Local Reactive Oxygen Species Production Research Articles

NADPH oxidase-derived reactive oxygen species regulates adipose tissue lipolysis in humans

There is mounting evidence suggesting that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) regulate physiological processes, as NOX has been shown to promote increases in lipolysis in cultured adipocytes. PURPOSE: To determine the mechanisms by which NOX stimulates adipose tissue lipolysis. METHODS: Young healthy females (n = 17; mean ± SD; age: 21.7 ± 5 years; body mass index: 22.2 ± 3.8 kg/m2) participated in this study. Participants underwent microdialysis and hyperinsulinemic-euglycemic clamp (clamp) procedures. Three microdialysis probes were inserted into subcutaneous abdominal adipose tissue to monitor local adipose tissue ROS (H2O2) production and lipolysis (as indicated by dialysate glycerol). To determine whether NOX stimulates lipolysis via distinct signaling pathways, microdialysis probes were perfused with either isoproterenol (Iso; β-adrenergic agonist to stimulate canonical lipolysis signaling) or atrial natriuretic peptide (ANP to stimulate non-canonical lipolysis signaling), then perfused with a local NOX inhibitor (apocynin). A control probe was perfused with apocynin but without any agonists of lipolysis. The microdialysis procedures were then repeated under hyperinsulinemic conditions during the same study visit. RESULTS: In the fasted state, dialysate H2O2 concentrations were lower in the control and Iso probes upon apocynin perfusion (control probe without apocynin: 1.06 ± 0.5 μM, with apocynin: 0.62 ± 0.2 μM; p = 0.005; Iso probe without apocynin: 1.01 ± 0.4 μM, with apocynin: 0.66 ± 0.2 μM; p = 0.003). Apocynin perfusion did not alter dialysate H2O2 concentrations in the ANP probe under the fasted state (p = 0.11). Dialysate glycerol concentrations did not change upon apocynin perfusion under any of the microdialysis probe conditions in the fasted state (p > 0.05). During the clamp, apocynin perfusion resulted in lower dialysate H2O2 concentrations in the three conditions (control probe without apocynin: 1.22 ± 0.6 μM, with apocynin: 0.75 ± 0.3 μM; p = 0.03; Iso probe without apocynin: 1.55 ± 0.8 μM, with apocynin: 0.79 ± 0.3 μM; p = 0.01; ANP probe without apocynin: 1.55 ± 0.6 μM, with apocynin: 0.92 ± 0.3 μM; p = 0.03). Dialysate glycerol concentrations were lower in the Iso probe upon apocynin perfusion during the clamp (Iso probe without apocynin: 124.7 ± 38 μM, with apocynin: 90.9 ± 25 μM; p = 0.003). CONCLUSIONS: This study presents adipose tissue NOX as a source of in vivo ROS production in human participants. In addition, we reveal a novel mechanism of NOX in adipose tissue function whereby NOX exerts increases in β-adrenergic-mediated lipolysis during hyperinsulinemia. Our findings highlight the roles of ROS as signaling mediators and suggest that an imbalance between NOX activity and insulin action on adipose tissue could negatively affect systemic fatty acid metabolism. Porter Pre-doctoral Fellowship from the American Physiological Society and an NIH National Research Service Award (F31HL154642). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alpha lipoic acid–loaded electrospun fibrous patch films protect heart in acute myocardial infarction mice by inhibiting oxidative stress

Oxidative stress, characterized by excessive accumulation of reactive oxygen species (ROS), is involved in acute myocardial infarction (AMI)-related pathological processes and vascular reperfusion therapy injury. Alpha lipoic acid (LA) exhibits excellent antioxidant properties, however, its application is limited by inherent characteristics, including rapid clearance and extensive volume distribution. In this study, we hypothesized that scavenging cardiac ROS using adequately delivered LA could promote heart repair. Here, we report a new strategy for dynamic-release LA to treat AMI disease. In particular, this involves using poly(lactic-co-glycolic) (PLGA) copolymers as carriers to form a thin film (LA@PLGA) via electrospinning technology to achieve controlled release of LA, which essentially blocking local ROS production in damaged hearts. The drug-loading capacity and capsulation efficiency of this compound film could be regulated by determining the dose proportions of LA and PLGA. The incubation of LA@PLGA showed strong anti-oxidative activity and anti-apoptosis effect in hydrogen peroxide–administered primary cardiomyocytes. Patching LA@PLGA on the infarcted cardiac surfaces of AMI mice dramatically improved heart functions and reduced cardiac fibrosis throughout ventricular remodeling process. Importantly, the attenuation of detrimental pathologies was observed, including oxidative stress, senescence, DNA damage, cytokine-related processes, apoptosis, and ferroptosis. These results suggest that PLGA-carried LA can reduce ROS damage and restore heart function after myocardial damage, demonstrating a great potential for LA drugs in treating AMI disease.

