Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology.

'Reactive oxygen species' (ROS) is a generic term that defines a wide variety of oxidant molecules with vastly different properties and biological functions that range from signalling to causing cell damage. Consequently, the description of oxidants needs to be chemically precise to translate research on their biological effects into therapeutic benefit in redox medicine. This Expert Recommendation article pinpoints key issues associated with identifying the physiological roles of oxidants, focusing on H2O2 and O2.-. The generic term ROS should not be used to describe specific molecular agents. We also advocate for greater precision in measurement of H2O2, O2.- and other oxidants, along with more specific identification of their signalling targets. Future work should also consider inter-organellar communication and the interactions of redox-sensitive signalling targets within organs and whole organisms, including the contribution of environmental exposures. To achieve these goals, development of tools that enable site-specific and real-time detection and quantification of individual oxidants in cells and model organisms are needed. We also stress that physiological O2 levels should be maintained in cell culture to better mimic in vivo redox reactions associated with specific cell types. Use of precise definitions and analytical tools will help harmonize research among the many scientific disciplines working on the common goal of understanding redox biology.

Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans.

The extensive oxygen gradient between the air we breathe (Po2 ~21 kPa) and its ultimate distribution within mitochondria (as low as ~0.5-1 kPa) is testament to the efforts expended in limiting its inherent toxicity. It has long been recognized that cell culture undertaken under room air conditions falls short of replicating this protection in vitro. Despite this, difficulty in accurately determining the appropriate O2 levels in which to culture cells, coupled with a lack of the technology to replicate and maintain a physiological O2 environment in vitro, has hindered addressing this issue thus far. In this review, we aim to address the current understanding of tissue Po2 distribution in vivo and summarize the attempts made to replicate these conditions in vitro. The state-of-the-art techniques employed to accurately determine O2 levels, as well as the issues associated with reproducing physiological O2 levels in vitro, are also critically reviewed. We aim to provide the framework for researchers to undertake cell culture under O2 levels relevant to specific tissues and organs. We envisage that this review will facilitate a paradigm shift, enabling translation of findings under physiological conditions in vitro to disease pathology and the design of novel therapeutics.

Interactions between zinc and NRF2 in vascular redox signalling.

Recent evidence highlights the importance of trace metal micronutrients such as zinc (Zn) in coronary and vascular diseases. Zn2+ plays a signalling role in modulating endothelial nitric oxide synthase and protects the endothelium against oxidative stress by up-regulation of glutathione synthesis. Excessive accumulation of Zn2+ in endothelial cells leads to apoptotic cell death resulting from dysregulation of glutathione and mitochondrial ATP synthesis, whereas zinc deficiency induces an inflammatory phenotype, associated with increased monocyte adhesion. Nuclear factor-E2-related factor 2 (NRF2) is a transcription factor known to target hundreds of different genes. Activation of NRF2 affects redox metabolism, autophagy, cell proliferation, remodelling of the extracellular matrix and wound healing. As a redox-inert metal ion, Zn has emerged as a biomarker in diagnosis and as a therapeutic approach for oxidative-related diseases due to its close link to NRF2 signalling. In non-vascular cell types, Zn has been shown to modify conformations of the NRF2 negative regulators Kelch-like ECH-associated Protein 1 (KEAP1) and glycogen synthase kinase 3ß (GSK3ß) and to promote degradation of BACH1, a transcriptional suppressor of select NRF2 genes. Zn can affect phosphorylation signalling, including mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinases and protein kinase C, which facilitate NRF2 phosphorylation and nuclear translocation. Notably, several NRF2-targeted proteins have been suggested to modify cellular Zn concentration via Zn exporters (ZnTs) and importers (ZIPs) and the Zn buffering protein metallothionein. This review summarises the cross-talk between reactive oxygen species, Zn and NRF2 in antioxidant responses of vascular cells against oxidative stress and hypoxia/reoxygenation.

Oxygen gradients can determine epigenetic asymmetry and cellular differentiation via differential regulation of Tet activity in embryonic stem cells.

