Dependent Reactive Oxygen Species Research Articles

CAX1 suppresses Cd-induced generation of reactive oxygen species in Arabidopsis halleri.

The molecular analysis of metal hyperaccumulation in species such as Arabidopsis halleri offers the chance to gain insights into metal homeostasis and into the evolution of adaptation to extreme habitats. A prerequisite of metal hyperaccumulation is metal hypertolerance. Genetic analysis of a backcross population derived from Arabidopsis lyrata×A. halleri crosses revealed three quantitative trait loci for Cd hypertolerance. A candidate gene for Cdtol2 is AhCAX1, encoding a vacuolar Ca2+ /H+ antiporter. We developed a method for the transformation of vegetatively propagated A.halleri plants and generated AhCAX1-silenced lines. Upon Cd2+ exposure, several-fold higher accumulation of reactive oxygen species (ROS) was detectable in roots of AhCAX1-silenced plants. In accordance with the dependence of Cdtol2 on external Ca2+ concentration, this phenotype was exclusively observed in low Ca2+ conditions. The effects of external Ca2+ on Cd accumulation cannot explain the phenotype as they were not influenced by the genotype. Our data strongly support the hypothesis that higher expression of CAX1 in A.halleri relative to other Arabidopsis species represents a Cd hypertolerance factor. We propose a function of AhCAX1 in preventing a positive feedback loop of Cd-elicited ROS production triggering further Ca2+ -dependent ROS accumulation.

Intermittent Hypoxia Induced Formation of "Endothelial Cell-Colony Forming Units (EC-CFUs)" Is Affected by ROS and Oxidative Stress.

Intermittent hypoxia (IH)—the hallmark of obstructive sleep apnea (OSA)—increases leukocyte activation, production of NADPH-oxidase dependent reactive oxygen species (ROS) and oxidative stress, affecting endothelial function. However, IH and oxidative stress can also stimulate adaptive-protective mechanisms by inducing the development of Endothelial Cell-Colony Forming Units (EC-CFUs), which are considered as a good surrogate marker for endothelial progenitor cells (EPCs), and likely reflect a reparatory response to vascular damage or tissue ischemia by leukocytes. Blood samples were obtained from 15 healthy consenting volunteers to evaluate the effects of IH and sustained hypoxia (SH) in vitro on EC-CFUs development and functions. The variables measured included: their numbers, the area, the proliferative capacity and ROS production. Additionally, NADPH-oxidase, VEGF and nuclear factor-erythroid 2 related factor 2 (Nrf2) expression, as well as their paracrine effects on endothelial tube formation were determined. The involvement of ROS was probed using the anti-oxidant N-acetylcysteine (NAC) and NADPH-oxidase inhibitors apocynin and diphenyl-iodide. Compared to normoxia, IH-dependent increases in EC-CFUs numbers were observed, showing an individual donor-dependent trait. Also, the expression of VEGF and gp91phox, a subunit of NADPH-oxidase, were significantly increased. ROS production and oxidative stress markers were also significantly increased, but Nrf2 expression and colony size were unaffected by IH. Additionally, conditioned media harvested from IH- and SH-treated mature EC-CFUs, significantly increased endothelial tube formation. These effects were markedly attenuated or diminished by the ROS and NADPH-oxidase inhibitors employed. In conclusion, we show here for the first time that IH-associated oxidative stress promotes EC-CFUs' vascular and paracrine capacities through ROS. However, the large inter-individual variability expressed in EC-CFUs numbers and functions to a given IH stimulus, may represent an individual trait with a potential clinical significance.

