Transient Reactive Oxygen Species Production Research Articles

Shockwave Treatment Enhanced Extracellular Matrix Production in Articular Chondrocytes Through Activation of the ROS/MAPK/Nrf2 Signaling Pathway.

Shockwave application is a potential treatment for osteoarthritis (OA), but the underlying mechanism remains unknown. Oxidative stress and a counterbalancing antioxidant system might be the key to understanding this mechanism. We hypothesized that reactive oxygen species (ROS) and the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2),which is an important regulator of cellular redox homeostasis, are plausible elements. Porcine chondrocytes were cultured in a 3-dimensional pellet model and subjected to shockwaves. The effects of shockwaves with various energy-flux densities on optimal extracellular matrix (ECM) synthesis were assessed. ROS, mitogen-activated protein kinase (MAPK) signaling, and the redox activity of Nrf2 were measured. To investigate the signaling mechanism involved in the shockwave treatment in chondrocytes, specific inhibitors of ROS, MAPK signaling, and Nrf2 activity were targeted. Shockwaves increased ECM synthesis without affecting cell viability or proliferation. Furthermore, they induced transient ROS production mainly through xanthine oxidase. The phosphorylation of ERK1/2 and p38 and the nuclear translocation of Nrf2 were activated by shockwaves. By contrast, suppression of ROS signaling mitigated shockwave-induced MAPK phosphorylation, Nrf2 nuclear translocation, and ECM synthesis. Pretreatment of chondrocytes with the specific inhibitors of MEK1/2 and p38, respectively, mitigated the shockwave-induced nuclear translocation of Nrf2 and ECM synthesis. Nrf2 inhibition by both small hairpin RNA knockdown and brusatol reduced the shockwave-enhanced ECM synthesis. Shockwaves activated Nrf2 activity through the induction of transient ROS signaling and subsequently enhanced ECM synthesis in chondrocytes. This study provided fundamental evidence confirming the potential of shockwaves for OA management.

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

Correction: Reversion of resistance to oxaliplatin by inhibition of p38 MAPK in colorectal cancer cell lines: involvement of the calpain / Nox1 pathway.

[This corrects the article DOI: 10.18632/oncotarget.21780.].

Respiratory Burst Oxidase Homolog Gene A Is Crucial for Rhizobium Infection and Nodule Maturation and Function in Common Bean.

Reactive oxygen species (ROS) produced by respiratory burst oxidase homologs (RBOHs) regulate numerous plant cell processes, including the symbiosis between legumes and nitrogen-fixing bacteria. Rapid and transient ROS production was reported after Phaseolus vulgaris root hairs were treated with Nod factors, indicating the presence of a ROS-associated molecular signature in the symbiosis signaling pathway. Rboh is a multigene family containing nine members (RbohA–I) in P. vulgaris. RNA interference of RbohB suppresses ROS production and attenuates rhizobial infection thread (IT) progression in P. vulgaris root hairs. However, the roles of other Rboh members in symbiotic interactions are largely unknown. In this study, we characterized the role of the NADPH oxidase-encoding gene RbohA (Phvulv091020621) in the P. vulgaris–Rhizobium tropici symbiosis. The spatiotemporal activity of the RbohA promoter colocalized with growing ITs and was associated with vascular bundles in developing nodules. Subcellular localization studies indicated that RBOHA was localized in the plasma membrane of P. vulgaris root hairs. After rhizobial inoculation, PvRBOHA was mainly distributed in the infection pocket and, to a lesser extent, throughout the IT. In PvRbohA RNAi lines, the rhizobial infection events were significantly reduced and, in successful infections, IT progression was arrested within the root hair, but did not impede cortical cell division. PvRbohA-RNAi nodules failed to fix nitrogen, since the infected cells in the few nodules formed were empty. RbohA-dependent ROS production and upregulation of several antioxidant enzymes was attenuated in rhizobia-inoculated PvRbohA-RNAi roots. These combined results indicate that PvRbohA is crucial for effective Rhizobium infection and its release into the nodule cells. This oxidase is partially or indirectly required to promote nodule organogenesis, altering the expression of auxin- and cyclin-related genes and genes involved in cell growth and division.

Reversion of resistance to oxaliplatin by inhibition of p38 MAPK in colorectal cancer cell lines: involvement of the calpain / Nox1 pathway.