Graded levels of molecular oxygen (O2) exist within developing mammalian embryos and can differentially regulate cellular specification pathways. During differentiation, cells acquire distinct epigenetic landscapes, which determine their function, however the mechanisms which regulate this are poorly understood. The demethylation of 5-methylcytosine (5mC) is achieved via successive oxidation reactions catalysed by the Ten-Eleven-Translocation (Tet) enzymes, yielding the 5-hydroxymethylcytosine (5hmC) intermediate. These require O2 as a co-factor, and hence may link epigenetic processes directly to O2 gradients during development. We demonstrate that the activities of Tet enzymes display distinct patterns of [O2]-dependency, and that Tet1 activity, specifically, is subject to differential regulation within a range of O2 which is physiologically relevant in embryogenesis. Further, differentiating embryonic stem cells displayed a transient burst of 5hmC, which was both dependent upon Tet1 and inhibited by low (1%) [O2]. A GC-rich promoter region within the Tet3 locus was identified as a significant target of this 5mC-hydroxylation. Further, this region was shown to associate with Tet1, and display the histone epigenetic marks, H3K4me3 and H3K27me3, which are characteristic of a bivalent, developmentally 'poised' promoter. We conclude that Tet1 activity, determined by [O2] may play a critical role in regulating cellular differentiation and fate in embryogenesis.

Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.

Unregulated increases in cellular Ca2+ homeostasis are a hallmark of pathophysiological conditions and a key trigger of cell death. Endothelial cells cultured under physiologic O2 conditions (5% O2) exhibit a reduced cytosolic Ca2+ response to stimulation. The mechanism for reduced plateau [Ca2+]i upon stimulation was due to increased sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-mediated reuptake rather than changes in Ca2+ influx capacity. Agonist-stimulated phosphorylation of the SERCA regulatory protein phospholamban was increased in cells cultured under 5% O2. Elevation of cytosolic and mitochondrial [Ca2+] and cell death after prolonged ionomycin treatment, as a model of Ca2+ overload, were lower when cells were cultured long-term under 5% compared with 18% O2. This protection was abolished by cotreatment with the SERCA inhibitor cyclopiazonic acid. Taken together, these results demonstrate that culturing cells under hyperoxic conditions reduces their ability to efficiently regulate [Ca2+]i, resulting in greater sensitivity to cytotoxic stimuli.-Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.

A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen.

Intracellular O2 is a key regulator of NO signaling, yet most in vitro studies are conducted in atmospheric O2 levels, hyperoxic with respect to the physiologic milieu. We investigated NO signaling in endothelial cells cultured in physiologic (5%) O2 and stimulated with histamine or shear stress. Culture of cells in 5% O2 (>5 d) decreased histamine- but not shear stress-stimulated endothelial (e)NOS activity. Unlike cells adapted to a hypoxic environment (1% O2), those cultured in 5% O2 still mobilized sufficient Ca2+ to activate AMPK. Enhanced expression and membrane targeting of PP2A-C was observed in 5% O2, resulting in greater interaction with eNOS in response to histamine. Moreover, increased dephosphorylation of eNOS in 5% O2 was Ca2+-sensitive and reversed by okadaic acid or PP2A-C siRNA. The present findings establish that Ca2+ mobilization stimulates both NO synthesis and PP2A-mediated eNOS dephosphorylation, thus constituting a novel negative feedback mechanism regulating eNOS activity not present in response to shear stress. This, coupled with enhanced NO bioavailability, underpins differences in NO signaling induced by inflammatory and physiologic stimuli that are apparent only in physiologic O2 levels. Furthermore, an explicit delineation between physiologic normoxia and genuine hypoxia is defined here, with implications for our understanding of pathophysiological hypoxia.-Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen.

The anti-ageing hormone klotho induces Nrf2-mediated antioxidant defences in human aortic smooth muscle cells.

