Nox2 dependent redox-regulation of microglial response to amyloid-β stimulation and microgliosis in aging

Microglia express constitutively a Nox2 enzyme that is involved in neuroinflammation by the generation of reactive oxygen species (ROS). Amyloid β (Aβ) plays a crucial role in Alzheimer’s disease. However, the mechanism of Aβ-induced microglial dysfunction and redox-regulation of microgliosis in aging remains unclear. In this study, we examined Nox2-derived ROS in mediating microglial response to Aβ peptide 1–42 (Aβ42) stimulation in vitro, in aging-associated microgliosis in vivo and in post-mortem human samples. Compared to controls, Aβ42 markedly induced BV2 cell ROS production, Nox2 expression, p47phox and ERK1/2 phosphorylation, cell proliferation and IL-1β secretion. All these changes could be inhibited to the control levels in the presence of Nox2 inhibitor or superoxide scavenger. Compared to young (3–4 months) controls, midbrain tissues from wild-type aging mice (20– 22 months) had significantly higher levels of Nox2-derived ROS production, Aβ deposition, microgliosis and IL-1β production. However, these aging-related changes were reduced or absent in Nox2 knockout aging mice. Clinical significance of aging-associated Nox2 activation, microgliosis and IL-1β production was investigated using post-mortem midbrain tissues of humans at young (25–38 years) and old age (61–85 years). In conclusion, Nox2-dependent redox-signalling is crucial in microglial response to Aβ42 stimulation and in aging-associated microgliosis and brain inflammation

In vivo and in silico characterization of apocynin in reducing organ oxidative stress: a pharmacokinetic and pharmacodynamic study

Apocynin has been widely used in vivo as a Nox2-contaninig NADPH oxidase inhibitor. However, its time-dependent tissue distribution and inhibition on organ reactive oxygen species (ROS) production remained unclear. In this study, we examined apocynin pharmacokinetics and pharmacodynamics (PKPD) after iv injection (bolus, 5 mg/kg) of mice (CD1, 12-week). Apocynin was detected using a HPLC coupled to a linear ion-trap tandem mass spectrometer. Apocynin peak concentrations were detected in plasma at 1 min (5494±400 ng/mL) (t1/2=0.05 h, clearance=7.76 L/h/kg), in urine at 15 min (14942±5977 ng/mL), in liver at 5 min (2853±35 ng/g), in heart at 5 min (3161±309 ng/g) and in brain at 1 min (4603±208 ng/g) after iv injection. These were accompanied with reduction of ROS production in the liver, heart and brain homogenates. Diapocynin was not detected in these samples. Therapeutic effect of apocynin was examined using a mouse model (C57BL/6J) of high-fat diet (HFD, 16 weeks)-induced obesity and accelerated aging. Apocynin (5 mM) was supplied in drinking water during the HFD period and was detected at the end of treatment in the brain (5369±1612 ng/g), liver (4818±1340 ng/g) and heart (1795±1487 ng/g) along with significant reductions of ROS production in these organs. In conclusion, apocynin PKPD is characterized by a short half-life, rapid clearance, good distribution and inhibition of ROS production in major organs. Diapocynin is not a metabolite of apocynin in vivo. Apocynin crosses easily the blood-brain barrier and reduces brain oxidative stress associated with metabolic disorders and aging

Aging-associated metabolic disorder induces Nox2 activation and oxidative damage of endothelial function