Anticancer activity study of A3 adenosine receptor agonists

AimsA3 adenosine receptor (A3AR) signalling activation seems to mediate anticancer effect, and it has been targeted for drug development. The identification of potent and selective A3AR agonists could be crucial for cancer drug development. Materials and methodsIn the present study was determined the in vitro activity of known 1–3 and newly 4–6 synthesized compounds with high A3AR affinity and selectivity (Ki in the low nanomolar range) in binding studies. Effect of known and novel A3AR agonists on human prostate cancer (PC3), hepatocellular carcinoma (Hep G2), and epithelial colorectal carcinoma (Caco-2) cells were analysed by cytotoxicity assay, dose and time dependent inhibitor assay, migration, apoptosis, autophagy and reactive oxygen species (ROS) assays. Key findingsResults show that the anticancer effect is not due to A3AR activation alone. In fact, the more active and selective agonist versus A3AR, compound 1, results inactive on cancer cells such as compounds 2–4. Moreover, results show that the novel compound 5, at micromolar concentration range (IC50 = 28.0 μM), inhibits the growth of PC3, Hep G2, and Caco-2 cells and their migration in time- and dose- dependent manner. The mechanism involved in cell death is attributable to apoptosis. At the same time compound 5 promotes autophagy, which induce apoptosis producing autophagic cell death. Further investigation revealed that compound 5 elevates the level of ROS in all cancer cells tested, suggesting the involvement of ROS in cell death. SignificanceThese results show that the new compound 5 exerts inhibitory effect on cancer cells through differential effect and may serve as a potential anticancer agent.

Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune inhibition

Abstract Neutrophils are a vital component of the immune system, however in cancer they often play major roles in tumour progression, partly by generating reactive oxygen species (ROS) which disrupts lymphocyte function. Metabolically, neutrophils are often overlooked as purely glycolytic, however we show that immature subsets, defined by c-Kit expression, possess the capacity for oxidative mitochondrial metabolism. In limited glucose, oxidative neutrophils use mitochondrial fatty acid oxidation to fuel NADPH-oxidase (NOX) dependent ROS production. In 4T1 tumour bearing mice, mitochondrial fitness is enhanced in circulating and splenic neutrophils and is associated with the expression of c-Kit. Concordantly, tumour-elicited oxidative neutrophils are able to maintain ROS production and suppression of T-cell when glucose utilization is restricted. In accordance with these findings, peripheral blood neutrophils from cancer patient are more immature, with increased mitochondrial content and higher subsequent oxidative phosphorylation. Together our data suggest that tumours elicit oxidative neutrophil populations which are adapted to maintain immune-suppressive activity in the glucose-restricted tumour microenvironment.

CD28 Induces Mitochondrial Respiration Dependent Reactive Oxygen Species (ROS) Signaling for Metabolic Fitness and Survival in Long-Lived Plasma Cells

Abstract Durable humoral immunity depends upon bone marrow resident long-lived plasma cell (LLPC) survival. We have shown that CD28, the canonical T-cell costimulatory molecule, is required for LLPC survival; however, the mechanistic basis for this is unclear. We have published that of two binding motifs in the CD28 cytoplasmic tail, only the Grb2/Vav moiety, and not the direct PI3k binding motif, supports CD28-mediated survival. CD28 is known to induce glycolysis in T-cells. In contrast, CD28 activation in both LLPCs and human PCs induces glucose metabolism through mitochondrial respiration. A major byproduct of mitochondrial respiration is the production of reactive oxygen species (ROS). Interestingly, the mitochondrial respiration-dependent production of ROS is required for CD28-mediated survival. Previously published data demonstrate the ability of ROS to signal through both NFkB and PI3k. CD28-induced ROS signal through PI3k in LLPCs for increased glucose uptake and the inhibition of PI3k prevents CD28-mediated survival. The ROS-PI3k signal drives the phosphorylation of both S6k and rpS6, downstream targets of mTORC1, and this regulation is independent of Blimp1 expression. Irf4, a critical PC survival factor, is upregulated by CD28-dependent NFkB activation. To our great surprise, ROS inhibition prevented the induction of mitochondrial respiration itself, demonstrating that signals emanating downstream of ROS govern the metabolic program for survival in LLPCs. Taken together, these data suggest a model wherein CD28 signals directly through Grb2/Vav to induce mitochondrial respiration and ROS production. ROS then reinforce signaling through PI3k and NFkB to increase Irf4 expression and metabolic fitness for LLPC survival.