Vascular ageing in conditions such as atherosclerosis, diabetes and chronic kidney disease, is associated with the activation of the renin angiotensin system (RAS) and diminished expression of antioxidant defences mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). The anti-ageing hormone klotho promotes longevity and protects against cardiovascular and renal diseases. Klotho has been shown to activate Nrf2 and attenuate oxidative damage in neuronal cells, however, the mechanisms by which it protects against vascular smooth muscle cell VSMC dysfunction elicited by Angiotensin II (AngII) remain to be elucidated. AngII contributes to vascular ageing and atherogenesis by enhancing VSMC oxidative stress, senescence and apoptosis. This study demonstrates that soluble klotho (1 nM, 24 hrs) significantly induces expression of Nrf2 and the antioxidant enzymes haeme oxygenase (HO-1) and peroxiredoxin-1 (Prx-1) and enhances glutathione levels in human aortic smooth muscle cells (HASMC). Silencing of Nrf2 attenuated the induction of HO-1 and Prx-1 expression by soluble klotho. Furthermore, soluble klotho protected against AngII-mediated HASMC apoptosis and senescence via activation of Nrf2. Thus, our findings highlight a novel Nrf2-mediated mechanism underlying the protective actions of soluble klotho in HAMSC. Targeting klotho may thus represent a therapeutic strategy against VSMC dysfunction and cardiovascular ageing.

Deficiency of p62/Sequestosome 1 causes hyperphagia due to leptin resistance in the brain.

The cytoplasmic regulatory protein p62 (Sequestosome 1/A170) is known to modulate various receptor-mediated intracellular signaling pathways. p62 deficiency was shown to result in mature-onset obesity in mice, but the mechanisms underlying this abnormality remained unclear. Here we report that hyperphagia due to central leptin resistance is the cause of obesity in p62(-/-) mice. We found that these mice show hyperphagia. Restriction of food to the amount eaten by wild-type mice prevented excess body weight gain and fat accumulation, suggesting that overfeeding is the primary cause of obesity in p62(-/-) mice. Brain-specific p62 deficiency caused mature-onset obesity to the same extent as in p62(-/-) mice, further supporting a neuronal mechanism as the major cause of obesity in these mice. Immunohistochemical analysis revealed that p62 is highly expressed in hypothalamic neurons, including POMC neurons in the arcuate nucleus. Central leptin resistance was observed even in young preobese p62(-/-) mice. We found a defect in intracellular distribution of the transcription factor Stat3, which is essential for the action of leptin, in p62(-/-) mice. These results indicate that brain p62 plays an important role in bodyweight control by modulating the central leptin-signaling pathway and that lack of p62 in the brain causes leptin resistance, leading to hyperphagia. Thus, p62 could be a clinical target for treating obesity and metabolic syndrome.

Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions.

The complex interplay between hydrogen peroxide (H2O2) and nitric oxide (NO) in endothelial cells presents challenges due to technical limitations in simultaneous measurement, hindering the elucidation of their direct relationship. Previous studies have yielded conflicting findings regarding the impact of H2O2 on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for H2O2 and geNOps for NO, allowing simultaneous imaging in single endothelial cells. Optimization strategies were implemented to enhance biosensor performance, including camera binning, temperature regulation, and environmental adjustments to mimic physiological normoxia. Our results demonstrate that under ambient oxygen conditions, H2O2 exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O2) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced H2O2 scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between H2O2 and NO under varying oxygen conditions revealed a lack of NO response to H2O2 under hyperoxia (18 kPa O2) but a modest NO response under physiological normoxia (5 kPa O2). Importantly, the NO response was attenuated by l-NAME, suggesting activation of eNOS by endogenous H2O2 generation upon auranofin treatment. Our study highlights the intricate interplay between H2O2 and NO within the endothelial EA.hy926 cell line, emphasizing the necessity for additional research within physiological contexts due to differential response observed under physiological normoxia (5 kPa O2). This further investigation is essential for a comprehensive understanding of the H2O2 and NO signaling considering the physiological effects of ambient O2 levels involved.

Opto-Lipidomics of Tissues.

