NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

Göllner, Monika; Ihrig-Biedert, Irmgard; Petermann, Victoria; Saurin, Sabrina; Oelze, Matthias; Kröller‐Schön, Swenja; Kuntic, Marin; Pautz, Andrea; Daiber, Andreas; Kleinert, Hartmut

doi:10.3390/antiox9111043

Cited by 3 publications

(5 citation statements)

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“…In this respect, Nox2 mRNA deficiency may be potentially associated with the presence of mutated Nox2 mRNA encoding an inactive NOX2 protein, which, however, cross-reacts with standard detection antibodies. 39 Our findings also confirm and extend previous observations that endothelial cell-targeted overexpression of Nox2 in mice potentiates Ang II-induced endothelial dysfunction and hypertension, 40 while Nox2 deletion results in improved endothelial function in a mouse model of renovascular hypertension. 41 However, only a marginal difference in the hypertensive response to Ang II between WT and Nox2-KO mice has also been reported.…”

Section: Discussionsupporting

confidence: 90%

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Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1

Kamhieh-Milz,

Chen,

Goettsch

et al. 2023

ATVB

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BACKGROUND: Increasing evidence suggests that superoxide ions produced by NOX (nicotinamide adenine dinucleotide phosphate oxidases) mediate vascular effects of Ang II (angiotensin II) evoked by atherogenic diets. Here, we analyzed the mechanism by which NOX2 contributes to Ang II–induced ET-1 (endothelin 1) production in human microvascular endothelial cells. METHODS: The effects of high-fat diet were compared between WT (wild type) and Nox2 ( mouse NOX2 gene )-deficient mice. ET-1 production and NOX2 expression by human microvascular endothelial cells in vitro were analyzed by ELISA, reverse transcription quantitative polymerase chain reaction, electrophoretic mobility shift assay, promoter deletions, RNA interference, and pharmacological inhibition. Production of superoxide anions was visualized by fluorescent cell labeling. RESULTS: Feeding mice high-fat diet for 10 weeks increased cardiac expression and plasma levels of Ang II and ET-1 in WT but not in Nox2 -deficient animals. Exposure of human microvascular endothelial cells to Ang II resulted in increased ET-1 production, which could be blocked by silencing NOX2 ( human NOX2 gene ). Ang II promoted NOX2 expression through induction of the Oct-1 (human/mouse octamer binding transcription factor 1 protein) and activation of the NOX2 promoter region containing Oct-1–binding sites. Stimulation of NOX2 expression by Ang II was associated with increased production of superoxide anions. Inhibition of Oct-1 by small interfering RNA reduced Ang II–induced NOX2 expression and superoxide anion production, and neutralization of superoxide by SOD (superoxide dismutase) abolished Ang II–stimulated ET1 ( human ET-1 gene ) promoter activity, ET1 mRNA expression, and ET-1 release. CONCLUSIONS: Ang II may promote ET-1 production in the endothelium in response to atherogenic diets through a mechanism that involves the transcription factor Oct-1 and the increased formation of superoxide anions by NOX2.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1

Kamhieh-Milz,

Chen,

Goettsch

et al. 2023

ATVB

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“…50,51 The lucigenin electrochemiluminescence method has previously been compared to other superoxide detection methods and tested by specific inhibition, revealing a good correlation with other methods of superoxide detection. [52][53][54] Notwithstanding, these methods can be associated with artifacts. 50 Besides, the clear detection of O 2…”

Section: Discussionmentioning

confidence: 99%

“…Both ROS levels in the skin and the neutrophilic oxidative burst were detected by lucigenin and luminol assays, respectively 50,51 . The lucigenin electrochemiluminescence method has previously been compared to other superoxide detection methods and tested by specific inhibition, revealing a good correlation with other methods of superoxide detection 52–54 …”

Section: Discussionmentioning

confidence: 99%

Reactive oxygen species produced by myeloid cells in psoriasis as a potential biofactor contributing to the development of vascular inflammation

et al. 2023

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Psoriasis is an immune‐mediated inflammatory skin disease driven by interleukin‐17A (IL‐17A) and associated with cardiovascular dysfunction. We used a severe psoriasis mouse model of keratinocyte IL‐17A overexpression (K14‐IL‐17Aind/+, IL‐17Aind/+ control mice) to investigate the activity of neutrophils and a potential cellular interconnection between skin and vasculature. Levels of dermal reactive oxygen species (ROS) and their release by neutrophils were measured by lucigenin‐/luminol‐based assays, respectively. Quantitative RT‐PCR determined neutrophilic activity and inflammation‐related markers in skin and aorta. To track skin‐derived immune cells, we used PhAM‐K14‐IL‐17Aind/+ mice allowing us to mark all cells in the skin by photoconversion of a fluorescent protein to analyze their migration into spleen, aorta, and lymph nodes by flow cytometry. Compared to controls, K14‐IL‐17Aind/+ mice exhibited elevated ROS levels in the skin and a higher neutrophilic oxidative burst accompanied by the upregulation of several activation markers. In line with these results psoriatic mice displayed elevated expression of genes involved in neutrophil migration (e.g., Cxcl2 and S100a9) in skin and aorta. However, no direct immune cell migration from the psoriatic skin into the aortic vessel wall was observed. Neutrophils of psoriatic mice showed an activated phenotype, but no direct cellular migration from the skin to the vasculature was observed. This suggests that highly active vasculature‐invading neutrophils must originate directly from the bone marrow. Hence, the skin‐vasculature crosstalk in psoriasis is most likely based on the systemic effects of the autoimmune skin disease, emphasizing the importance of a systemic therapeutic approach for psoriasis patients.

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“…The family of superoxide-generating enzymes, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), is considered to be a primary contributor to oxidative stress in various human pathologies [ 10 ]. NOX2, the primary NOX isoform of phagocytes, consists of membrane-bound (gp91 phox , p22 phox ) and cytoplasmic (p40 phox , p47 phox , and p67 phox ) subunits [ 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway

Shi,

Li,

Herb

et al. 2024

J Neuroinflammation

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Background NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. Methods Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2−/− mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. Results We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. Conclusions Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

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NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein

Cited by 3 publications

References 70 publications

Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1

Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1

Reactive oxygen species produced by myeloid cells in psoriasis as a potential biofactor contributing to the development of vascular inflammation

Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway

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