Downregulating p22phox ameliorates inflammatory response in Angiotensin II-induced oxidative stress by regulating MAPK and NF-?B pathways in ARPE-19 cells.
ABSTRACT: Oxidative stress and inflammation are two interrelated biological events implicated in the pathogenesis of many diseases. Reactive oxygen species (ROS) produced under oxidative stress play a key role in pathological conditions. Inhibition of p22phox, an indispensable component of the NADPH oxidase (NOX) complex comprising the main source of ROS, plays a protective role in many ocular conditions by inhibiting the activation of NOXs and the generation of ROS. However, little is understood regarding the role of p22phox in oxidative stress-related inflammation in the eye. We used a p22phox small interfering RNA (siRNA) to transfect the retinal pigment epithelium (RPE)-derived cell line ARPE-19, and human primary RPE (hRPE) cells, then stimulated with Ang II. We observed a potent anti-inflammatory effect and studied the underlying mechanism. Downregulating p22phox resulted in decreased ROS generation, a reduction of NOXs (NOX1, 2, 4) and a decrease in inflammatory cytokine. In addition, p22phox downregulation reduced the activation of the MAPK and NF-?B signaling pathways. We conclude that inhibition of p22phox has an anti-inflammatory effect in Ang II-induced oxidative stress. Suppressing the MAPK and NF-?B pathways is involved in this protective effect. These results suggest that p22phox may provide a promising therapeutic target for oxidative stress-induced ocular inflammation.
Project description:<h4>Purpose</h4>To determine the effect of decorin on oxidative stress and apoptosis of human lens epithelial (HLE) cells under high glucose condition.<h4>Methods</h4>HLE cell line (HLEB3) was incubated in normal glucose (5.5 mM) or high glucose (60 mM) medium. Decorin (50 nM) was applied 2 hours before high glucose medium was added. Apoptosis detection was executed by flow cytometry and western blotting (analysis of bcl-2 and bax). Oxidative stress level was measured by the generation of reactive oxygen species (ROS), glutathione peroxidase (GSH) and superoxide dismutase (SOD). P38 mitogen-activated protein kinase (MAPK) phosphorylation, the expression of p22phox of HLE cells and human lens anterior capsules were detected by western blotting. Small interfering RNA transfection to p22phox and p38 MAPK was also carried out on HLEB3.<h4>Results</h4>High glucose caused HLE cells oxidative stress and apoptosis exhibiting the increase of apoptotic cells and ROS production and decrease of bcl-2/bax ratio, GSH/GSSG ration and SOD activity. P22phox and phospho-p38 MAPK were upregulated in high glucose treated HLEB3 cells. Knocking down p22phox or p38 by siRNAs can reduce high glucose induced cell apoptosis and oxidative stress level. Silencing p22phox by siRNA can downregulate the phosphorylation of p38 MAPK. Decorin can inhibit the apoptosis, oxidative stress level and the induction of p22phox and phospho-p38 of HLEB3 induced by high glucose. Furthermore, the expression of p22phox and p38 were found significantly increased in lens anterior capsules of diabetic cataract patients compared to that of normal age-related cataract patients.<h4>Conclusions</h4>Results showed that p22phox-p38 pathway may be participated in high glucose induced lens epithelial cell injury, decorin may inhibit the high glucose induced apoptosis and oxidative stress injury by suppressing this pathway in part.
Project description:The retinal pigment epithelium (RPE) plays a key role in retinal health, being essential for the protection against reactive oxygen species (ROS). Nevertheless, excessive oxidative stress can induce RPE dysfunction, promoting visual loss. Our aim is to clarify the possible implication of CYP2E1 in ethanol (EtOH)-induced oxidative stress in RPE alterations. Despite the increase in the levels of ROS, measured by fluorescence probes, the RPE cells exposed to the lowest EtOH concentrations were able to maintain cell survival, measured by the Cell Proliferation Kit II (XTT). However, EtOH-induced oxidative stress modified inflammation and angiogenesis biomarkers, analyzed by proteome array, ELISA, qPCR and Western blot. The highest EtOH concentration used stimulated a large increase in ROS levels, upregulating the cytochrome P450-2E1 (CYP2E1) and promoting cell death. The use of antioxidants such as N-acetylcysteine (NAC) and diallyl sulfide (DAS), which is also a CYP2E1 inhibitor, reverted cell death and oxidative stress, modulating also the upstream angiogenesis and inflammation regulators. Because oxidative stress plays a central role in most frequent ocular diseases, the results herein support the proposal that CYP2E1 upregulation could aggravate retinal degeneration, especially in those patients with high baseline oxidative stress levels due to their ocular pathology and should be considered as a risk factor.
