Effects of Nox4 upregulation on PECAM-1 expression in a mouse model of diabetic retinopathy.

Diabetic Retinopathy (DR) is the leading cause of vision loss in working-age adults. The hallmark features of DR include vascular leakage, capillary loss, retinal ischemia, and aberrant neovascularization. Although the pathophysiology is not fully understood, accumulating evidence supports elevated reactive oxygen species associated with increased activity of NADPH oxidase 4 (Nox4) as major drivers of disease progression. Previously, we have shown that Nox4 upregulation in retinal endothelial cells by diabetes leads to increased vascular leakage by an unknown mechanism. Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell surface molecule that is highly expressed in endothelial cells and regulates endothelial barrier function. In the present study, using endothelial cell-specific human Nox4 transgenic (TG) mice and endothelial cell-specific Nox4 conditional knockout (cKO) mice, we investigated the impact of Nox4 upregulation on PECAM-1 expression in mouse retinas and brain microvascular endothelial cells (BMECs). Additionally, cultured human retinal endothelial cells (HRECs) transduced with adenovirus overexpressing human Nox4 were used in the study. We found that overexpression of Nox4 increases PECAM-1 mRNA but has no effect on its protein expression in the mouse retina, BMECs, or HRECs. Furthermore, PECAM-1 mRNA and protein expression was unchanged in BMECs isolated from cKO mice compared to wild type (WT) mice with or without 2 months of diabetes. Together, these findings do not support a significant role of Nox4 in the regulation of PECAM-1 expression in the diabetic retina and endothelial cells. Further studies are warranted to elucidate the mechanism of Nox4-induced vascular leakage by investigating other intercellular junctional proteins in endothelial cells and their implications in the pathophysiology of diabetic retinopathy.

Activation of PPARδ in bone marrow endothelial progenitor cells improves their hematopoiesis-supporting ability after myelosuppressive injury.

Dysfunctional bone marrow (BM) endothelial progenitor cells (EPCs) with high levels of reactive oxygen species (ROS) are responsible for defective hematopoiesis in poor graft function (PGF) patients with acute leukemia or myelodysplastic neoplasms post-allotransplant. However, the underlying mechanism by which BM EPCs regulate their intracellular ROS levels and the capacity to support hematopoiesis have not been well clarified. Herein, we demonstrated decreased levels of peroxisome proliferator-activated receptor delta (PPARδ), a lipid-activated nuclear receptor, in BM EPCs of PGF patients compared with those with good graft function (GGF). In vitro assays further identified that PPARδ knockdown contributed to reduced and dysfunctional BM EPCs, characterized by the impaired ability to support hematopoiesis, which were restored by PPARδ overexpression. Moreover, GW501516, an agonist of PPARδ, repaired the damaged BM EPCs triggered by 5-fluorouracil (5FU) in vitro and in vivo. Clinically, activation of PPARδ by GW501516 benefited the damaged BM EPCs from PGF patients or acute leukemia patients in complete remission (CR) post-chemotherapy. Mechanistically, we found that increased expression of NADPH oxidases (NOXs), the main ROS-generating enzymes, may lead to elevated ROS level in BM EPCs, and insufficient PPARδ may trigger BM EPC damage via ROS/p53 pathway. Collectively, we found that defective PPARδ contributes to BM EPC dysfunction, whereas activation of PPARδ in BM EPCs improves their hematopoiesis-supporting ability after myelosuppressive therapy, which may provide a potential therapeutic target not only for patients with leukemia but also for those with other cancers.

Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2.

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple ß-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.

Simvastatin attenuates silica-induced pulmonary inflammation and fibrosis in rats via the AMPK-NOX pathway.