A reversible mitochondrial complex I thiol switch mediates hypoxic avoidance behavior in C. elegans

C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.

TrpML-mediated astrocyte microdomain Ca2+ transients regulate astrocyte-tracheal interactions.

Astrocytes exhibit spatially-restricted near-membrane microdomain Ca2+transients in their fine processes. How these transients are generated and regulate brain function in vivo remains unclear. Here we show that Drosophila astrocytes exhibit spontaneous, activity-independent microdomain Ca2+ transients in their fine processes. Astrocyte microdomain Ca2+ transients are mediated by the TRP channel TrpML, stimulated by reactive oxygen species (ROS), and can be enhanced in frequency by the neurotransmitter tyramine via the TyrRII receptor. Interestingly, many astrocyte microdomain Ca2+ transients are closely associated with tracheal elements, which dynamically extend filopodia throughout the central nervous system (CNS) to deliver O2 and regulate gas exchange. Many astrocyte microdomain Ca2+ transients are spatio-temporally correlated with the initiation of tracheal filopodial retraction. Loss of TrpML leads to increased tracheal filopodial numbers, growth, and increased CNS ROS. We propose that local ROS production can activate astrocyte microdomain Ca2+ transients through TrpML, and that a subset of these microdomain transients promotes tracheal filopodial retraction and in turn modulate CNS gas exchange.

Food-borne Lactiplantibacillus plantarum protect normal intestinal cells against inflammation by modulating reactive oxygen species and IL-23/IL-17 axis

Food-associated Lactiplantibacillus plantarum (Lpb. plantarum) strains, previously classified as Lactobacillus plantarum, are a promising strategy to face intestinal inflammatory diseases. Our study was aimed at clarifying the protective role of food-borne Lpb. plantarum against inflammatory damage by testing the scavenging microbial ability both in selected strains and in co-incubation with normal mucosa intestinal cells (NCM460). Here, we show that Lpb. plantarum endure high levels of induced oxidative stress through partially neutralizing reactive oxygen species (ROS), whereas they elicit their production when co-cultured with NCM460. Moreover, pre-treatment with food-borne Lpb. plantarum significantly reduce pro-inflammatory cytokines IL-17F and IL-23 levels in inflamed NCM460 cells. Our results suggest that food-vehicled Lpb. plantarum strains might reduce inflammatory response in intestinal cells by directly modulating local ROS production and by triggering the IL-23/IL-17 axis with future perspectives on health benefits in the gut derived by the consumption of functional foods enriched with selected strains.

Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

Reactive oxygen species (ROS) localized at the precise subcellular compartments are essential for regulating the activity of signaling proteins. Furthermore, ROS are master regulators of tumor malignant progression that respond to a diverse set of environmental stress, especially hypoxia. NADPH oxidases (NOXs) appear to be activated within discrete subcellular compartments to facilitate local ROS production. However, the subcellular function of NOXs in hypoxic tumor is still unclear. In this study, we demonstrated that NOX5 was greatly upregulated in clinical esophageal squamous cell carcinoma (ESCC) tumors, ESCC cell lines or primary ESCC cells, and elevated NOX5 was correlated to malignancy of ESCC tumors and poor prognosis. NOX5 induced the malignant progression of ESCC by activating Src, especially under hypoxic condition. Mechanistically, we showed that hypoxia promoted the interaction between NOX5 and Pyk2 on cell membrane via facilitating Ca2+-mediated Pyk2 Tyr402 site phosphorylation. Subsequently, Pyk2 acted as a scaffold for c-Abl phosphorylating the catalytic domain of NOX5 Tyr476/478 sites, which in turn upregulated hydrogen peroxide (H2O2) inside the Pyk2/NOX5 complex to oxidize and activate local Src. These findings provide insights into the biological significance of NOX5 in the development of ESCC.

Cross-Talk between NADPH Oxidase and Mitochondria: Role in ROS Signaling and Angiogenesis.

