bims-noxint 2022-04-03 papers

Issue of 2022–04–03
five papers selected by
Laia Caja Puigsubira, Uppsala University

J Cancer Res Clin Oncol. 2022 Mar 29.

Context-specific effects of NOX4 inactivation in acute myeloid leukemia (AML).

Muhammed Burak Demircan, Tina M Schnoeder, Peter C Mgbecheta, Katrin Schröder, Frank-D Böhmer, Florian H Heidel.

PURPOSE: Oxidative stress has been linked to initiation and progression of cancer and recent studies have indicated a potential translational role regarding modulation of ROS in various cancers, including acute myeloid leukemia (AML). Detailed understanding of the complex machinery regulating ROS including its producer elements in cancer is required to define potential translational therapeutic use. Based on previous studies in acute myeloid leukemia (AML) models, we considered NADPH oxidase (NOX) family members, specifically NOX4 as a potential target in AML.
METHODS: Pharmacologic inhibition and genetic inactivation of NOX4 in murine and human models of AML were used to understand its functional role. For genetic inactivation, CRISPR-Cas9 technology was used in human AML cell lines in vitro and genetically engineered knockout mice for Nox4 were used for deletion of Nox4 in hematopoietic cells via Mx1-Cre recombinase activation.
RESULTS: Pharmacologic NOX inhibitors and CRISPR-Cas9-mediated inactivation of NOX4 and p22-phox (an essential NOX component) decreased proliferative capacity and cell competition in FLT3-ITD-positive human AML cells. In contrast, conditional deletion of Nox4 enhanced the myeloproliferative phenotype of an FLT3-ITD induced knock-in mouse model. Finally, Nox4 inactivation in normal hematopoietic stem and progenitor cells (HSPCs) caused a minor reduction in HSC numbers and reconstitution capacity.
CONCLUSION: The role of NOX4 in myeloid malignancies appears highly context-dependent and its inactivation results in either enhancing or inhibitory effects. Therefore, targeting NOX4 in FLT3-ITD positive myeloid malignancies requires additional pre-clinical assessment.

Keywords: Acute myeloid leukemia (AML); CRISPR-Cas9; FLT3-ITD; NADPH oxidases (NOX); Nox4; Oxidative stress; Reactive oxygen species (ROS)

DOI: https://doi.org/10.1007/s00432-022-03986-3

Arterioscler Thromb Vasc Biol. 2022 Mar 31. 101161ATVBAHA121317239

NADPH Oxidase 1 Mediates Acute Blood Pressure Response to Angiotensin II by Contributing to Calcium Influx in Vascular Smooth Muscle Cells.

Jung-Min Park, Van Quan Do, Yoon-Seok Seo, Hyun Jong Kim, Joo Hyun Nam, Ming Zhe Yin, Hae Jin Kim, Sung Joon Kim, Kathy K Griendling, Moo-Yeol Lee.

BACKGROUND: Reactive oxygen species (ROS) and calcium ions (Ca2+) are among the major effectors of Ang II (angiotensin II) in vascular smooth muscle cells. ROS are related to Ca2+ signaling or contraction induced by Ang II, but little is known about their detailed functions. Here, NOX (NADPH oxidase), a major ROS source responsive to Ang II, was investigated regarding its contribution to Ca2+ signaling.
METHODS: Vascular smooth muscle cells were primary cultured from rat aorta. Ca2+ and ROS were monitored mainly using fura-2 and HyPer family probes. Signals activating NOX were examined with relevant pharmacological inhibitors and genetic manipulation techniques.
RESULTS: Ang II-induced ROS generation was found to be biphasic: the first phase of ROS production, which was mainly mediated by NOX1, was small and transient, preceding a rise in Ca2+, and the second phase of ROS generation, mediated by NOX1 and NOX4, was slow but sizeable, continuing over tens of minutes. NOX1-derived superoxide in the first phase is required for Ca2+ influx through nonselective cation channels. AT1R (Ang II type 1 receptor)-Gβγ-PI3Kγ (phosphoinositide 3-kinase γ) signaling pathway was responsible for the rapid activation of NOX1 in the first phase, while in the second phase, NOX1 was further activated by a separate AT1R-Gαq/11-PLC (phospholipase C)-PKCβ (protein kinase Cβ) signaling axis. Consistent with these observations, aortas from NOX1-knockout mice exhibited reduced contractility in response to Ang II, and thus the acute pressor response to Ang II was also attenuated in NOX1-knockout mice.
CONCLUSIONS: NOX1 mediates Ca2+ signal generation and thereby contributes to vascular contraction and blood pressure elevation by Ang II.

Keywords: NADPH oxidases; angiotensin II; calcium; muscle cells; reactive oxygen species

DOI: https://doi.org/10.1161/ATVBAHA.121.317239

Blood. 2022 Mar 23. pii: blood.2021015365. [Epub ahead of print]

Macrophage NOX2 NADPH oxidase maintains alveolar homeostasis in mice.

Sourav Bhattacharya, Rachel Idol, Wei Yang, Jorge David Rojas Marquez, Yanan Li, Guangming Huang, Wandy Beatty, Jeffrey J Atkinson, John Brumell, Juhi Bagaitkar, Jeffrey A Magee, Mary C Dinauer.