HDAC1 Mediates High Salt‐Induced α‐Adrenergic Sensitivity in Mouse Aorta

Clincal studies have shown that high salt intake can have a negative impact on vascular function and induce hypertension in some individuals; however, the mechanism for these effects is not well understood. We previously found that histone deacetylase 1 (HDAC1) is upregulated in response to a high salt diet and have reported that HDAC1 deacetylates endothelial nitric oxide synthase (NOS3) leading to reduced NO production. Adrenergic vasoconstriction may be enhanced by the loss of NO. We therefore hypothesized that high salt induces enhanced adrenergic vasoconstriction via HDAC1. In order to test this, we isolated aortic segments from both male and female mice after 1 week of either normal salt diet (NS; 0.49% NaCl) or high salt diet (HS; 4% NaCl). Aortic constriction was measured in response to 31.62 μM phenylephrine, an α1‐adrenergic receptor agonist, in the presence of vehicle or 300 nM MS‐275, an HDAC1 inhibitor, pre‐incubated for 40 min. Aorta from mice on HS displayed greater constriction in response to phenylephrine compared to aorta from mice on NS (HS: 13.16 ± 0.61 mN vs. NS: 8.01 ± 0.51 mN, n=3–5, p<0.001). MS‐275 pre‐treatment did not change PE‐induced vasoconstriction in aorta from mice on NS (Veh: 8.01 ± 0.51 mN vs. MS‐275: 7.04 ± 2.42 mN, n=2–3). While MS‐275 pre‐treatment reduced PE‐induced vasoconstriction in aorta from mice on HS (Veh: 13.16 ± 0.61 mN vs. MS‐275: 10.78 ± 0.73 mN, n=3–5, p<0.05). HS and NS had similar vasoconstriction induced by 60 mM KCl (NS: 4.85 ± 0.69 mN vs. HS: 5.29 ± 0.83 mN, n=3–5). These data suggest that HS induces enhanced adrenergic vasoconstriction via an HDAC1‐dependent mechanism. Because, adrenergic vasoconstriction may be enhanced by the loss of NO and/or increased reactive oxygen species (ROS), we hypothesized that HDAC1 leads to NOS3 uncoupling, reducing NO and increasing ROS. Endothelial cells were transfected with HDAC1 plasmid and Western blot confirmed HDAC1 over‐expression. Basal and stimulated ROS production was detected by luminol luminescence. HDAC1 overexpression increased both basal NOS dependent ROS production (Vector: 66.35 ± 4.91 RLU/sec/g vs. HDAC1: 111.46 ± 11.98 RLU/sec/g, n=3, p<0.05) and stimulated NOS dependent ROS production (Vector: 856.38 ± 196.10 RLU/sec/g vs. HDAC1: 1578.93 ± 29.59 RLU/sec/g, n=3, p<0.05). These data suggest that HDAC1 deacetylation of NOS3 results in NOS‐dependent ROS. Taken together, these findings suggest that HDAC1 contributes indirectly to the enhanced adrenergic mediated signaling observed in high salt fed animals through the uncoupling of NOS3.Support or Funding InformationThis research was funded by Translational and Molecular Sciences (TMS) Training Grant 5 T32 GM 109780‐3 awarded to Luke Dunaway, KO1‐DK‐105038 awarded to Dr. Kelly A. Hyndman, and HL 136936 awarded to Dr. Jennifer S. Pollock and Dr. David M. Pollock.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Doxorubicin‐induced fetal glomerular mesangial cell apoptosis involves NADPH oxidase‐dependent reactive oxygen species generation

Animal studies have shown that chemotherapeutic agent doxorubicin (DOX) causes fetal toxicity, including renal abnormalities. Although DOX‐associated nephropathy includes oxyradical stress and glomerular injury, its effect on fetal glomerular cells remains unclear. This study aimed to examine the effect of DOX on fetal glomerular mesangial cell (FE‐GMC) survival. Furthermore, we tested the hypothesis that NADPH oxidase (NOX)‐dependent reactive oxygen species generation contributes to DOX‐induced alterations in FE‐GMCs. Confocal microscopy and high‐performance liquid chromatography revealed that the level of 2‐hydroxyethidium was increased in DOX‐treated cells when compared to DMSO controls, which was reversed by selective NOX inhibitor, apocynin. Apocynin also attenuated DOX‐induced caspase 3/7 activation in FE‐GMCs. These data suggest that DOX induces apoptosis in porcine FE‐GMCs by promoting NOX‐dependent oxidative stress. Caspase‐mediated cell death may contribute to the toxic effects of DOX on the developing kidney.Support or Funding InformationR01DK101668; R01DK101668‐04S1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