Lipid metabolism and signaling play pivotal functions in biology and disease development. Despite this, currently available optical techniques are limited in their ability to directly visualize the lipidome in tissues. In this study, opto-lipidomics, a new approach to optical molecular tissue imaging is introduced. The capability of vibrational Raman spectroscopy is expanded to identify individual lipids in complex tissue matrices through correlation with desorption electrospray ionization (DESI) - mass spectrometry (MS) imaging in an integrated instrument. A computational pipeline of inter-modality analysis is established to infer lipidomic information from optical vibrational spectra. Opto-lipidomic imaging of transient cerebral ischemia-reperfusion injury in a murine model of ischemic stroke demonstrates the visualization and identification of lipids in disease with high molecular specificity using Raman scattered light. Furthermore, opto-lipidomics in a handheld fiber-optic Raman probe is deployed and demonstrates real-time classification of bulk brain tissues based on specific lipid abundances. Opto-lipidomics opens a host of new opportunities to study lipid biomarkers for diagnostics, prognostics, and novel therapeutic targets.

Temporal and spatial distribution of Nrf2 in rat brain following stroke: quantification of nuclear to cytoplasmic Nrf2 content using a novel immunohistochemical technique.

Activation of the redox-sensitive transcription factor NF-E2 related factor 2 (Nrf2) affords protection against cerebral ischaemia-reperfusion injury via the upregulation of antioxidant defence genes. We have quantified for the first time Nrf2 content in brains from rats subjected to stroke and from cultured bEnd.3 brain endothelial cells using a novel immunohistochemical technique. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 70 min followed by reperfusion for 4, 24 or 72 h. Coronal brain sections were incubated with anti-Nrf2 primary and biotinylated-horseradish peroxidase-conjugated secondary antibody, after which sections were reacted with 3,3-diaminobenzidine (DAB) in the presence of hydrogen peroxide. The initial rates of DAB polymer formation were directly proportional to the Nrf2 protein concentration. Image processing was used to determine the temporal and spatial distribution of Nrf2 in nuclear and cytoplasmic compartments in stroke-affected and contralateral hemispheres. Nuclear to cytoplasmic Nrf2 ratios were increased in the stroke region after 24 h reperfusion and declined after 72 h reperfusion. Pretreatment with the Nrf2 inducer sulforaphane reduced total cellular Nrf2 levels in peri-infarct and core regions of the stroke hemisphere after 24 h reperfusion. Treatment of cultured murine brain endothelial cells with sulforaphane (2.5 µm) increased nuclear accumulation of Nrf2 over 1-4 h. We report the first quantitative measurements of spatial and temporal nuclear Nrf2 expression in rat brains following stroke, and show that sulforaphane pretreatment affects Nrf2 distribution in the brain of naïve rats and animals subjected to cerebral ischaemia. Our findings provide novel insights for targeting endogenous redox-sensitive antioxidant pathways to ameliorate the damaging consequences of stroke.

Stress Activated MAP Kinases and Cyclin-Dependent Kinase 5 Mediate Nuclear Translocation of Nrf2 via Hsp90α-Pin1-Dynein Motor Transport Machinery.

