our methodological and statistical approaches. Noteworthy, this is in stark contrast to the great advances in analytical redox chemistry, which aims to identify and quantify the precise reactive species involved in particular settings. In fact, many studies on the topic follow suboptimal methodological approaches to quantify individual responses as well as to specify statistical or clinical thresholds of effectiveness for precise redox-dependent measures (i.e., ‘minimal clinically important difference’ or ‘smallest worthwhile change’). Hence, the interpretational potential of any finding at the individual level commonly lies in the eye of the beholder, while any causative association between redox biology and physiology becomes tricky. Methodological and statistical practices from other fields that seem to provide a more straightforward approach for personalized studies will be presented. Hopefully, similar approaches will be applied in a redox biology context in future studies as well.
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p53 triggers necroptosis through loss of sulfiredoxin in acute pancreatitis
Dr. Sergio Rius-Pérez | University of Valencia (UV) | Spain
Necroptosis activation is highly dependent on the generation of mitochondrial reactive oxygen species. In this study, we assess the contribution of p53 and sulfiredoxin to necroptosis in pancreas with acute pancreatitis. Acute pancreatitis was induced by seven hourly intraperitoneal injections of cerulein in C57BL6/J wild-type mice, p53 knockout mice and sulfiredoxin knockout mice. Necroptosis occurred intensely in pancreas of wild-type mice 24 h after the first cerulein injection. At this time point p53 was upregulated and translocated into pancreatic mitochondria. However, necroptosis was abrogated in p53 knockout mice with pancreatitis. In the pancreas of p53 knockout mice, PGC-1α protein levels as well as those of its transcriptional target peroxiredoxin 3 increased and remained high upon pancreatitis induction. In addition, sulfiredoxin protein levels also increased in p53-deficient mice with pancreatitis, which prevented peroxiredoxin-3 hyperoxidation. During the early stages of pancreatitis, when necroptosis was still absent in the pancreas of wild-type mice, sulfiredoxin was upregulated and located into the mitochondria, protecting peroxiredoxin 3 from hyperoxidation. The absence of sulfirredoxin caused peroxiredoxin 3 hyperoxidation, p53 mitochondrial translocation and necroptosis early in the course of acute pancreatitis. Mito-TEMPO treatment in acute pancreatitis abrogated p53 mitochondrial translocation and necroptosis in pancreas. In conclusion, p53 is required for necroptosis in pancreas during acute pancreatitis through loss of sulfiredoxin and peroxiredoxin 3.
SFRR-E YIA L 4:
Culturing bEnd.3 brain microvascular endothelial cells in normoxic conditions has direct consequences to hypoxia-reoxygenation injury
Dr Gabriela Warpsinski | King's College London | United Kingdom
Treatments available for ischaemic stroke remain limited due to failures in clinical translation and to improve this, physiological oxygen encountered in vivo need to be considered in cell culture. As cells in vivo experience O2 levels ranging from ~13kPa to ~1kPa, cells cultured under room air – 18kPa O2 – are hyperoxic. Using an oxygen-sensitive probe (MitoXpress-INTRA, Agilent), we have identified that long-term culture under 5kPa O2 is needed to recapitulate reported intracellular O2 levels in the brain. Long-term culture under 5kPa O2 demonstrates a phenotype different to cultures under 18kPa O2, as evidenced by downregulation of specific Nrf2 target antioxidant genes. Superoxide production measured using luminescent L-012 and mitochondrial-specific superoxide indicator MitoSOX™ corroborated findings that long-term culture under 5kPa O2 prevented superoxide production associated with hypoxia-reoxygenation injury. Similarly, real-time labile Fe2+ measurements revealed less Fe2+ release following reoxygenation in bEnd.3 cells adapted to 5kPa O2. Exaggerated superoxide production in cultures exposed to hyperoxic environment may create misleading insights in screening potential therapies for pathology involving hypoxia-reoxygenation injury. The present study provides evidence that adapting cells to physiological normoxia has direct consequence for hypoxia-reoxygenation injury. Future in vitro studies should consider a paradigm shift by conducting cell culture studies under their respective physiological O2 levels to enhance translation to the clinic.