Oxidative stress attributable to the activation of a Nox2-containing NADPH oxidase is involved in the development of vascular diseases and in aging. However, the mechanism of Nox2 activation in normal aging remains unclear. In this study, we used age-matched wild-type (WT) and Nox2 knockout (KO) mice at 3–4 months (young); 11–12 months (middle-aged) and 21–22 months (aging) to investigate age-related metabolic disorders, Nox2 activation and endothelial dysfunction. Compared to young mice, middle-aged and aging WT mice had significant hyperglycaemia, hyperinsulinaemia, increased systemic oxidative stress and higher blood pressure. Endothelium-dependent vessel relaxation to acetylcholine was significantly impaired in WT aging aortas, and this was accompanied by increased Nox2 and ICAM-1 expressions, MAPK activation and decreased insulin receptor expression and signaling. However, these aging-associated disorders were significantly reduced or absent in Nox2KO aging mice. The effect of metabolic disorder on Nox2 activation and endothelial dysfunction was further confirmed using high-fat diet-induced obesity and insulin resistance in middle-aged WT mice treated with apocynin (a Nox2 inhibitor). In vitro experiments showed that in response to high glucose plus high insulin challenge, WT coronary microvascular endothelial cells increased significantly the levels of Nox2 expression, activation of stress signaling pathways and the cells were senescent, e.g. increased p53 and β–galactosidase activity. However,these changes were absent in Nox2KO cells. In conclusion, Nox2 activation in response to aging-associated hyperglycaemia and hyperinsulinaemia plays a key role in the oxidative damage of vascular function. Inhibition or knockout of Nox2 preserves endothelial function and improves global metabolism in old age

Exploration of alcohol consumption behaviours and health-related influencing factors of young adults in the UK

Hazardous alcohol consumption is ranked above illicit drug use with regards to health deterioration and social and economic burden. This study sought to clarify the factors influencing alcohol consumption and its prevalence in young adults. Demographics, alcohol consumption and lifestyle information were gathered via anonymous questionnaires during 2011–2019, crossing Reading, Surrey and Farnborough universities, UK. Controlling for confounders, a multinomial logistic regression was performed using SAS® 9.4 software. A total of 1440 students (43.5% males, 56.5% females; 54.4% Caucasians) with a mean (SD) age of 19.9 (2.73) were included. Among them, 68.9% consumed alcohol frequently and 31.7% had �12 units/week. Statistical analysis revealed that males consumed twice more alcohol than females, odds ratio (OR) 1.67 (95% confidence interval (CI) = 1.34–2.09), p-value < 0.01. Caucasians consumed up to five times more alcohol than other ethnicities, OR 4.55 (3.57–5.56), p-value < 0.01. Smokers consumed three times more alcohol than non-smokers, OR 2.69 (1.82, 3.99), p-value < 0.01. In general, the levels of alcohol consumption were positively associated with the levels of physical activity, OR 2.00 (1.17–3.42), p-value < 0.05 and negatively associated with recreational sedentary screen-time activities in males, OR 0.31 (0.12–0.86), p-value = 0.03. Focusing alcohol interventions toward Caucasians, smokers and physically active students, particularly males, may guide university strategies to reduce alcohol-related societal harm and risks of morbidity and mortality

p47phox-dependent oxidant signalling through ASK1, MKK3/6 and MAPKs in Angiotensin II-induced cardiac hypertrophy and apoptosis

The p47phox is a key regulatory subunit of Nox2-containing NADPH oxidase (Nox2) that by generating reactive oxygen species (ROS) plays an important role in Angiotensin II (AngII)-induced cardiac hypertrophy and heart failure. However, the signalling pathways of p47phox in the heart remains unclear. In this study, we used wild-type (WT) and p47phox knockout (KO) mice (C57BL/6, male, 7-month-old, n = 9) to investigate p47phox-dependent oxidant-signalling in AngII infusion (0.8 mg/kg/day, 14 days)-induced cardiac hypertrophy and cardiomyocyte apoptosis. AngII infusion resulted in remarkable high blood pressure and cardiac hypertrophy in WT mice. However, these AngII-induced pathological changes were significantly reduced in p47phox KO mice. In WT hearts, AngII infusion increased significantly the levels of superoxide production, the expressions of Nox subunits, the expression of PKCα and C-Src and the activation of ASK1 (apoptosis signal-regulating kinase 1), MKK3/6, ERK1/2, p38 MAPK and JNK signalling pathways together with an elevated expression of apoptotic markers, i.e., γH2AX and p53 in the cardiomyocytes. However, in the absence of p47phox, although PKCα expression was increased in the hearts after AngII infusion, there was no significant activation of ASK1, MKK3/6 and MAPKs signalling pathways and no increase in apoptosis biomarker expression in cardiomyocytes. In conclusion, p47phox-dependent redox-signalling through ASK1, MKK3/6 and MAPKs plays a crucial role in AngII-induced cardiac hypertrophy and cardiomyocyte apoptosis