Non-lethal low levels of oxidative stress leads to rapid activation of the transcription factor nuclear factor-E2-related factor 2 (Nrf2), which upregulates the expression of genes important for detoxification, glutathione synthesis, and defense against oxidative damage. Stress-activated MAP kinases p38, ERK, and JNK cooperate in the efficient nuclear accumulation of Nrf2 in a cell-type-dependent manner. Activation of p38 induces membrane trafficking of a glutathione sensor neutral sphingomyelinase 2, which generates ceramide upon depletion of cellular glutathione. We previously proposed that caveolin-1 in lipid rafts provides a signaling hub for the phosphorylation of Nrf2 by ceramide-activated PKCζ and casein kinase 2 to stabilize Nrf2 and mask a nuclear export signal. We further propose a mechanism of facilitated Nrf2 nuclear translocation by ERK and JNK. ERK and JNK phosphorylation of Nrf2 induces the association of prolyl cis/trans isomerase Pin1, which specifically recognizes phosphorylated serine or threonine immediately preceding a proline residue. Pin1-induced structural changes allow importin-α5 to associate with Nrf2. Pin1 is a co-chaperone of Hsp90α and mediates the association of the Nrf2-Pin1-Hsp90α complex with the dynein motor complex, which is involved in transporting the signaling complex to the nucleus along microtubules. In addition to ERK and JNK, cyclin-dependent kinase 5 could phosphorylate Nrf2 and mediate the transport of Nrf2 to the nucleus via the Pin1-Hsp90α system. Some other ERK target proteins, such as pyruvate kinase M2 and hypoxia-inducible transcription factor-1, are also transported to the nucleus via the Pin1-Hsp90α system to modulate gene expression and energy metabolism. Notably, as malignant tumors often express enhanced Pin1-Hsp90α signaling pathways, this provides a potential therapeutic target for tumors.

Control of Redox Homeostasis by Short-Chain Fatty Acids: Implications for the Prevention and Treatment of Breast Cancer.

Breast cancer is the leading cause of death among women worldwide, and certain subtypes are highly aggressive and drug resistant. As oxidative stress is linked to the onset and progression of cancer, new alternative therapies, based on plant-derived compounds that activate signaling pathways involved in the maintenance of cellular redox homeostasis, have received increasing interest. Among the bioactive dietary compounds considered for cancer prevention and treatment are flavonoids, such as quercetin, carotenoids, such as lycopene, polyphenols, such as resveratrol and stilbenes, and isothiocyanates, such as sulforaphane. In healthy cells, these bioactive phytochemicals exhibit antioxidant, anti-apoptotic and anti-inflammatory properties through intracellular signaling pathways and epigenetic regulation. Short-chain fatty acids (SCFAs), produced by intestinal microbiota and obtained from the diet, also exhibit anti-inflammatory and anti-proliferative properties related to their redox signaling activity-and are thus key for cell homeostasis. There is evidence supporting an antioxidant role for SCFAs, mainly butyrate, as modulators of Nrf2-Keap1 signaling involving the inhibition of histone deacetylases (HDACs) and/or Nrf2 nuclear translocation. Incorporation of SCFAs in nutritional and pharmacological interventions changes the composition of the the intestinal microbiota, which has been shown to be relevant for cancer prevention and treatment. In this review, we focused on the antioxidant properties of SCFAs and their impact on cancer development and treatment, with special emphasis on breast cancer.

The NRF2/Keap1 pathway as a therapeutic target in inflammatory bowel disease.

Oxidative stress (OS) is an important pathophysiological mechanism in inflammatory bowel disease (IBD). However, clinical trials investigating compounds directly targeting OS in IBD yielded mixed results. The NRF2 (nuclear factor erythroid 2-related factor 2)/Keap1 (Kelch-like ECH-associated protein 1) pathway orchestrates cellular responses to OS, and dysregulation of this pathway has been implicated in IBD. Activation of the NRF2/Keap1 pathway may enhance antioxidant responses. Although this approach could help to attenuate OS and potentially improve clinical outcomes, an overview of human evidence for modulating the NRF2/Keap1 axis and more recent developments in IBD is lacking. This review explores the NRF2/Keap1 pathway as potential therapeutic target in IBD and presents compounds activating this pathway for future clinical applications.

Redox and metal profiles in human coronary endothelial and smooth muscle cells under hyperoxia, physiological normoxia and hypoxia: Effects of NRF2 signaling on intracellular zinc.