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Structural and redox-metabolic remodelling of brown adipose tissue in mice lacking nuclear factor erythroid 2-related factor 2 under basal conditions and cold acclimation
Tamara Zakic | Institute for Biological Research “Sinisa Stankovic”-National Institute of Republic of Serbia, University of Belgrade | Serbia
Interscapular brown adipose tissue (IBAT) is a highly metabolically active, thermogenic tissue essential for the maintenance of total energy homeostasis, with a remarkable ability for remodelling in response to exogenous stimuli. Given that nuclear factor erythroid 2-related factor 2 (NRF2) has a pivotal role in redox-metabolic homeostasis, we aimed to investigate its role in IBAT homeostasis under basal conditions or upon cold stimulation. Therefore, we analysed structural and redox-metabolic profiles of IBAT in wild-type (WT) and mice lacking functional Nrf2 (Nrf2KO) maintained at room (RT, 24±1°C) or low temperature (4±1°C). Our results show that both WT and Nrf2KO mice appear to be acclimated to cold, showing characteristics of thermogenically active IBAT, including increased gene and protein expression of uncoupling protein 1 (UCP1). Surprisingly, light and electron microscopy revealed that Nrf2KO mice at RT displayed distinct structural features of activated IBAT, together with the presence of vasodilated blood vessels, while the expression of thermogenic marker UCP1 did not show a corresponding cold-induced change, thus indicating IBAT functional inactivity. This lack of IBAT thermogenic activity in Nrf2KO mice at RT is consistent with its altered redox-metabolic profile, whereby protein expression of the main antioxidant defence and key metabolic enzymes either remained the same or was decreased compared to WT mice at RT. Accordingly, circulatory levels of triglycerides and cholesterol were decreased while glucose, urea and creatinine remained unchanged. Moreover, gene and/or protein expression of important redox-metabolic transcriptional factors - erythroid NRF1, NFkB, PGC-1α and PPARγ, as well as eNOS and AMPKα were increased, suggesting compensatory molecular mechanisms leading to altered IBAT phenotype in Nrf2KO mice at RT. In conclusion, the lack of functional Nrf2 leads to marked structural characteristics of active IBAT in Nrf2KO mice at RT, which are only followed by its functional activation through distinct redox-metabolic reprogramming after cold stimulation.
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Protective role of plasma EVs cargo released before and after endurance exercise on human iPS- derived cardiomyocytes in prooxidant conditions.
Dr. Veronica Lisi | University of Rome Foro Italico | Italy
Cardiovascular diseases (CVDs) result in several conditions such as increase in ROS level that lead to decrease nitric oxide availability and vasoconstriction, promoting arterial hypertension. Physical exercise (PE) has been shown to be protective against CVD. PE can lead to a maintenance of redox homeostasis, with a decrease of ROS by increased expression of antioxidant enzymes and HSPs modulation. These molecules can be shuttled between the cells by Extracellular Vesicle (EVs). EVs are lipids bound vesicles secreted by cells with a crosstalk function, carrying bioactive molecules (i.g.proteins). Our preliminary studies, show the presence of Hsp70, Hsp27 and SOD2 in plasma EVs isolated from trained and untrained healthy young males, with higher enrichment of SOD2 in untrained respect trained. Moreover, Hsp27 phosphorylation increased by acute endurance exercise (70% HRmax for 30’). These proteins are of particular interest for their protective role in cardiomyocyte (Vicencio et al., Bartz et al.) thus the aim of this work is to test the cardioprotective effect of plasma-EVs isolated from 2 groups of healthy young males with different VO2max levels (Untrained: 44.35 ± 3.0, Trained 52.27 ± 4.21) before and after a single bout of endurance exercise. Trained subject showed a significant decrease in EVs number (p=0,05) and an increase in size (p=0,0001) after training, the same trend is observed in untrained. Isolated small and large EVs were used to treat human iPS-derived cardiomyocyte exposed to a prooxidant condition (Doxorubicin, 0.2 uM for 3h and H2O2, 100 uM for 24h). ROS level (ROS detection kit far-red), apoptosis (caspase-3 detection) and senescence phenotype (p16 and Beta-galactosidase detection) were used as readout for cardioprotection (Milano et al.). Protein cargo of plasma EVs are also in depth characterized with proteomic (Mass spectrometry) and target specific (western blot) approach in order to identify the putative PE-related proteins involved in cardioprotection.