418 Protection against AhR-induced oxidative stress and premature skin aging

Pollution is a major concern in our urbanized and industrialized countries. As a result, the skin is in contact with particulate matter (PM), which are tiny pollution particles associated to polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HM). Deleterious effects of these pollutants on the skin are now well described. Key players induced by pollutants following activation of Aryl hydrocarbon receptor (AhR) are the noxious Reactive Oxygen Species (ROS). We showed in keratinocytes that AhR was activated following application of calibrated HM or PAHs and in the mean time we measured an increase of ROS production. A more close-to-reality model, ex vivo skin explant, was then exposed to a mix of HM and PAHs to check for pollutants damaging effects. After HM/PAHs treatment, we measured an increase of malonaldehyde (MDA), a lipid peroxidation product. This oxidative stress causes dramatic biological consequences occurring during skin aging: a deterioration of dermo-epidermal junction (DEJ), an overexpression of MMP-1 protein, and a decrease of collagen type I expression. AhR activation regulates the expression of the NADPH oxidases (NOX) that are major contributors of ROS production. Intracellular ROS activate AP-1 signalling which in turn stimulates inflammatory pathways evidenced here by COX2 and MMP1 expression. Camellia Japonica flower extract exhibits an interesting antioxidant activity measured by DPPH assay (radical scavenging), thus constituting a good candidate for skin protection from harmful pollutants. We showed in keratinocytes, that this extract limits AhR activation and abrogates HM and PAHs dependent ROS induction. In addition, we showed in ex vivo model that this extract limits NOX related inflammatory pathways activation by inhibiting MMP1 and COX2 induction but stimulates collagen type I expression and preserves the global tissue morphology. Moreover, clinical results confirmed this anti-aging effect. In conclusion, we propose Camellia japonica flower extract as a powerful active ingredient fighting against pollutants-induced premature skin aging.

Ferroptosis and autophagy induced cell death occur independently after siramesine and lapatinib treatment in breast cancer cells.

Ferroptosis is a cell death pathway characterized by iron-dependent accumulation of reactive oxygen species (ROS) within the cell. The combination of siramesine, a lysosome disruptor, and lapatinib, a dual tyrosine kinase inhibitor, has been shown to synergistically induce cell death in breast cancer cells mediated by ferroptosis. These treatments also induce autophagy but its role in this synergistic cell death is unclear. In this study, we determined that siramesine and lapatinib initially induced ferroptosis but changes to an autophagy induced cell death after 24 hours. Furthermore, we found that intracellular iron level increased in a time dependent manner following treatment accompanied by an increase in ROS. Using the iron chelator deferoxamine (DFO) or the ROS scavenger alpha-tocopherol decreased both autophagy flux and cell death. We further discovered that decreased expression of the iron storage protein, ferritin was partially dependent upon autophagy degradation. In contrast, the expression of transferrin, which is responsible for the transport of iron into cells, is increased following treatment with lapatinib alone or in combination with siramesine. This indicates that ferroptosis and autophagy induced cell death occur independently but both are mediated by iron dependent ROS generation in breast cancer cells.

Signaling at the Crossroads: Matrix-Derived Proteoglycan and Reactive Oxygen Species Signaling.

Proteoglycans (PGs), besides their structural contribution, have emerged as dynamic components that mediate a multitude of cellular events. The various roles of PGs are attributed to their structure, spatial localization, and ability to act as ligands and receptors. Reactive oxygen species (ROS) are small mediators that are generated in physiological and pathological conditions. Besides their reactivity and ability to induce oxidative stress, a growing body of data suggests that ROS signaling is more relevant than direct radical damage in development of human pathologies. Recent Advances: Cell surface transmembrane PGs (syndecans, cluster of differentiation 44) represent receptors in diverse and complex transduction networks, which involve redox signaling with implications in cancer, fibrosis, renal dysfunction, or Alzheimer's disease. Through NADPH oxidase (NOX)-dependent ROS, the extracellular PG, hyaluronan is involved in osteoclastogenesis and cancer. The ROS sources, NOX1 and NOX4, increase biglycan-induced inflammation, while NOX2 is a negative regulator. The complexity of the mechanisms that bring ROS into the light of PG biology might be the foundation of a new research area with significant promise for understanding health and disease. Important aspects need to be investigated in PG/ROS signaling: the discovery of specific targets of ROS, the precise ROS-induced chemical modifications of these targets, and the study of their pathological relevance. As we become more and more aware of the interactions between PG and ROS signaling underlying intracellular communication and cell fate decisions, it is quite conceivable that this field will allow to identify new therapeutic targets.-Antioxid. Redox Signal. 27, 855-873.