Zinc is an important component of cellular antioxidant defenses and dysregulation of zinc homeostasis is a risk factor for coronary heart disease and ischemia/reperfusion injury. Intracellular homeostasis of metals, such as zinc, iron and calcium are interrelated with cellular responses to oxidative stress. Most cells experience significantly lower oxygen levels in vivo (2-10 kPa O2) compared to standard in vitro cell culture (18kPa O2). We report the first evidence that total intracellular zinc content decreases significantly in human coronary artery endothelial cells (HCAEC), but not in human coronary artery smooth muscle cells (HCASMC), after lowering of O2 levels from hyperoxia (18 kPa O2) to physiological normoxia (5 kPa O2) and hypoxia (1 kPa O2). This was paralleled by O2-dependent differences in redox phenotype based on measurements of glutathione, ATP and NRF2-targeted protein expression in HCAEC and HCASMC. NRF2-induced NQO1 expression was attenuated in both HCAEC and HCASMC under 5 kPa O2 compared to 18 kPa O2. Expression of the zinc efflux transporter ZnT1 increased in HCAEC under 5 kPa O2, whilst expression of the zinc-binding protein metallothionine (MT) decreased as O2 levels were lowered from 18 to 1 kPa O2. Negligible changes in ZnT1 and MT expression were observed in HCASMC. Silencing NRF2 transcription reduced total intracellular zinc under 18 kPa O2 in HCAEC with negligible changes in HCASMC, whilst NRF2 activation or overexpression increased zinc content in HCAEC, but not HCASMC, under 5 kPa O2. This study has identified cell type specific changes in the redox phenotype and metal profile in human coronary artery cells under physiological O2 levels. Our findings provide novel insights into the effect of NRF2 signaling on Zn content and may inform targeted therapies for cardiovascular diseases.

Vascular protection afforded by zinc supplementation in human coronary artery smooth muscle cells mediated by NRF2 signaling under hypoxia/reoxygenation.

Zinc (Zn) has antioxidant, anti-inflammatory and anti-proliferative actions, with Zn dysregulation associated with coronary ischemia/reperfusion injury and smooth muscle cell dysfunction. As the majority of studies concerning Zn have been conducted under non-physiological hyperoxic conditions, we compare the effects of Zn chelation or supplementation on total intracellular Zn content, antioxidant NRF2 targeted gene transcription and hypoxia/reoxygenation-induced reactive oxygen species generation in human coronary artery smooth muscle cells (HCASMC) pre-adapted to hyperoxia (18 kPa O2) or normoxia (5 kPa O2). Expression of the smooth muscle marker SM22-α was unaffected by lowering pericellular O2, whereas calponin-1 was significantly upregulated in cells under 5 kPa O2, indicating a more physiological contractile phenotype under 5 kPa O2. Inductively coupled plasma mass spectrometry established that Zn supplementation (10 µM ZnCl2 + 0.5 µM pyrithione) significantly increased total Zn content in HCASMC under 18 but not 5 kPa O2. Zn supplementation increased metallothionein mRNA expression and NRF2 nuclear accumulation in cells under 18 or 5 kPa O2. Notably, NRF2 regulated HO-1 and NQO1 mRNA expression in response to Zn supplementation was only upregulated in cells under 18 but not 5 kPa. Furthermore, whilst hypoxia increased intracellular glutathione (GSH) in cells pre-adapted to 18 but not 5 kPa O2, reoxygenation had negligible effects on GSH or total Zn content. Reoxygenation-induced superoxide generation in cells under 18 kPa O2 was abrogated by PEG-superoxide dismutase but not by PEG-catalase, and Zn supplementation, but not Zn chelation, attenuated reoxygenation-induced superoxide generation in cells under 18 but not 5kPaO2, consistent with a lower redox stress under physiological normoxia. Our findings highlight that culture of HCASMC under physiological normoxia recapitulates an in vivo contractile phenotype and that effects of Zn on NRF2 signaling are altered by oxygen tension.

Sulforaphane induced NRF2 activation in obese pregnancy attenuates developmental redox imbalance and improves early-life cardiovascular function in offspring.