BACKGROUND: Simvastatin (Sim), a hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been widely used in prevention and treatment of cardiovascular diseases. Studies have suggested that Sim exerts anti-fibrotic effects by interfering fibroblast proliferation and collagen synthesis. This study was to determine whether Sim could alleviate silica-induced pulmonary fibrosis and explore the underlying mechanisms. METHODS: The rat model of silicosis was established by the tracheal perfusion method and treated with Sim (5 or 10 mg/kg), AICAR (an AMPK agonist), and apocynin (a NOX inhibitor) for 28 days. Lung tissues were collected for further analyses including pathological histology, inflammatory response, oxidative stress, epithelial mesenchymal transformation (EMT), and the AMPK-NOX pathway. RESULTS: Sim significantly reduced silica-induced pulmonary inflammation and fibrosis at 28 days after administration. Sim could reduce the levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α and transforming growth factor-ß1 in lung tissues. The expressions of hydroxyproline, α-SMA and vimentin were down-regulated, while E-cad was increased in Sim-treated rats. In addition, NOX4, p22pox, p40phox, p-p47phox/p47phox expressions and ROS levels were all increased, whereas p-AMPK/AMPK was decreased in silica-induced rats. Sim or AICAR treatment could notably reverse the decrease of AMPK activity and increase of NOX activity induced by silica. Apocynin treatment exhibited similar protective effects to Sim, including down-regulating of oxidative stress and inhibition of the EMT process and inflammatory reactions. CONCLUSIONS: Sim attenuates silica-induced pulmonary inflammation and fibrosis by downregulating EMT and oxidative stress through the AMPK-NOX pathway.

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.

Duox activation in Drosophila Malpighian tubules stimulates intestinal epithelial renewal through a countercurrent flow.

The gut must perform a dual role of protecting the host against toxins and pathogens while harboring mutualistic microbiota. Previous studies suggested that the NADPH oxidase Duox contributes to intestinal homeostasis in Drosophila by producing reactive oxygen species (ROS) in the gut that stimulate epithelial renewal. We find instead that the ROS generated by Duox in the Malpighian tubules leads to the production of Upd3, which enters the gut and stimulates stem cell proliferation. We describe in Drosophila the existence of a countercurrent flow system, which pushes tubule-derived Upd3 to the anterior part of the gut and stimulates epithelial renewal at a distance. Thus, our paper clarifies the role of Duox in gut homeostasis and describes the existence of retrograde fluid flow in the gut, collectively revealing a fascinating example of inter-organ communication.

Proteasome inhibitors induce apoptosis by superoxide anion generation via NADPH oxidase 5 in human neuroblastoma SH-SY5Y cells.

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.

Salmonella Typhimurium employs spermidine to exert protection against ROS-mediated cytotoxicity and rewires host polyamine metabolism to ameliorate its survival in macrophages.

Salmonella infection entails a cascade of attacks and defence measures. After breaching the intestinal epithelial barrier, Salmonella is phagocytosed by macrophages, where the bacteria encounter multiple stresses, to which it employs relevant countermeasures. Our study shows that, in Salmonella, the polyamine spermidine activates a stress response mechanism by regulating critical antioxidant genes. Salmonella Typhimurium mutants for spermidine transport and synthesis cannot mount an antioxidative response, resulting in high intracellular ROS levels. These mutants are also compromised in their ability to be phagocytosed by macrophages. Furthermore, it regulates a novel enzyme in Salmonella, Glutathionyl-spermidine synthetase (GspSA), which prevents the oxidation of proteins in E. coli. Moreover, the spermidine mutants and the GspSA mutant show significantly reduced survival in the presence of hydrogen peroxide in vitro and reduced organ burden in the mouse model of Salmonella infection. Conversely, in macrophages isolated from gp91phox-/- mice, we observed a rescue in the attenuated fold proliferation previously observed upon infection. We found that Salmonella upregulates polyamine biosynthesis in the host through its effectors from SPI-1 and SPI-2, which addresses the attenuated proliferation observed in spermidine transport mutants. Thus, inhibition of this pathway in the host abrogates the proliferation of Salmonella Typhimurium in macrophages. From a therapeutic perspective, inhibiting host polyamine biosynthesis using an FDA-approved chemopreventive drug, D, L-α-difluoromethylornithine (DFMO), reduces Salmonella colonisation and tissue damage in the mouse model of infection while enhancing the survival of infected mice. Therefore, our work provides a mechanistic insight into the critical role of spermidine in stress resistance of Salmonella. It also reveals a bacterial strategy in modulating host metabolism to promote their intracellular survival and shows the potential of DFMO to curb Salmonella infection.