Angiogenesis, a new vessel formation from the pre-existing ones, is essential for embryonic development, wound repair and treatment of ischemic heart and limb diseases. However, dysregulated angiogenesis contributes to various pathologies such as diabetic retinopathy, atherosclerosis and cancer. Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) as well as mitochondria play an important role in promoting the angiogenic switch from quiescent endothelial cells (ECs). However, how highly diffusible ROS produced from different sources and location can communicate with each other to regulate angiogenesis remains unclear. To detect a localized ROS signal in distinct subcellular compartments in real time in situ, compartment-specific genetically encoded redox-sensitive fluorescence biosensors have been developed. Recently, the intercellular communication, “cross-talk”, between ROS derived from NOX and mitochondria, termed “ROS-induced ROS release”, has been proposed as a mechanism for ROS amplification at distinct subcellular compartments, which are essential for activation of redox signaling. This “ROS-induced ROS release” may represent a feed-forward mechanism of localized ROS production to maintain sustained signaling, which can be targeted under pathological conditions with oxidative stress or enhanced to promote therapeutic angiogenesis. In this review, we summarize the recent knowledge regarding the role of the cross-talk between NOX and mitochondria organizing the sustained ROS signaling involved in VEGF signaling, neovascularization and tissue repair.

Proteomic and metabolomic basis for improved textural quality in crisp grass carp (Ctenopharyngodon idellus C.et V) fed with a natural dietary pro-oxidant

Reactive oxygen species (ROS) regulate metabolism and chemical composition of various tissues. To understand how ROS affect the textural quality of fish muscle, we performed a multi-omics analysis on an established crisp grass carp model fed with a natural pro-oxidant faba bean. ROS levels were systemically and significantly increased up to three-fold in crisp grass carp, improving the muscle texture. Lipid metabolism was significantly enhanced up to five-fold in muscle and liver possibly to compensate the impaired carbohydrate metabolism of these tissues, but this caused further local ROS production. Mitochondrial damage associated with autophagy was evident in crisp grass carp. Proteomics revealed that elevated ROS likely disturbed the actin-myosin interaction and collagen turnover inducing fragmentation of myofibrillar proteins, all of which could have positively impacted the textural quality. The systemic metabolic changes that lead to the partial collapse of redox regulation likely underlie the ROS-induced improvement of textural quality.

SCHENGEN receptor module drives localized ROS production and lignification in plant roots

Production of reactive oxygen species (ROS) by NADPH oxidases (NOXs) impacts many processes in animals and plants, and many plant receptor pathways involve rapid, NOX‐dependent increases of ROS. Yet, their general reactivity has made it challenging to pinpoint the precise role and immediate molecular action of ROS. A well‐understood ROS action in plants is to provide the co‐substrate for lignin peroxidases in the cell wall. Lignin can be deposited with exquisite spatial control, but the underlying mechanisms have remained elusive. Here, we establish a kinase signaling relay that exerts direct, spatial control over ROS production and lignification within the cell wall. We show that polar localization of a single kinase component is crucial for pathway function. Our data indicate that an intersection of more broadly localized components allows for micrometer‐scale precision of lignification and that this system is triggered through initiation of ROS production as a critical peroxidase co‐substrate.

In vivo assessment of silver nanoparticle induced reactive oxygen species reveals tissue specific effects on cellular redox status in the nematode Caenorhabditis elegans

The current study provides an in vivo analysis of the production of reactive oxygen species (ROS) and oxidative stress in the nematode Caenorhabditis elegans following exposure to EU reference silver nanoparticles NM300K and AgNO3. Induction of antioxidant defenses was measured through the application of a SOD-1 reporter, and the HyPer and GRX biosensor strains to monitor changes in the cellular redox state. Both forms of Ag resulted in an increase in sod-1 expression, elevated H2O2 levels and an imbalance in the cellular GSSG/GSH redox status. Microscopy analysis of the strains revealed that AgNO3 induced ROS-related effects in multiple tissues, including the pharynx, intestinal cells and muscle tissues. In contrast, NM300K resulted in localized ROS production and oxidative stress, specifically in tissues surrounding the intestinal lumen. This indicates that Ag from AgNO3 exposure was readily transported across the whole body, while Ag or ROS from NM300K exposure was predominantly confined within the luminal tissues. Concentrations resulting in an increase in ROS production and changes in GSSG/GSH ratio were in line with the levels associated with observed physiological toxic effects. However, sod-1 was not induced at the lowest Ag concentrations, although reprotoxicity was seen at these levels. While both forms of Ag caused oxidative stress, impaired development, and reprotoxicity, the results suggest different involvement of ROS production to the toxic effects of AgNO3versus NM300K.