Project description:Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-?), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved.In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-? antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-? protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-? or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and ?-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-? and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of ?-catenin, VEGF, and p22phox and coimmunoprecipitation of ?-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-? following pretreatment with ?-catenin transcriptional inhibitors, XAV939 or JW67, or transfection with p22phox siRNA and compared to appropriate controls.Compared to the non-lasered control, TNF-? and VEGF protein were increased in the RPE/choroids in a murine laser-induced CNV model (p<0.05). An intravitreal neutralizing antibody to mouse TNF-? reduced CNV volume, and VEGF protein in the RPE/choroids (p<0.01) and oxidized phospholipids within CNV compared to IgG control (p<0.05). In cultured RPE cells and compared to controls, TNF-? induced ROS generation and increased activation of NOX4, an isoform of NADPH oxidase; both were prevented by pretreatment with the apocynin or VAS2870 or knockdown of p22phox, a subunit of NADPH oxidase. TNF-? treatment increased VEGF expression (p<0.001) and the formation of a transcriptional complex of ?-catenin and T-cell transcriptional factor; both were prevented by pretreatment with apocynin or knockdown of p22phox. Inhibition of ?-catenin by XAV939, but not the nuclear factor kappa B inhibitor, Bay 11-7082, prevented TNF-?-induced VEGF upregulation.Our results support the thinking that TNF-? contributes to CNV by upregulating VEGF production in RPE cells through ROS-dependent activation of ?-catenin signaling. These results provide mechanisms of crosstalk between inflammatory mediator, TNF-?, and ROS in RPE cells.
Project description:Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease linked to oxidative stress, which is associated with significant morbidity. The NADPH oxidase complex (NOX) produces reactive oxygen species (ROS) that are among the key markers for determining RA's pathophysiology. Therefore, understanding ROS-regulated molecular pathways and their interaction is necessary for developing novel therapeutic approaches for RA. Here, by combining mouse genetics and biochemistry with clinical tissue analysis, we reveal that in vivo Rubicon interacts with the p22phox subunit of NOX, which is necessary for increased ROS-mediated RA pathogenesis. Furthermore, we developed a series of new aryl propanamide derivatives consisting of tetrahydroindazole and thiadiazole as p22phox inhibitors and selected 2-(tetrahydroindazolyl)phenoxy-N-(thiadiazolyl)propanamide 2 (TIPTP, M.W. 437.44), which showed considerably improved potency, reaching an IC50 value up to 100-fold lower than an inhibitor that we previously synthesized reported N8 peptide-mimetic small molecule (blocking p22phox-Rubicon interaction). Notably, TIPTP treatment showed significant therapeutic effects a mouse model for RA. Furthermore, TIPTP had anti-inflammatory effects ex vivo in monocytes from healthy individuals and synovial fluid cells from RA patients. These findings may have clinical applications for the development of TIPTP as a small molecule inhibitor of the p22phox-Rubicon axis for the treatment of ROS-driven diseases such as RA.
Project description:The run/cysteine-rich-domain-containing Beclin1-interacting autophagy protein (Rubicon) is essential for the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by interacting with p22phox to trigger the production of reactive oxygen species (ROS) in immune cells. In a previous study, we demonstrated that the interaction of Rubicon with p22phox increases cellular ROS levels. The correlation between Rubicon and mitochondrial ROS (mtROS) is poorly understood. Here, we report that Rubicon interacts with p22phox in the outer mitochondrial membrane in macrophages and patients with human ulcerative colitis. Upon lipopolysaccharide (LPS) activation, the binding of Rubicon to p22phox was elevated, and increased not only cellular ROS levels but also mtROS, with an impairment of mitochondrial complex III and mitochondrial biogenesis in macrophages. Furthermore, increased Rubicon decreases mitochondrial metabolic flux in macrophages. Mito-TIPTP, which is a p22phox inhibitor containing a mitochondrial translocation signal, enhances mitochondrial function by inhibiting the association between Rubicon and p22phox in LPS-primed bone-marrow-derived macrophages (BMDMs) treated with adenosine triphosphate (ATP) or dextran sulfate sodium (DSS). Remarkably, Mito-TIPTP exhibited a therapeutic effect by decreasing mtROS in DSS-induced acute or chronic colitis mouse models. Thus, our findings suggest that Mito-TIPTP is a potential therapeutic agent for colitis by inhibiting the interaction between Rubicon and p22phox to recover mitochondrial function.
Project description:The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-? inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.
Project description:Age-related macular degeneration (AMD) involves the loss of retinal pigment epithelium (RPE) and photoreceptors and is one of the leading causes of blindness in the elderly. Oxidative damage to proteins, lipids, and DNA has been associated with RPE dysfunction and AMD. In this study, we evaluated oxidative stress in AMD and the efficacy of antioxidant, N-acetyl-L-cysteine (NAC), in protecting RPE from oxidative damage. To test this idea, primary cultures of RPE from human donors with AMD (n = 32) or without AMD (No AMD, n = 21) were examined for expression of NADPH oxidase (NOX) genes, a source of reactive oxygen species (ROS). Additionally, the cells were pretreated with NAC for 2 hours and then treated with either hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (t-BHP) to induce cellular oxidation. Twenty-four hours after treatment, ROS production, cell survival, the content of glutathione (GSH) and adenosine triphosphate (ATP), and cellular bioenergetics were measured. We found increased expression of p22phox, a NOX regulator, in AMD cells compared to No AMD cells (p = 0.02). In both AMD and No AMD cells, NAC pretreatment reduced t-BHP-induced ROS production and protected from H2O2-induced cell death and ATP depletion. In the absence of oxidation, NAC treatment improved mitochondrial function in both groups (p < 0.01). Conversely, the protective response exhibited by NAC was disease-dependent for some parameters. In the absence of oxidation, NAC significantly reduced ROS production (p < 0.001) and increased GSH content (p = 0.02) only in RPE from AMD donors. Additionally, NAC-mediated protection from H2O2-induced GSH depletion (p = 0.04) and mitochondrial dysfunction (p < 0.05) was more pronounced in AMD cells compared with No AMD cells. These results demonstrate the therapeutic benefit of NAC by mitigating oxidative damage in RPE. Additionally, the favorable outcomes observed for AMD RPE support NAC's relevance and the potential therapeutic value in treating AMD.