Membrane potential dynamics of C5a-stimulated neutrophil granulocytes.

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3-4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47phox Membrane Anchoring.

As a key component for NADPH oxidase 2 (NOX2) activation, the peripheral membrane protein p47phox translocates a cytosolic activating complex to the membrane through its PX domain. This study elucidates a potential regulatory mechanism of p47phox recruitment and NOX2 activation by inositol hexaphosphate (IP6). Through NMR, fluorescence polarization, and FRET experimental results, IP6 is shown to be capable of breaking the lipid binding and membrane anchoring events of p47phox-PX with low micromolar potency. Other phosphorylated inositol species such as IP5(1,3,4,5,6), IP4(1,3,4,5), and IP3(1,3,4) show weaker binding and no ability to inhibit lipid interactions in physiological concentration ranges. The low micromolar potency of IP6 inhibition of the p47phox membrane anchoring suggests that physiologically relevant concentrations of IP6 serve as regulators, as seen in other membrane anchoring domains. The PX domain of p47phox is known to be promiscuous to a variety of phosphatidylinositol phosphate (PIP) lipids, and this regulation may help target the domain only to the membranes most highly enriched with the highest affinity PIPs, such as the phagosomal membrane, while preventing aberrant binding to other membranes with high and heterogeneous PIP content, such as the plasma membrane. This study provides insight into a potential novel regulatory mechanism behind NOX2 activation and reveals a role for small-molecule regulation in this important NOX2 activator.

Reactive oxygen species impair Na+ transport and renal components of the renin-angiotensin-aldosterone system after paraquat poisoning.

Paraquat (1,1'-dimethyl-4,4'-bipyridyl dichloride) is an herbicide widely used worldwide and officially banned in Brazil in 2020. Kidney lesions frequently occur, leading to acute kidney injury (AKI) due to exacerbated reactive O2 species (ROS) production. However, the consequences of ROS exposure on ionic transport and the regulator local renin-angiotensin-aldosterone system (RAAS) still need to be elucidated at a molecular level. This study evaluated how ROS acutely influences Na+-transporting ATPases and the renal RAAS. Adult male Wistar rats received paraquat (20 mg/kg; ip). After 24 h, we observed body weight loss and elevation of urinary flow and serum creatinine. In the renal cortex, paraquat increased ROS levels, NADPH oxidase and (Na++K+)ATPase activities, angiotensin II-type 1 receptors, tumor necrosis factor-α (TNF-α), and interleukin-6. In the medulla, paraquat increased ROS levels and NADPH oxidase activity but inhibited (Na++K+)ATPase. Paraquat induced opposite effects on the ouabain-resistant Na+-ATPase in the cortex (decrease) and medulla (increase). These alterations, except for increased serum creatinine and renal levels of TNF-α and interleukin-6, were prevented by 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (tempol; 1 mmol/L in drinking water), a stable antioxidant. In summary, after paraquat poisoning, ROS production culminated with impaired medullary function, urinary fluid loss, and disruption of Na+-transporting ATPases and angiotensin II signaling.

Composition and Function of Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) are intricate fibrous structures released by neutrophils in response to specific stimuli. These structures are composed of depolymerized chromatin adorned with histones, granule proteins, and cytosolic proteins. NETs are formed via two distinct pathways known as suicidal NETosis, which involves NADPH oxidase (NOX), and vital NETosis, which is independent of NOX. Certain proteins found within NETs exhibit strong cytotoxic effects against both pathogens and nearby host cells. While NETs play a defensive role against pathogens, they can also contribute to tissue damage and worsen inflammation. Despite extensive research on the pathophysiological role of NETs, less attention has been paid to their components, which form a unique structure containing various proteins that have significant implications in a wide range of diseases. This review aims to elucidate the components of NETs and provide an overview of their impact on host defense against invasive pathogens, autoimmune diseases, and cancer.