In adverse pregnancy a perturbed redox environment is associated with abnormal early-life cardiovascular development and function. Previous studies have noted alterations in the expression and/or activity of Nuclear Factor E2 Related Factor 2 (NRF2) and its antioxidant targets during human gestational diabetic (GDM) pregnancy, however to our knowledge the functional role of NRF2 in fetal 'priming' of cardiovascular dysfunction in obese and GDM pregnancy has not been investigated. Using a murine model of obesity-induced glucose dysregulated pregnancy, we demonstrate that NRF2 activation by maternal sulforaphane (SFN) supplementation normalizes NRF2-linked NQO1, GCL and CuZnSOD expression in maternal and fetal liver placental and fetal heart tissue by gestational day 17.5. Activation of NRF2 in utero in wild type but not NRF2 deficient mice improved markers of placental efficiency and partially restored fetal growth. SFN supplementation was associated with reduced markers of fetal cardiac oxidative stress, including Nox2 and 3-nitrotyrosine, as well as attenuation of cardiac mass and cardiomyocyte area in male offspring by postnatal day 52 and improved vascular function in male and female offspring by postnatal day 98. Our findings are the first to highlight the functional consequences of NRF2 modulation in utero on early-life cardiovascular function in offspring, demonstrating that activation of NRF2 affords cardiovascular protection in offspring of pregnancies affected by redox dysregulation.

Human blood vessel organoids reveal a critical role for CTGF in maintaining microvascular integrity.

The microvasculature plays a key role in tissue perfusion and exchange of gases and metabolites. In this study we use human blood vessel organoids (BVOs) as a model of the microvasculature. BVOs fully recapitulate key features of the human microvasculature, including the reliance of mature endothelial cells on glycolytic metabolism, as concluded from metabolic flux assays and mass spectrometry-based metabolomics using stable tracing of 13C-glucose. Pharmacological targeting of PFKFB3, an activator of glycolysis, using two chemical inhibitors results in rapid BVO restructuring, vessel regression with reduced pericyte coverage. PFKFB3 mutant BVOs also display similar structural remodelling. Proteomic analysis of the BVO secretome reveal remodelling of the extracellular matrix and differential expression of paracrine mediators such as CTGF. Treatment with recombinant CTGF recovers microvessel structure. In this work we demonstrate that BVOs rapidly undergo restructuring in response to metabolic changes and identify CTGF as a critical paracrine regulator of microvascular integrity.

Mechanisms underlying Nrf2 nuclear translocation by non-lethal levels of hydrogen peroxide: p38 MAPK-dependent neutral sphingomyelinase2 membrane trafficking and ceramide/PKCζ/CK2 signaling.

Hydrogen peroxide is an aerobic metabolite playing a central role in redox signaling and oxidative stress. H2O2 could activate redox sensitive transcription factors, such as Nrf2, AP-1 and NF-κB by different manners. In some cells, treatment with non-lethal levels of H2O2 induces rapid activation of Nrf2, which upregulates expression of a set of genes involved in glutathione (GSH) synthesis and defenses against oxidative damage. It depends on two steps, the rapid translational activation of Nrf2 and facilitation of Nrf2 nuclear translocation. We review the molecular mechanisms by which H2O2 induces nuclear translocation of Nrf2 in cultured cells by highlighting the role of neutral sphingomyelinase 2 (nSMase2), a GSH sensor. H2O2 enters cells through aquaporin channels in the plasma membrane and is rapidly reduced to H2O by GSH peroxidases to consume cellular GSH, resulting in nSMase2 activation to generate ceramide. H2O2 also activates p38 MAP kinase, which enhances transfer of nSMase2 from perinuclear regions to plasma membrane lipid rafts to accelerate ceramide generation. Low levels of ceramide activate PKCζ, which then activates casein kinase 2 (CK2). These protein kinases are able to phosphorylate Nrf2 to stabilize and activate it. Notably, Nrf2 also binds to caveolin-1 (Cav1), which protects Nrf2 from Keap1-mediated degradation and limits Nrf2 nuclear translocation. We propose that Cav1serves as a signaling hub for the control of H2O2-mediated phosphorylation of Nrf2 by kinases, which results in release of Nrf2 from Cav1 to facilitate nuclear translocation. In summary, H2O2 induces GSH depletion which is recovered by Nrf2 activation dependent on p38/nSMase2/ceramide signaling.