Inhibition of endothelial Nox2 activation by LMH001 protects mice from angiotensin II-induced vascular oxidative stress, hypertension and aortic aneurysm

Endothelial oxidative stress and inflammation attributable to the activation of a Nox2-NADPH oxidase are key features of many cardiovascular diseases. Here, we report a novel small chemical compound (LMH001, MW=290.079), by blocking phosphorylated p47phox interaction with p22phox, inhibited effectively angiotensin II (AngII)-induced endothelial Nox2 activation and superoxide production at a small dose (IC50=0.25µM) without effect on peripheral leucocyte oxidative response to pathogens. The therapeutic potential of LMH001 was tested using a mouse model (C57BL/6J, 7-month-old) of AngII infusion (0.8mg/kg/d, 14 days)-induced vascular oxidative stress, hypertension and aortic aneurysm. Age-matched littermates of p47phox knockout mice were used as controls of Nox2 inhibition. LMH001 (2.5mg/kg/d, ip. once) showed no effect on control mice, but inhibited completely AngII infusion-induced excess ROS production in vital organs, hypertension, aortic walls inflammation and reduced incidences of aortic aneurysm. LMH001 effects on reducing vascular oxidative stress was due to its inhibition of Nox2 activation and was abrogated by knockout of p47phox. LMH001 has the potential to be developed as a novel drug candidate to treat oxidative stress-related cardiovascular diseases

Intra-coronary physiology in contemporary percutaneous coronary intervention and anginal therapy with a focus on microvascular disease

Coronary physiological measurements have transformed the treatment of coronary artery disease (CAD), with increasing evidence supporting the use of pressure wire guided revascularisation. Advances in microvascular assessment have enabled clinicians to discern angina aetiology even in patients without obstructive epicardial coronary artery disease, paving the way for more effective tailored therapy. In this article, the authors will examine pressure wire indices, their role in influencing clinical outcomes and future directions

Molecular insights of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox_/_ coronary microvascular cells. Compared with wild-type p47phoxcDNAtransfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2 . production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells

Nox2 contributes to age-related oxidative damage to neurons and the cerebral vasculature

Oxidative stress plays an important role in aging-related neurodegeneration. This study used littermates of WT and Nox2-knockout (Nox2KO) mice plus endothelial cell-specific human Nox2 overexpression-transgenic (HuNox2Tg) mice to investigate Nox2-derived ROS in brain aging. Compared with young WT mice (3-4 months), aging WT mice (20-22 months) had obvious metabolic disorders and loss of locomotor activity. Aging WT brains had high levels of angiotensin II (Ang II) and ROS production; activation of ERK1/2, p53, and γH2AX; and losses of capillaries and neurons. However, these abnormalities were markedly reduced in aging Nox2KO brains. HuNox2Tg brains at middle age (11-12 months) already had high levels of ROS production and activation of stress signaling pathways similar to those found in aging WT brains. The mechanism of Ang II-induced endothelial Nox2 activation in capillary damage was examined using primary brain microvascular endothelial cells. The clinical significance of Nox2-derived ROS in aging-related loss of cerebral capillaries and neurons was investigated using postmortem midbrain tissues of young (25-38 years) and elderly (61-85 years) adults. In conclusion, Nox2 activation is an important mechanism in aging-related cerebral capillary rarefaction and reduced brain function, with the possibility of a key role for endothelial cells