The leukocyte NADPH oxidase 2 (NOX2) plays a key role in pathogen killing and immunoregulation. Genetic defects in NOX2 result in chronic granulomatous disease (CGD), associated with microbial infections and inflammatory disorders, often involving the lung. Alveolar macrophages (AM) are the predominant immune cell in the airways at steady state, and limiting their activation is important given constant exposure to inhaled materials, yet the importance of NOX2 in this process is not well-understood. Here, we show a previously undescribed role for NOX2 in maintaining lung homeostasis by suppressing AM activation, as studied using CGD mice or mice with selective loss of NOX2 preferentially in macrophages. AM lacking NOX2 have increased cytokine responses to TLR2 and TLR4 stimulation ex vivo. Moreover, between 4 and 12 weeks of age, mice with global NOX2 deletion developed an activated CD11bhigh subset of AM with epigenetic and transcriptional profiles reflecting immune activation compared to WT AM. The presence of CD11bhigh AM in CGD mice correlated with increased numbers of alveolar neutrophils and proinflammatory cytokines at steady state as well as increased lung inflammation following insults. Moreover, deletion of NOX2 preferentially in macrophages was sufficient for mice to develop an activated CD11bhigh AM subset and accompanying pro-inflammatory sequela. Additionally, we showed that the altered resident macrophage transcriptional profile in the absence of NOX2 is tissue-specific as these changes were not seen in resident peritoneal macrophages. Thus, these data demonstrate that absence of NOX2 in alveolar macrophages leads to their pro-inflammatory remodeling and dysregulates alveolar homeostasis.

DOI: https://doi.org/10.1182/blood.2021015365

Biochim Biophys Acta Mol Cell Res. 2022 Mar 25. pii: S0167-4889(22)00053-2. [Epub ahead of print]1869(7): 119262

GRK2 selectively attenuates the neutrophil NADPH-oxidase response triggered by β-arrestin recruiting GPR84 agonists.

Johanna Fredriksson, André Holdfeldt, Jonas Mårtensson, Lena Björkman, Thor C Møller, Erik Müllers, Claes Dahlgren, Martina Sundqvist, Huamei Forsman.

In order to avoid a prolonged pro-inflammatory neutrophil response, signaling downstream of an agonist-activated G protein-coupled receptor (GPCR) has to be rapidly terminated. Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, GRK2, which is highly expressed by immune cells, plays an important role. The medium chain fatty acid receptor GPR84 as well as formyl peptide receptor 2 (FPR2), receptors expressed in neutrophils, play a key role in regulating inflammation. In this study, we investigated the effects of GRK2 inhibitors on neutrophil functions induced by GPR84 and FPR2 agonists. GRK2 was shown to be expressed in human neutrophils and analysis of subcellular fractions revealed a cytosolic localization. The GRK2 inhibitors enhanced and prolonged neutrophil production of reactive oxygen species (ROS) induced by GPR84- but not FPR2-agonists, suggesting a receptor selective function of GRK2. This suggestion was supported by β-arrestin recruitment data. The ROS production induced by a non β-arrestin recruiting GPR84 agonist was not affected by the GRK2 inhibitor. Termination of this β-arrestin independent response relied, similar to the response induced by FPR2 agonists, primarily on the actin cytoskeleton. In summary, we show that GPR84 utilizes GRK2 in concert with β-arrestin and actin cytoskeleton dependent processes to fine-tune the activity of the ROS generating NADPH-oxidase in neutrophils.

Keywords: FPR2; GPR84; GRK2; Neutrophils; Reactive oxygen species; β-Arrestin

DOI: https://doi.org/10.1016/j.bbamcr.2022.119262

Redox Biol. 2022 Mar 25. pii: S2213-2317(22)00059-3. [Epub ahead of print]52 102287

The amyloid peptide β disrupts intercellular junctions and increases endothelial permeability in a NADPH oxidase 1-dependent manner.

Anuradha Tarafdar, Nina Wolska, Christoph Krisp, Hartmut Schlüter, Giordano Pula.

Alzheimer's disease is the most common form of dementia and is associated with the accumulation of amyloid peptide β in the brain parenchyma. Vascular damage and microvascular thrombosis contribute to the neuronal degeneration and the loss of brain function typical of this disease. In this study, we utilised a murine model of Alzheimer's disease to evaluate the neurovascular effects of this disease. Upon detection of an increase in the phosphorylation of the endothelial surface receptor VE-cadherin, we focused our attention on endothelial cells and utilised two types of human endothelial cells cultured in vitro: 1) human umbilical vein endothelial cells (HUVECs) and 2) human brain microvascular endothelial cells (hBMECs). Using an electrical current impedance system (ECIS) and FITC-albumin permeability assays, we discovered that the treatment of human endothelial cells with amyloid peptide β causes a loss in their barrier function, which is oxidative stress-dependent and similarly to our observation in mouse brain associates with VE-cadherin phosphorylation. The activation of the superoxide anion-generating enzyme NADPH oxidase 1 is responsible for the oxidative stress that leads to the disruption of barrier function in human endothelial cells in vitro. In summary, we have identified a novel molecular mechanism explaining how the accumulation of amyloid peptide β in the brain parenchyma may induce the loss of neurovascular barrier function, which has been observed in patients. Neurovascular leakiness plays an important role in brain inflammation and neuronal degeneration driving the progression of the Alzheimer's disease. Therefore, this study provides a novel and promising target for the development of a pharmacological treatment to protect neurovascular function and reduce the progression of the neurodegeneration in Alzheimer's patients.

Keywords: Alzheimer; Endothelial; NADPH oxidase; Neuroinflammation; Oxidative stress; Permeability

DOI: https://doi.org/10.1016/j.redox.2022.102287