Radical Stress Is More Cytotoxic in the Nucleus than in Other Organelles.

Cells are exposed to reactive oxygen species (ROS) as a by-product of mitochondrial metabolism, especially under hypoxia. ROS are also enzymatically generated at the plasma membrane during inflammation. Radicals cause cellular damage leading to cell death, as they react indiscriminately with surrounding lipids, proteins, and nucleotides. However, ROS are also important for many physiological processes, including signaling, pathogen killing and chemotaxis. The sensitivity of cells to ROS therefore likely depends on the subcellular location of ROS production, but how this affects cell viability is poorly understood. As ROS generation consumes oxygen, and hypoxia-mediated signaling upregulates expression of antioxidant transcription factor Nrf2, it is difficult to discern hypoxic from radical stress. In this study, we developed an optogenetic toolbox for organelle-specific generation of ROS using the photosensitizer protein SuperNova which produces superoxide anion upon excitation with 590 nm light. We fused SuperNova to organelle specific localization signals to induce ROS with high precision. Selective ROS production did not affect cell viability in most organelles except for the nucleus. SuperNova is a promising tool to induce locally targeted ROS production, opening up new possibilities to investigate processes and organelles that are affected by localized ROS production.

A gold nanoparticle system for the enhancement of radiotherapy and simultaneous monitoring of reactive-oxygen-species formation

Gold nanoparticles (AuNPs) are known to sensitize cancer cells to radiation therapy (RT) by increasing the deposition of ionizing energy in their immediate vicinity. However, this process of dose enhancement is challenging to monitor because it is heterogeneous at the sub-cellular scale. Furthermore, radiation damage is primarily mediated by reactive oxygen species (ROS) that are produced following water radiolysis. Here, radiation-responsive PEGylated gold nanoparticles (RPAuNPs) were synthesized for the enhanced generation and concurrent detection of ROS in cancer cells and tumors. PEGylated gold particles (20 nm diameter) were functionalized with dihydrorhodamine 123 (DHR-123), a known ROS sensor, to monitor ROS generation in their immediate vicinity. These NPs were able to effectively radiosensitize cells, as measured by increased cell apoptosis following RT. Furthermore, the fluorescence of these RPAuNPs was 7-fold higher after 6 Gy RT due to the local production of ROS near the surface of the NP. Finally, multispectral fluorescence imaging was used to monitor NP-induced ROS in vivo, following conformal RT, in a xenograft model of breast cancer. This theranostic NP system provides a novel approach for monitoring the nanoscale enhancement of RT by high-Z metal NPs.

75 - VEGFR2 Signaling Driven by Cellular Redox Status

Angiogenesis (a process of new vessel formation) is triggered by tissue ischemia. Paradoxically, angiogenesis is diminished in cardiovascular diseases (CVDs) due to a decrease in cellular glutathione (GSH) and increase in reactive oxygen species (ROS). Interestingly, our laboratory has demonstrated that a small decrease in cellular GSH promotes vascular endothelial growth factor (VEGF)-driven ischemic angiogenesis while a larger decrease in cellular GSH does not. Thus, we hypothesize VEGF receptor2 (VEGFR2) activation is regulated by intracellular GSH redox state. To specifically study the role of altered GSH redox state on angiogenesis, we used diamide to oxidize GSH to GSSG in human aortic endothelial cells. GSSG can react with protein cysteine thiols to form S-glutathionylated proteins that in turn affect protein function and stability. Our preliminary experiments show that drastic changes in GSH/GSSG activate VEGFR2 independent of VEGF while a moderate change does not activate the receptor. Interestingly, the addition of H2O2 alone did not activate VEGFR2 suggesting a role for GSH/GSSG in regulating VEGFR2. To understand the mechanism of S-glutathionylation in VEGFR2 activation, we used dimedone, a sulfenic acid trap. Pretreatment with dimedone abolished VEGFR2 activation after diamide treatment suggesting that cysteine sulfenic acid formation precedes S-glutathionylation. Local ROS production in the vicinity of proteins can effectively mediate cellular signaling. On that note, O2●– derived from endothelial nitric oxide synthase (eNOS) could activate VEGFR2 in altered GSH/GSSG. S-glutathionylation of eNOS leads to its uncoupling and drives O2●– generation. To test the role of O2●– in VEGFR2 activation in altered GSH redox state, we inhibited eNOS using L-NAME or siRNA and then treated with diamide. VEGFR2 activation decreased after eNOS inhibition suggesting a potential role for eNOS in VEGFR2 activation under altered GSH redox state. We believe a small decrease in GSH/GSSG would result in priming of VEGFR2 signaling pathway while a larger decrease in GSH/GSSG will result in dysfunctional signaling. In conclusion, the GSH/GSSH ratio is important in triggering and promoting VEGFR2 signaling.