Hv1 proton channel facilitates production of ROS and pro-inflammatory cytokines in microglia and enhances oligodendrocyte progenitor cells damage from oxygen-glucose deprivation in vitro

The contribution of microglial activation to oligodendrocyte precursor cell (OPC) damage in the brain is considered to be a principal pathophysiological feature of periventricular leukomalacia (PVL). Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-dependent reactive oxygen species (ROS) produced in microglia has been shown to be significantly toxic to OPCs. The voltage-gated proton channel Hv1 is selectively expressed in microglia and is essential for NOX-dependent ROS production in the central nervous system. This study aimed to investigate the effects of microglial Hv1 deficiency on the protection of OPCs from oxygen-glucose deprivation (OGD)-induced injury in vitro. In the present study, the levels of OGD-induced ROS and pro-inflammatory cytokine production were dramatically lower in Hv1-deficient microglia (Hv1−/−) than in wild-type (WT) microglia. Following OGD, OPCs co-cultured with WT microglia had increased apoptosis and decreased proliferation and maturation, while those co-cultured with Hv1−/− microglia had attenuated apoptosis and greater proliferation and differentiation. Furthermore, the attenuated damage and enhanced regeneration of OPCs were associated with decreases in extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase phosphorylation. These results indicate that the protective effects of Hv1 deficiency on OPCs are due to the suppression of ROS and pro-inflammatory cytokine production in microglia. We thus suggest that the microglial proton channel Hv1 may be a potential therapeutic target in PVL.

Differential cytotoxic activity of Quercetin on colonic cancer cells depends on ROS generation through COX-2 expression

Quercetin is a bioactive compound with anti-inflammatory, antioxidant and anticancer properties. This study exemplifies the differential cytotoxic activity of Quercetin on two human colonic cancer cell lines, HT29 and HCT15. IC50 of Quercetin for HT29 and HCT15 cells were 42.5 μM and 77.4 μM, respectively. Activation of caspase-3, increased level of cytosolic cytochrome c, decreased levels of pAkt, pGSK-3β and cyclin D1 in 40 μM Quercetin treated HT29 cells alone. Though, nuclear translocation of NFkB was increased in 40 μM Quercetin treated HT29 and HCT15 cells, over expression of COX-2 was observed in 40 μM Quercetin treated HT29 cells, whereas, Quercetin treated HCT15 cells did not expressed COX-2. Increased generation of reactive oxygen species (ROS) was observed only in Quercetin treated HT29 cells, which is due to over expression of COX-2, as COX-2 silencing inhibited Quercetin induced apoptosis and ROS generation. Insilico analysis provided evidence that Quercetin could partially inhibit COX-2 enzyme by binding to subunit A which has peroxidase activity and serves as source of ROS. However, Quercetin showed minimal effect on normal intestinal epithelial cells i,e IEC-6. To conclude, differential sensitivity of two cancer cells, HT29 and HCT15, to Quercetin depends on COX-2 dependent ROS generation that induces apoptosis and inhibits cell survival.

Reactive Oxygen Species-Producing Myeloid Cells Act as a Bone Marrow Niche for Sterile Inflammation-Induced Reactive Granulopoiesis.