Project description:ANG II is thought to increase sympathetic outflow by increasing oxidative stress and promoting local inflammation in the paraventricular nucleus (PVN) of the hypothalamus. However, the relative contributions of inflammation and oxidative stress to sympathetic drive remain poorly understood, and the underlying cellular and molecular targets have yet to be examined. ANG II has been shown to enhance Toll-like receptor (TLR)4-mediated signaling on microglia. Thus, in the present study, we aimed to determine whether ANG II-mediated activation of microglial TLR4 signaling is a key molecular target initiating local oxidative stress in the PVN. We found TLR4 and ANG II type 1 (AT1) receptor mRNA expression in hypothalamic microglia, providing molecular evidence for the potential interaction between these two receptors. In hypothalamic slices, ANG II induced microglial activation within the PVN (?65% increase, P < 0.001), an effect that was blunted in the absence of functional TLR4. ANG II increased ROS production, as indicated by dihydroethidium fluorescence, within the PVN of rats and mice (P < 0.0001 in both cases), effects that were also dependent on the presence of functional TLR4. The microglial inhibitor minocycline attenuated ANG II-mediated ROS production, yet ANG II effects persisted in PVN single-minded 1-AT1a knockout mice, supporting the contribution of a non-neuronal source (likely microglia) to ANG II-driven ROS production in the PVN. Taken together, these results support functional interactions between AT1 receptors and TLR4 in mediating ANG II-dependent microglial activation and oxidative stress within the PVN. More broadly, our results support a functional interaction between the central renin-angiotensin system and innate immunity in the regulation of neurohumoral outflows from the PVN.
Project description:Oxidative stress in the rostral ventrolateral medulla (RVLM), a sympathetic center in the brainstem, was implicated in the regulation of sympathetic activity in various hypertensive models including stroke-prone spontaneously hypertensive rats (SHRSP). In this study, we evaluated the role of the NADPH oxidases (NOX) in the blood pressure (BP) regulation in RVLM in SHRSP. The P22PHOX-depleted congenic SHRSP (called SP.MES) was constructed by introducing the mutated p22phox gene of Matsumoto Eosinophilic Shinshu rat. BP response to glutamate (Glu) microinjection into RVLM was compared among SHRSP, SP.MES, SHR and Wistar Kyoto (WKY); the response to Glu microinjection was significantly greater in SHRSP than in SP.MES, SHR and WKY. In addition, tempol, losartan and apocynin microinjection reduced the response to Glu significantly only in SHRSP. The level of oxidative stress, measured in the brainstem using lucigenin and dihydroethidium, was reduced in SP.MES than in SHRSP. BP response to cold stress measured by telemetry system was also blunted in SP.MES when compared with SHRSP. The results suggested that oxidative stress due to the NOX activation in RVLM potentiated BP response to Glu in SHRSP, which might contribute to the exaggerated response to stress in this strain.
Project description:Vascular oxidative injury accompanies many common conditions associated with hypertension. In the present study, we employed mouse models with excessive vascular production of ROS (tg(sm/p22phox) mice, which overexpress the NADPH oxidase subunit p22(phox) in smooth muscle, and mice with vascular-specific deletion of extracellular SOD) and have shown that these animals develop vascular collagen deposition, aortic stiffening, renal dysfunction, and hypertension with age. T cells from tg(sm/p22phox) mice produced high levels of IL-17A and IFN-?. Crossing tg(sm/p22phox) mice with lymphocyte-deficient Rag1(-/-) mice eliminated vascular inflammation, aortic stiffening, renal dysfunction, and hypertension; however, adoptive transfer of T cells restored these processes. Isoketal-protein adducts, which are immunogenic, were increased in aortas, DCs, and macrophages of tg(sm/p22phox) mice. Autologous pulsing with tg(sm/p22phox) aortic homogenates promoted DCs of tg(sm/p22phox) mice to stimulate T cell proliferation and production of IFN-?, IL-17A, and TNF-?. Treatment with the superoxide scavenger tempol or the isoketal scavenger 2-hydroxybenzylamine (2-HOBA) normalized blood pressure; prevented vascular inflammation, aortic stiffening, and hypertension; and prevented DC and T cell activation. Moreover, in human aortas, the aortic content of isoketal adducts correlated with fibrosis and inflammation severity. Together, these results define a pathway linking vascular oxidant stress to immune activation and aortic stiffening and provide insight into the systemic inflammation encountered in common vascular diseases.