Switching on a transient endogenous ROS production in mammalian cells and tissues

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide.We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems.

Type-1 angiotensin receptor signaling in central nervous system myeloid cells is pathogenic during fatal alphavirus encephalitis in mice.

BackgroundAlphaviruses can cause fatal encephalitis in humans. Natural infections occur via the bite of infected mosquitos, but aerosol transmissibility makes some of these viruses potential bioterrorism agents. Central nervous system (CNS) host responses contribute to alphavirus pathogenesis in experimental models and are logical therapeutic targets. We investigated whether reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activity within the CNS contributes to fatal alphavirus encephalitis in mice.MethodsInfected animals were treated systemically with the angiotensin receptor-blocking drug, telmisartan, given its ability to cross the blood-brain barrier, selectively block type-1 angiotensin receptors (AT1R), and inhibit Nox-derived ROS production in vascular smooth muscle and other extraneural tissues. Clinical, virological, biochemical, and histopathological outcomes were followed over time.ResultsThe importance of the angiotensin II (Ang II)/AT1R axis in disease pathogenesis was confirmed by demonstrating increased Ang II levels in the CNS following infection, enhanced disease survival when CNS Ang II production was suppressed, increased AT1R expression on microglia and tissue-infiltrating myeloid cells, and enhanced disease survival in AT1R-deficient mice compared to wild-type (WT) controls. Systemic administration of telmisartan protected WT mice from lethal encephalitis caused by two different alphaviruses in a dose-dependent manner without altering virus replication or exerting any anti-inflammatory effects in the CNS. Infection triggered up-regulation of multiple Nox subunits in the CNS, while drug treatment inhibited local Nox activity, ROS production, and oxidative neuronal damage. Telmisartan proved ineffective in Nox-deficient mice, demonstrating that this enzyme is its main target in this experimental setting.ConclusionsNox-derived ROS, likely arising from CNS myeloid cells triggered by AT1R signaling, are pathogenic during fatal alphavirus encephalitis in mice. Systemically administered telmisartan at non-hypotensive doses targets Nox activity in the CNS to exert a neuroprotective effect. Disruption of this pathway may have broader implications for the treatment of related infections as well as for other CNS diseases driven by oxidative injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0683-7) contains supplementary material, which is available to authorized users.

Photoactivation of ROS Production In Situ Transiently Activates Cell Proliferation in Mouse Skin and in the Hair Follicle Stem Cell Niche Promoting Hair Growth and Wound Healing

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism.

Dectin-1 deficiency does not affect atherosclerosis development in mice

ObjectiveRecent data suggest the involvement of dectin-1 in atherosclerosis through regulation of local reactive oxygen species production. The aim of the current study was to assess the effect of dectin-1 deficiency on atherosclerotic plaque development. MethodsUsing immunohistochemistry dectin-1 expression was observed on foamy macrophages in atherosclerotic lesions in mice. Following lethal irradiation LDLR−/− mice were reconstituted with bone marrow from either wild type or dectin-1−/− mice. After recovery, mice were fed a high fat diet for 9 weeks and atherosclerotic lesions were analyzed. Results and conclusionOverall, we found no significant differences in plaque size or severity between the groups. Also no differences were observed in granulocyte or macrophage composition of the plaques or in the ability to produce reactive oxygen species by macrophages from both groups. Dectin-1 is dispensable for the development of atherosclerotic lesions in mice.

Beneficial Effects of High S100A9 Expression On Graft Infiltrating Myeloid Cells in Kidney Transplantation.