Reactive oxygen species augment contractile responses of saphenous artery in 10-15-day-old but not adult rats: Substantial role of NADPH oxidases.

Reactive oxygen species (ROS) produced by NADPH oxidases (NOX, a key source of ROS in vascular cells) are involved in the regulation of vascular tone, but this has been explored mainly for adult organisms. Importantly, the mechanisms of vascular tone regulation differ significantly in early postnatal ontogenesis and adulthood, while the vasomotor role of ROS in immature systemic arteries is poorly understood. We tested the hypothesis that the functional contribution of NADPH oxidase-derived ROS to the regulation of peripheral arterial tone is higher in the early postnatal period than in adulthood. We studied saphenous arteries from 10- to 15-day-old ("young") and 3- to 4-month-old ("adult") male rats using lucigenin-enhanced chemiluminescence, quantitative PCR, Western blotting, and isometric myography. We demonstrated that both basal and NADPH-stimulated superoxide anion radical (O2•-) production was significantly higher in the arteries from young in comparison to adult rats. Importantly, pan-inhibitor of NADPH oxidase VAS2870 (10 µM) reduced NADPH-induced O2•- production in arteries of young rats. Saphenous arteries of both young and adult rats demonstrated high levels of Nox2 and Nox4 mRNAs, while Nox1 and Nox3 mRNAs were not detected. The protein contents of NOX2 and NOX4 were significantly higher in arterial tissue of young compared to adult animals. Moreover, VAS2870 (10 µM) had no effect on methoxamine-induced contractile responses of adult arteries but decreased them significantly in young arteries; such effect of VAS2870 persisted after removal of the endothelium. Finally, NOX2 inhibitor GSK2795039 (10 µM), but not NOX1/4 inhibitor GKT137831 (10 µM) weakened methoxamine-induced contractile responses of arteries from young rats. Thus, ROS produced by NOX2 have a pronounced contractile influence in saphenous artery smooth muscle cells of young, but not adult rats, which is associated with the increased vascular content of NOX2 protein at this age.

Gene Expression and Prognostic Value of NADPH Oxidase Enzymes in Breast Cancer.

NADPH oxidase enzymes (NOX) are involved in all stages of carcinogenesis, but their expression levels and prognostic value in breast cancer (BC) remain unclear. Thus, we aimed to assess the expression and prognostic value of NOX enzymes in BC samples using online databases. For this, mRNA expression from 290 normal breast tissue samples and 1904 BC samples obtained from studies on cBioPortal, Kaplan-Meier Plotter, and The Human Protein Atlas were analyzed. We found higher levels of NOX2, NOX4, and Dual oxidase 1 (DUOX1) in normal breast tissue. NOX1, NOX2, and NOX4 exhibited higher expression in BC, except for the basal subtype, where NOX4 expression was lower. DUOX1 mRNA levels were lower in all BC subtypes. NOX2, NOX4, and NOX5 mRNA levels increased with tumor progression stages, while NOX1 and DUOX1 expression decreased in more advanced stages. Moreover, patients with low expression of NOX1, NOX4, and DUOX1 had lower survival rates than those with high expression of these enzymes. In conclusion, our data suggest an overexpression of NOX enzymes in breast cancer, with certain isoforms showing a positive correlation with tumor progression.

ER-tethered RNA-binding protein controls NADPH oxidase translation for hydrogen peroxide homeostasis.

Hydrogen peroxide (H2O2) functions as a signaling molecule in diverse cellular processes. While cells have evolved the capability to detect and manage changes in H2O2 levels, the mechanisms regulating key H2O2-producing enzymes to maintain optimal levels, especially in pancreatic beta cells with notably weak antioxidative defense, remain unclear. We found that the protein EI24 responds to changes in H2O2 concentration and regulates the production of H2O2 by controlling the translation of NOX4, an enzyme that is constitutively active, achieved by recruiting an RNA-binding protein, RTRAF, to the 3'-UTR of Nox4. Depleting EI24 results in RTRAF relocating into the nucleus, releasing the brake on NOX4 translation. The excessive production of H2O2 by liberated NOX4 further suppresses the translation of the key transcription factor MafA, ultimately preventing its binding to the Ins2 gene promoter and subsequent transcription of insulin. Treatment with a specific NOX4 inhibitor or the antioxidant NAC reversed these effects and alleviated the diabetic symptoms in beta-cell specific Ei24-KO mice. This study revealed a new mechanism through which cells regulate oxidative stress at the translational level, involving an ER-tethered RNA-binding protein that controls the expression of the key H2O2-producing enzyme NOX4.