Oxaliplatin is a major treatment for metastatic colorectal cancer, however its effectiveness is greatly diminished by the development of resistances. Our previous work has shown that oxaliplatin efficacy depends on the reactive oxygen species (ROS) produced by Nox1. In this report, we investigated Nox1 involvement in the survival mechanisms of oxaliplatin resistant cell lines that we have selected. Our results show that basal ROS production by Nox1 is increased in resistant cells. Whereas the transitory Nox1-dependent production of superoxide contributes to the cytotoxicity of oxaliplatin in sensitive cells, oxaliplatin treatment of resistant cells leads to a decrease in the production of superoxide associated with an increase of H2O2 and a decreased cytotoxicity of oxaliplatin. We have shown that calpains regulate differently Nox1 according to the sensitivity of the cells to oxaliplatin. In sensitive cells, calpains inhibit Nox1 by cleaving NoxA1 leading to a transient ROS production necessary for oxaliplatin cytotoxic effects. In contrast, in resistant cells calpain activation is associated with an increase of Nox1 activity through Src kinases, inducing a strong and maintained ROS production responsible for cell survival. Using a kinomic study we have shown that this overactivation of Nox1 results in an increase of p38 MAPK activity allowing the resistant cells to escape apoptosis. Our results show that the modulation of Nox1 activity in the context of anticancer treatment remains complex. However, a strategy to maximize Nox1 activation while inhibiting the p38 MAPK-dependent escape routes appears to be an option of choice to optimize oxaliplatin efficiency.

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.

Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Mediated Redox Signaling and Vascular Remodeling by 16α-Hydroxyestrone in Human Pulmonary Artery Cells: Implications in Pulmonary Arterial Hypertension.

Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16α-hydroxyestrone (16αOHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)-induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen-Nox-dependent processes was studied in female Nox1(-/-) and Nox4(-/-) mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16αOHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-α, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16αOHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16αOHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid-related factor 2 activity and expression of nuclear factor erythroid-related factor 2-regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1(-/-) but not Nox4(-/-) mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16αOHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1(-/-) mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid-related factor 2 whereby 16αOHE1 influences hPASMC function, which when upregulated may contribute to vascular injury in PAH, particularly important in women.

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.

Computational Modeling Predicts Phosphatase Oxidation as an Important Axis of Redox Regulation in IL-4 Signaling

Reactive oxygen species (ROS) are produced following activation of several types of cell surface receptors and can play an important role in modulating cell signaling. How simultaneous oxidative modifications of multiple proteins, sometimes with potentially opposite effects, regulate cell signaling is not well understood at the system level. We are using computational modeling based on quantitative experimental measurements to develop a systemic understanding of redox regulation of cell signaling in the context of the IL-4 signaling pathway. We observe that IL-4 signaling in Jurkat cells is accompanied by transient ROS production, and ROS augment signaling activity as measured by STAT6 phosphorylation. A number of candidate redox-regulated mechanisms exist in the IL-4 pathway that could contribute to the observed outcomes; however, it is technically challenging to directly measure redox modifications of the possibly redox-regulated proteins. To circumvent this issue, we have developed kinetic models of IL-4 signaling that incorporate competing hypotheses regarding redox regulatory mechanisms. With the guidance of measurable experimental data we are using innovative model selection strategies to determine the best candidate models. We have also acquired time course data for processes not directly related to redox regulation, such as transcriptional negative feedback regulation and proteasomal degradation as mechanisms for downregulating IL-4 signaling, that aid model selection and validation. Our results so far indicate that reversible oxidative inhibition of phosphatases and compartmentation of phosphatase activity between subcellular compartments may be the primary redox regulatory mechanisms in IL-4 signaling. These studies will help evolve an understanding of how oxidative modifications of different components of the signaling pathway operate in parallel along with better studied post-translational modification mechanisms of protein regulation to determine the overall dynamics of cell signaling.

Ferritin up-regulation and transient ROS production in cultured brain astrocytes after loading with iron oxide nanoparticles

To investigate the cellular consequences of a prolonged cellular presence of large amounts of iron oxide nanoparticles (IONPs) as well as the fate of such particles in brain cells, cultured primary astrocytes were loaded for 4h with dimercaptosuccinate-coated IONPs. Subsequently, the IONP-treated cells were incubated for up to 7days in IONP-free medium and the cell viability, metabolic parameters and iron metabolism of the cells were investigated. Despite an up to 100-fold elevated specific cellular iron content, IONP-loaded cells remained viable throughout the 7day main incubation and did not show any substantial alteration in glucose and glutathione metabolism. During the incubation, the high cellular iron content of IONP-loaded astrocytes remained almost constant. Electron microscopy revealed that after 7days of incubation most of the cellular iron was still present in IONP-filled vesicles. However, the transient appearance of reactive oxygen species (ROS) as well as a strong increase in cellular levels of the iron storage protein ferritin suggest that at least some low-molecular-weight iron was liberated from the accumulated IONPs. These results demonstrate that even the prolonged presence of large amounts of accumulated IONPs does not harm astrocytes and that these cells store IONP-derived iron in ferritin.