Both microbial infection and sterile inflammation augment bone marrow (BM) neutrophil production, but whether the induced accelerated granulopoiesis is mediated by a common pathway and the nature of such a pathway are poorly defined. We recently established that BM myeloid cell-derived reactive oxygen species (ROS) externally regulate myeloid progenitor proliferation and differentiation in bacteria-elicited emergency granulopoiesis. In this article, we show that BM ROS levels are also elevated during sterile inflammation. Similar to in microbial infection, ROS were mainly generated by the phagocytic NADPH oxidase in Gr1+ myeloid cells. The myeloid cells and their ROS were uniformly distributed in the BM when visualized by multiphoton intravital microscopy, and ROS production was both required and sufficient for sterile inflammation-elicited reactive granulopoiesis. Elevated granulopoiesis was mediated by ROS-induced phosphatase and tensin homolog oxidation and deactivation, leading to upregulated PtdIns(3,4,5)P3 signaling and increased progenitor cell proliferation. Collectively, these results demonstrate that, although infection-induced emergency granulopoiesis and sterile inflammation-elicited reactive granulopoiesis are triggered by different stimuli and are mediated by distinct upstream signals, the pathways converge to NADPH oxidase-dependent ROS production by BM myeloid cells. Thus, BM Gr1+ myeloid cells represent a key hematopoietic niche that supports accelerated granulopoiesis in infective and sterile inflammation. This niche may be an excellent target in various immune-mediated pathologies or immune reconstitution after BM transplantation.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (Gϕ). Unlike neutrophils, Gϕ are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. Gϕ also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing Gϕ in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes <em>In Vitro</em>

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (Gϕ). Unlike neutrophils, Gϕ are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. Gϕ also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing Gϕ in order to further investigate their significance and functions.

The NOX2-derived reactive oxygen species damaged endothelial nitric oxide system via suppressed BKCa/SKCa in preeclampsia

The endothelial nitric oxide (NO) system may be damaged in preeclampsia; however, the involved mechanisms are unclear. In this study, we used primary human umbilical vein endothelial cells (HUVECs) to evaluate the endothelial NO system in preeclampsia and to determine the underlying mechanisms that are involved. We isolated and cultured HUVECs from normal and preeclamptic pregnancies and evaluated endothelial NO synthase enzyme (eNOS) expression and NO production. Whole-cell K+ currents and oxidative stress were also determined in normal and preeclamptic HUVECs. Compared with normal HUVECs, eNOS expression, NO production and whole-cell K+ currents in preeclamptic HUVECs were markedly decreased, whereas oxidative stress was significantly increased. The decreased K+ currents were associated with damaged Ca2+-activated K+ (KCa) channels, especially the large (BKCa) and small (SKCa) conductance KCa channels, and were involved in the downregulated eNOS expression in preeclamptic HUVECs. Moreover, the increased oxidative stress detected in preeclamptic HUVECs was mediated by NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2)-dependent reactive oxygen species overproduction that could downregulate whole-cell K+ currents, eNOS expression and NO production. Taken together, our study indicated that the increased oxidative stress in preeclamptic HUVECs could downregulate the NO system by suppressing BKCa and SKCa channels. Because the damaged NO system was closely related to endothelial dysfunction, this study provides important information to further understand the pathological process of endothelial cell dysfunction in preeclampsia.

Hydrogen-Rich Saline Attenuates Acute Hepatic Injury in Acute Necrotizing Pancreatitis by Inhibiting Inflammation and Apoptosis, Involving JNK and p38 Mitogen-Activated Protein Kinase-dependent Reactive Oxygen Species.

The objective of this study was to study the role of hydrogen-rich saline (HRS) on acute hepatic injury (AHI) in acute necrotizing pancreatitis (ANP). Rats were used for this study and an ANP model was induced by injecting 5% sodium taurocholate into the biliary-pancreatic duct. Experiments were performed in 3 groups: sham, ANP, and ANP + HRS (HRS). Animals were killed at 3, 12, and 24 hours after operation, and then blood and tissue samples were harvested. Various physiological, histological, and cellular and molecular parameters were analyzed. Analyses of serum, lipase, alanine transaminase, and aspartate aminotransferase indicated that ANP-induced AHI model was established successfully and HRS attenuated hepatic dysfunction. Hepatic superoxide dismutase and malondialdehyde levels showed HRS against oxidative stress. Cellular and molecular analyses including p-p38, p-JNK, p-ERK, and caspase-3, caspase-9, NF-κB, and TNF-α in hepatic tissues revealed that HRS attenuated ANP-induced AHI by inhibiting apoptosis and phosphorylation of JNK and p38, as well as NF-κB activation. Hydrogen-rich saline plays a protective role in ANP-induced AHI through inhibiting inflammation and apoptosis, involving JNK and p38 MAPK-dependent reactive oxygen species.