Our goal is to predict the impact of acute rejection (AR) on kidney graft outcome using molecular markers. In patients suffering from AR (n=28) we initially identified by microarray S100A9 expression as a prognostic indicator of graft outcome. In a second cohort, we confirmed that patients with high S100A9 expression during AR (n=36) have significantly better 12-year graft survival (91.5%) than low expressers (n=61; 69.2%). Next, we studied the mechanism by which S100A9 exerts its biologic effects. In the biopsies, the S100A9+ cell population consisted of CD68+ myeloid cells of which 75% expressed HLA-DR. A minor fraction of S100A9+ cells represented CD66b+ granulocytes. S100A9 levels were related to expression of immune regulatory molecules IL-10, FoxP3/CD3, and p40 component of the reactive oxygen species (ROS)-producing machinery. Overexpression (10-35 fold) of S100A9 in cultured macrophages led to a dose-dependent increase in ROS production, whereas S100A9 inhibition by siRNA transfection resulted into decreased ROS production. The presence of low concentrations of the ROS metabolite H2O2 for 0.5h led to a significant downregulation of the proliferative response of activated T cells. S100A9 levels in the graft are specifically elevated during conditions of increased inflammation. The presence of distinct myeloid cell subsets early after transplantation provides prognostic information with respect to graft survival. The data suggest that an increased expression of S100A9 by myeloid cells leads to local ROS production in the immune synapse, and as a consequence, to downregulation of the alloimmune responses by T cells to the graft.

The Basic Biology of Redoxosomes in Cytokine-Mediated Signal Transduction and Implications for Disease-Specific Therapies

Redox reactions have been established as major biological players in many cellular signaling pathways. Here we review mechanisms of redox signaling with an emphasis on redox-active signaling endosomes. Signals are transduced by relatively few reactive oxygen species (ROS), through very specific redox modifications of numerous proteins and enzymes. Although ROS signals are typically associated with cellular injury, these signaling pathways are also critical for maintaining cellular health at homeostasis. An important component of ROS signaling pertains to localization and tightly regulated signal transduction events within discrete microenvironments of the cell. One major aspect of this specificity is ROS compartmentalization within membrane-enclosed organelles such as redoxosomes (redox-active endosomes) and the nuclear envelope. Among the cellular proteins that produce superoxide are the NADPH oxidases (NOXes), transmembrane proteins that are implicated in many types of redox signaling. NOXes produce superoxide on only one side of a lipid bilayer; as such, their orientation dictates the compartmentalization of ROS and the local control of signaling events limited by ROS diffusion and/or movement through channels associated with the signaling membrane. NOX-dependent ROS signaling pathways can also be self-regulating, with molecular redox sensors that limit the local production of ROS required for effective signaling. ROS regulation of the Rac-GTPase, a required co-activator of many NOXes, is an example of this type of sensor. A deeper understanding of redox signaling pathways and the mechanisms that control their specificity will provide unique therapeutic opportunities for aging, cancer, ischemia-reperfusion injury, and neurodegenerative diseases.

NOX2 interacts with podocyte TRPC6 channels and contributes to their activation by diacylglycerol: essential role of podocin in formation of this complex

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathophysiology of glomerular diseases. TRPC6 channels are typically activated by diacylglycerol (DAG) during PLC-dependent transduction cascades. TRPC6 channels can also be activated by reactive oxygen species (ROS). We previously showed that podocin is required for DAG analogs to produce robust activation of TRPC6 channels in podocytes. Here we show that endogenous TRPC6 channels in immortalized podocytes reciprocally coimmunoprecipitate with the catalytic subunit of the NADPH oxidase NOX2 (gp91(phox)). The NOX2-TRPC6 interaction was not detected in cells stably expressing a short hairpin RNA targeting podocin, although NOX2 and TRPC6 were present at normal levels. Application of a membrane-permeable DAG analog [1-oleoyl-2-acetyl-sn-glycerol (OAG)] increased generation of ROS in podocytes, but this effect was not detected in podocin knockdown cells. OAG also increased steady-state surface expression of the NOX2 regulatory subunit p47(phox). In whole cell recordings, TRPC6 activation by OAG was reduced in podocytes pretreated with the NOX2 inhibitor apocynin, by the pan-NOX inhibitor diphenylene iodonium, and by tempol, a ROS quencher. Cholesterol depletion and disruption of lipid rafts by methyl-β-cyclodextrin reduced activation of podocyte TRPC6 channels by OAG and also eliminated the NOX2-TRPC6 interaction as assessed by coimmunoprecipitation. These data suggest that active NOX2 assembles with TRPC6 at podocin-organized sterol-rich raft domains and becomes catalytically active in response to DAG. The localized production of ROS contributes to TRPC6 activation by chemical stimuli such as DAG. Podocin appears to be necessary for assembly of the NOX2-TRPC6 complex in lipid rafts.