Particulate matter stimulates the NADPH oxidase system via AhR-mediated epigenetic modifications.

Stimulation of human keratinocytes with particulate matter 2.5 (PM2.5) elicits complex signaling events, including a rise in the generation of reactive oxygen species (ROS). However, the mechanisms underlying PM2.5-induced ROS production remain unknown. Here, we show that PM2.5-induced ROS production in human keratinocytes is mediated via the NADPH oxidase (NOXs) system and the Ca2+ signaling pathway. PM2.5 treatment increased the expression of NOX1, NOX4, and a calcium-sensitive NOX, dual oxidase 1 (DUOX1), in human epidermal keratinocyte cell line. PM2.5 bound to aryl hydrocarbon receptor (AhR), and this complex bound to promoter regions of NOX1 and DUOX1, suggesting that AhR acted as a transcription factor of NOX1 and DUOX1. PM2.5 increased the transcription of DUOX1 via epigenetic modification. Moreover, a link between DNA demethylase and histone methyltransferase with the promoter regions of DUOX1 led to an elevation in the expression of DUOX1 mRNA. Interestingly, PM2.5 increased NOX4 expression and promoted the interaction of NOX4 and Ca2+ channels within the cytoplasmic membrane or endoplasmic reticulum, leading to Ca2+ release. The increase in intracellular Ca2+ concentration activated DUOX1, responsible for ROS production. Our findings provide evidence for a PM2.5-mediated ROS-generating system network, in which increased NOX1, NOX4, and DUOX1 expression serves as a ROS signal through AhR and Ca2+ activation.

The circular RNA circATP8B(2) regulates ROS production and antiviral immunity in Drosophila.

We identified and validated a collection of circular RNAs (circRNAs) in Drosophila melanogaster. We show that depletion of the pro-viral circRNA circATP8B(2), but not its linear siblings, compromises viral infection both in cultured Drosophila cells and in vivo. In addition, circATP8B(2) is enriched in the fly gut, and gut-specific depletion of circATP8B(2) attenuates viral replication in an oral infection model. Furthermore, circATP8B(2) depletion results in increased levels of reactive oxygen species (ROS) and enhanced expression of dual oxidase (Duox), which produces ROS. Genetic and pharmacological manipulations of circATP8B(2)-depleted flies that reduce ROS levels rescue the viral replication defects elicited by circATP8B(2) depletion. Mechanistically, circATP8B(2) associates with Duox, and circATP8B(2)-Duox interaction is crucial for circATP8B(2)-mediated modulation of Duox activity. In addition, Gαq, a G protein subunit required for optimal Duox activity, acts downstream of circATP8B(2). We conclude that circATP8B(2) regulates antiviral defense by modulating Duox expression and Duox-dependent ROS production.

Upregulation of NADPH-oxidase, inducible nitric oxide synthase and apoptosis in the hippocampus following impaired insulin signaling in the rats: Development of sporadic Alzheimer's disease.