Peptide screen identifies a new NADPH oxidase inhibitor: impact on cell migration and invasion

The NADPH oxidase proteins catalyse the formation of superoxide anion which act as signalling molecules in physiological and pathological processes. Nox1-dependent NADPH oxidase is expressed in heart, lung, colon, blood vessels and brain. Different strategies involving Nox1 inhibition based on diphenylene iodonium derivatives are currently tested for colorectal cancer therapy. Here, after peptides screening on Nox1-dependent NADPH oxidase assay in HT-29 cells, we identify a peptide (referred to as NF02), cell-active, that potently block Nox1-dependent reactive oxygen species generation. Study of DEPMPO adduct formation by electron paramagnetic resonance showed that NF02 has no superoxide scavenging activity and no impact on cellular reactive oxygen species-producing enzymes such xanthine oxidase. NF02 was not cytotoxic, inhibited reactive oxygen species production of reconstituted Nox1/Noxo1/Noxa1 complex in HEK293 and did not decrease Nox2 dependent cellular NADPH oxidase reactive oxygen species production. Finally, NF02 inhibited cell migration and invasion of colorectal cancer cells which is consistent with the described impact of Nox1 inhibitors on cell migration. NF02 peptide is a new NADPH oxidase inhibitor specific for Nox1 over Nox2 and xanthine oxidase which might represent a useful Nox1 tool with potential therapeutic insights.

Deficiency in the voltage‐gated proton channel Hv1 increases M2 polarization of microglia and attenuates brain damage from photothrombotic ischemic stroke

Microglia become activated during cerebral ischemia and exert pro-inflammatory or anti-inflammatory role dependent of microglial polarization. NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) production in microglia plays an important role in neuronal damage after ischemic stroke. Recently, NOX and ROS are consistently reported to participate in the microglial activation and polarization; NOX2 inhibition or suppression of ROS production are shown to shift the microglial polarization from M1 toward M2 state after stroke. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent ROS generation in the brain. However, the effect of Hv1 proton channel on microglial M1/M2 polarization state after cerebral ischemia remains unknown. In this study, we investigated the role of microglial Hv1 proton channel in modulating microglial M1/M2 polarization during the pathogenesis of ischemic cerebral injury using a mouse model of photothrombosis. Following photothrombotic ischemic stroke, wild-type mice presented obvious brain infarct, neuronal damage, and impaired motor coordination. However, mice lacking Hv1 (Hv1(-/-)) were partially protected from brain damage and motor deficits compared to wild-type mice. These rescued phenotypes in Hv1(-/-) mice in ischemic stroke is accompanied by reduced ROS production, shifted the microglial polarization from M1 to M2 state. Hv1 deficiency was also found to shift the M1/M2 polarization in primary cultured microglia. Our study suggests that the microglial Hv1 proton channel is a unique target for modulation of microglial M1/M2 polarization in the pathogenesis of ischemic stroke. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent generation of reactive oxygen species (ROS) in the brain. ROS participate in microglial activation and polarization. However, the effect of Hv1 on microglial M1/M2 polarization state after cerebral ischemia remains unknown. Hv1 deficiency was found to shift the microglial polarization from M1 to M2 state in ischemic stroke accompanied by reduced ROS production. Our study suggests that the microglial Hv1 proton channel is a unique target for modulation of microglial M1/M2 polarization in the pathogenesis of ischemic stroke.

Bacteriochlorin Dyads as Solvent Polarity Dependent Near-Infrared Fluorophores and Reactive Oxygen Species Photosensitizers.

Symmetrical, near-infrared absorbing bacteriochlorin dyads exhibit gradual reduction of their fluorescence (intensity and lifetime) and reactive oxygen species photosensitization efficiency (ROS) with increasing solvent dielectric constant ε. For the directly linked dyad, significant reduction is observed even in solvents of moderate ε, while for the dyad containing a 1,4-phenylene linker, reduction is more parallel to an increase in solvent ε. Bacteriochlorin dyads are promising candidates for development of environmentally responsive fluorophores and ROS sensitizers.