Oxidative Stress in Older Adults: Effects of Fitness and Lifetime Physical Activity

We previously demonstrated an age-related decline in the ability to constrain oxidative stress induced by forearm ischemia-reperfusion (I/R) and that this response could be improved with a dietary antioxidant intervention. Reactive oxygen species (ROS) at low concentrations are important in cell signaling with one important effect being upregulation of endogenous antioxidant enzymes. Therefore, repeated bouts of transient ROS production induced by acute exercise may also increase the ability to constrain oxidative stress and reduce oxidative damage. PURPOSE: This cross-sectional study tested the hypothesis that physical fitness and/or lifetime physical activity modulate the capacity to constrain acute oxidative stress and basal urinary excretion of oxidative damage products in healthy older men and women. METHODS: We compared 40 fit and 39 unfit men and women, ages 60-78y (mean age: 66 ± 5y). Fitness status was classified by measuring VO2 max and maximal leg power, using age- and gender appropriate norms. Lifetime physical activity (low, moderate, high) was assessed with the Historical Leisure Physical Activity (HLPA) questionnaire. The capacity to constrain oxidative stress was measured as plasma F2-isoprostane (F2isoP) responses to forearm I/R. Basal systemic levels of oxidative damage were assessed by measuring urinary excretion products of lipid and nucleic acid oxidation. RESULTS: VO2max, maximal leg power, and HLPA score were significantly different between groups (p<0.001). F2isoP levels were significantly increased in response to the I/R trial (p<0.001). Fit individuals who reported high lifetime physical activity had significantly lower I/R-induced F2isoP response compared to unfit individuals who reported low lifetime physical activity (p<0.05). Compared to the unfit, sedentary group, the fit active group also had significantly lower levels of urinary markers of oxidative damage (8-OHdG, 8-oxo-G; p<0.01, isoprostanes; p<0.05). CONCLUSION: These data indicate that higher physical fitness levels and lifetime physical activity of healthy older adults are associated with greater antioxidant defenses and lower oxidative damage to lipids and nucleic acids. Furthermore, these results suggest that "quality" (VO2 max) and "quantity" (lifetime physical activity) may both be important in determining antioxidant capacity as the differences were not as marked when analyzed by either factor alone. Supported by NIH Grant R03 AG030551.

Superoxide anion and hydrogen peroxide in the yeast antagonist–fruit interaction: A new role for reactive oxygen species in postharvest biocontrol?

The importance of reactive oxygen species (ROS) in plant defense responses against certain pathogens is well documented. There is some evidence that microbial biocontrol agents also induce a transient production of ROS in a host plant which triggers local and systemic defense responses to pathogens. The ability of biocontrol agents used to control postharvest diseases to induce defense-related oxidative responses in fruits, however, has not been explored. Here we show that the yeast antagonists, Metschnikowia fructicola (strain 277) and Candida oleophila (strain 182) generate greater levels of super oxide anion (O 2 −) on intact fruit surfaces (poor in nutrients) then those applied on a nutrient-poor agar medium. Even though yeast antagonists show a high level of O 2 − on nutrient-rich media, when applied on fruits around wounds (areas abundant in nutrients) accumulation of O 2 −, as detected by nitro blue tetrazolium staining, occurred much more rapidly on the latter. Using laser scanning confocal microscopy we observed that the application of M. fructicola and C. oleophila into citrus and apple fruit wounds correlated with an increase in H 2O 2 accumulation in host tissue. In citrus fruit, the level of H 2O 2 around inoculated wounds increased by 4-fold compared to controls (wounds inoculated with water) as early as 18 h after inoculation. Yeast continued to stimulate H 2O 2 production in citrus fruit up to 66 h after inoculation and H 2O 2 levels were still 3-fold above the control. Living yeast cells were detected in fruit wounds at this time point indicating the ability of M. fructicola to tolerate host ROS, which has been reported to be an intrinsic characteristic of efficient yeast antagonists. Similar increase in H 2O 2 accumulation around yeast-inoculated wounds was observed in apple fruit exocarp. The present data, together with our earlier discovery of the importance of H 2O 2 production in the defense response of citrus flavedo to postharvest pathogens, indicate that the yeast-induced oxidative response in fruit exocarp may be associated with the ability of specific yeast species to serve as biocontrol agents for the management of postharvest diseases.