NADPH-oxidase (NOX) is a multi-subunit enzyme complex. The upregulation of NOX causes massive production of superoxide (O2¯), which avidly reacts with nitric oxide (NO) and increases cellular reactive oxygen/nitrogen species (ROS/RNS). Increased ROS/RNS plays pivotal role in the sporadic Alzheimer's disease (sAD) development and brain damage following impaired insulin signaling. Hence, this study aimed to examine early-time course of changes in NOX and NOS expression, and apoptotic proteins in the rats hippocampi following insulin signaling impairment [induced by STZ injection; intraperitoneal (IP) or in cerebral ventricles (ICV)]. Early effects (1, 3, or 6 weeks) on the NOX activity, translocation of NOX subunits from cytosol to the membrane, NO-synthases [neuronal-, inducible- and endothelial-NOS; nNOS, iNOS and eNOS], The Rac-1 protein expression, levels of NO and O2¯, cytochrome c release, caspase-3 and 9 activations (cleavage) were studied. STZ injection (in both models) increased NOX activity, O2¯ production, and enhanced cytosolic subunits translocation into membrane. The iNOS but not nNOS and eNOS expression and NO levels were increased in STZ treated rats. Finally, STZ injection increased cytochrome c release, caspase-3 and 9 activations in a manner that was significantly associated with levels of O2¯ and NO in the hippocampus. ICV-STZ administration resulted in significant profound changes over the IP route. In conclusion, impairment in insulin function induces early changes in ROS/RNS contents through NOX and iNOS upregulation and neuronal apoptosis in the hippocampus. Our results could mechanistically explain the role of impaired insulin function in the development of sAD.

Redox regulation of platelet function and thrombosis.

Platelets are well-known players in several cardiovascular diseases such as atherosclerosis and venous thrombosis. There is increasing evidence demonstrating that reactive oxygen species (ROS) are generated within activated platelets. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of ROS generation in platelets. Ligand binding to platelet receptor glycoprotein (GP) VI stimulates intracellular ROS generation consisting of a spleen tyrosine kinase-independent production involving NOX activation and a following spleen tyrosine kinase-dependent generation. In addition to GPVI, stimulation of platelet thrombin receptors (protease-activated receptors [PARs]) can also trigger NOX-derived ROS production. Our recent study found that mitochondria-derived ROS production can be induced by engagement of thrombin receptors but not by GPVI, indicating that mitochondria are another source of PAR-dependent ROS generation apart from NOX. However, mitochondria are not involved in GPVI-dependent ROS generation. Once generated, the intracellular ROS are also involved in modulating platelet function and thrombus formation; therefore, the site-specific targeting of ROS production or clearance of excess ROS within platelets is a potential intervention and treatment option for thrombotic events. In this review, we will summarize the signaling pathways involving regulation of platelet ROS production and their role in platelet function and thrombosis, with a focus on GPVI- and PAR-dependent platelet responses.

Curcumin Modulates NOX Gene Expression and ROS Production via P-Smad3C in TGF-ß-Activated Hepatic Stellate Cells.

Background: Liver fibrosis, associated with hepatic stellate cells (HSCs), occurs when a healthy liver sustains damage, thereby impairing its function. NADPH oxidases (NOXs), specifically isoforms 1, 2, and 4, play a role in reactive oxygen species (ROS) production during hepatic injuries, resulting in fibrosis. Curcumin has shown strong potential in mitigating liver fibrosis. Our research aimed to investigate the effects of curcumin on lowering NOX and ROS levels. This compound was also studied for its effects on NOXs, ROS concentrations through the inhibition of Smad3 phosphorylation in transforming growth factor beta (TGF-ß)-activated human HSCs. Methods: MTT assay investigated the cytotoxic effects of curcumin on HSCs. The cells were activated by exposure to TGF-ß (2 ng/mL) for 24 hours. After activating, the cells were treated with curcumin at 25-150 µM concentrations. After administering curcumin to the cells, we employed RT-PCR and Western blot techniques to evaluate the related gene and protein expression levels. This evaluation was primarily focused on the mRNA expression levels of NOX1, NOX2, NOX4 and phosphorylated Smad3C. Results: The mRNA expression level of aforesaid NOXs as well as α-smooth muscle actin (α-SMA), collagen1-α, and ROS levels were significantly reduced following 100 µM curcumin treatment. Furthermore, curcumin significantly decreased the p-Smad3C protein level in TGF-ß-activated cells, with fold changes of 3 and 2 observed at 75 and 100 µM, respectively. Conclusion: Curcumin decreased the levels of ROS and NOX, as well as the expression of α-SMA and collagen1-α. The primary mechanism for this reduction could be linked to the level of p-Smad3C. Hence, curcumin could serve as an effective therapeutic agent for liver fibrosis.