Role for Mitochondrial Reactive Oxygen Species in Brain Lipid Sensing

The ability for the brain to sense peripheral fuel availability is mainly accomplished within the hypothalamus, which detects ongoing systemic nutrients and adjusts food intake and peripheral metabolism as needed. Here, we hypothesized that mitochondrial reactive oxygen species (ROS) could trigger sensing of nutrients within the hypothalamus. For this purpose, we induced acute hypertriglyceridemia in rats and examined the function of mitochondria in the hypothalamus. Hypertriglyceridemia led to a rapid increase in the mitochondrial respiration in the ventral hypothalamus together with a transient production of ROS. Cerebral inhibition of fatty acids-CoA mitochondrial uptake prevented the hypertriglyceridemia-stimulated ROS production, indicating that ROS derived from mitochondrial metabolism. The hypertriglyceridemia-stimulated ROS production was associated with change in the intracellular redox state without any noxious cytotoxic effects, suggesting that ROS function acutely as signaling molecules. Moreover, cerebral inhibition of hypertriglyceridemia-stimulated ROS production fully abolished the satiety related to the hypertriglyceridemia, suggesting that hypothalamic ROS production was required to restrain food intake during hypertriglyceridemia. Finally, we found that fasting disrupted the hypertriglyceridemia-stimulated ROS production, indicating that the redox mechanism of brain nutrient sensing could be modulated under physiological conditions. Altogether, these findings support the role of mitochondrial ROS as molecular actors implied in brain nutrient sensing.

Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries

Myogenic vasoconstriction, an intrinsic response to elevated transmural pressure (TMP), requires the activation of sphingosine kinase (Sk1) and the generation of reactive oxygen species (ROS). We hypothesized that pressure-induced Sk1 signaling and ROS generation are functionally linked. Using a model of cannulated resistance arteries isolated from the hamster gracilis muscle, we monitored vessel diameter and smooth muscle cell (SMC) Ca2+i (Fura-2) or ROS production (dichlorodihydrofluorescein). Elevation of TMP stimulated the translocation of a GFP-tagged Sk1 fusion protein from the cytosol to the plasma membrane, indicative of enzymatic activation. Concurrently, elevation of TMP initiated a rapid and transient production of ROS, which was enhanced by expression of wild-type Sk1 (hSk(wt)) and inhibited by its dominant-negative mutant (hSk(G82D)). Exogenous sphingosine-1-phosphate (S1P) also stimulated ROS generation is isolated vessels. Chemical (1 micromol/L DPI), peptide (gp91ds-tat/gp91ds), and genetic (N17Rac) inhibition strategies indicated that NADPH oxidase was the source of the pressure-induced ROS. NADPH oxidase inhibition attenuated myogenic vasoconstriction and reduced the apparent Ca2+ sensitivity of the SMC contractile apparatus, without affecting Ca2+-independent, RhoA-mediated vasoconstriction in response to exogenous S1P. Our results indicate a mandatory role for Sk1/S1P in mediating pressure-induced, NADPH oxidase-derived ROS formation. In turn, ROS generation appears to increase Ca2+ sensitivity, necessary for full myogenic vasoconstriction.

Roles of reactive oxygen species in angiopoietin‐1/tie‐2 receptor signaling

In this study we identified the involvement of reactive oxygen species (ROS) in signaling and biological effects of the angiopoietin-1 (Ang-1)/tie-2 receptor pathway. Exposure of human umbilical vein endothelial cells to Ang-1 (50 ng/ml) induced rapid and transient production of ROS, particularly superoxide anions. ROS production was attenuated by preincubation with a peptide (gp91ds-tat) that inhibits the association of the gp91(phox) subunit with the p47(phox) subunit of NADPH oxidase and by the expression of a dominant-negative form of Rac-1 (Rac1N17). These results suggest that ROS production in response to Ang-1 exposure originates mainly from a Rac-1-dependent NADPH oxidase. Overexpression of antioxidants (superoxide dismutase and catalase) and Rac1N17, as well as preincubation with selective inhibitors of NADPH oxidase augmented basal p38 phosphorylation, inhibited Ang-1-induced PAK-1 phosphorylation and potentiated Ang-1-induced Erk1/2 phosphorylation but had no influence on AKT and SAPK/JNK phosphorylation by Ang-1. Exposure to Ang-1 (100 ng/ml) for 5 h induced a threefold increase in endothelial cell migration, a response that was strongly inhibited by overexpression of antioxidants, Rac1N17, and selective NADPH oxidase inhibitors. We conclude that activation of tie-2 receptors by Ang-1 triggers the production of ROS through activation of NADPH oxidase and that ROS generation by Ang-1 promotes endothelial cell migration while negatively regulating Erk1/2 phosphorylation.