Cigarette Smoke Initiates Oxidative Stress-Induced Cellular Phenotypic Modulation Leading to Cerebral Aneurysm Pathogenesis.

OBJECTIVE: Cigarette smoke exposure (CSE) is a risk factor for cerebral aneurysm (CA) formation, but the molecular mechanisms are unclear. Although CSE is known to contribute to excess reactive oxygen species generation, the role of oxidative stress on vascular smooth muscle cell (VSMC) phenotypic modulation and pathogenesis of CAs is unknown. The goal of this study was to investigate whether CSE activates a NOX (NADPH oxidase)-dependent pathway leading to VSMC phenotypic modulation and CA formation and rupture. APPROACH AND RESULTS: In cultured cerebral VSMCs, CSE increased expression of NOX1 and reactive oxygen species which preceded upregulation of proinflammatory/matrix remodeling genes (MCP-1, MMPs [matrix metalloproteinase], TNF-α, IL-1β, NF-κB, KLF4 [Kruppel-like factor 4]) and downregulation of contractile genes (SM-α-actin [smooth muscle α actin], SM-22α [smooth muscle 22α], SM-MHC [smooth muscle myosin heavy chain]) and myocardin. Inhibition of reactive oxygen species production and knockdown of NOX1 with siRNA or antisense decreased CSE-induced upregulation of NOX1 and inflammatory genes and downregulation of VSMC contractile genes and myocardin. p47phox-/- NOX knockout mice, or pretreatment with the NOX inhibitor, apocynin, significantly decreased CA formation and rupture compared with controls. NOX1 protein and mRNA expression were similar in p47phox-/- mice and those pretreated with apocynin but were elevated in unruptured and ruptured CAs. CSE increased CA formation and rupture, which was diminished with apocynin pretreatment. Similarly, NOX1 protein and mRNA and reactive oxygen species were elevated by CSE, and in unruptured and ruptured CAs. CONCLUSIONS: CSE initiates oxidative stress-induced phenotypic modulation of VSMCs and CA formation and rupture. These molecular changes implicate oxidative stress in the pathogenesis of CAs and may provide a potential target for future therapeutic strategies

NOX Inhibitors - A Promising Avenue for Ischemic Stroke.

NADPH-oxidase (NOX) mediated superoxide originally found on leukocytes, but now recognized in several types of cells in the brain. It has been shown to play an important role in the progression of stroke and related cerebrovascular disease. NOX is a multisubunit complex consisting of 2 membrane-associated and 4 cytosolic subunits. NOX activation occurs when cytosolic subunits translocate to the membrane, leading to transport electrons to oxygen, thus producing superoxide. Superoxide produced by NOX is thought to function in long-term potentiation and intercellular signaling, but excessive production is damaging and has been implicated to play an important role in the progression of ischemic brain. Thus, inhibition of NOX activity may prove to be a promising treatment for ischemic brain as well as an adjunctive agent to prevent its secondary complications. There is mounting evidence that NOX inhibition in the ischemic brain is neuroprotective, and targeting NOX in circulating immune cells will also improve outcome. This review will focus on therapeutic effects of NOX assembly inhibitors in brain ischemia and stroke. However, the lack of specificity and toxicities of existing inhibitors are clear hurdles that will need to be overcome before this class of compounds could be translated clinically

Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAc) induces vascular dysfunction via production of superoxide anion/reactive oxygen species

Overproduction of superoxide anion (•O2-) and O-linked β-N-acetylglucosamine (O-GlcNAc)-modification in the vascular system are contributors to endothelial dysfunction. This study tested the hypothesis that increased levels of O-GlcNAc-modified proteins contribute to •O2- production via activation of NADPH oxidase, resulting in impaired vasodilation. Rat aortic segments and vascular smooth muscle cell (VSMCs) were incubated with vehicle (methanol) or PUGNAc (100 µM). PUGNAc produced a time-dependent increase in O-GlcNAc levels in VSMC and decreased endothelium-dependent relaxation, which was prevented by apocynin and Tiron, suggesting that •O2- contributes to endothelial dysfunction under augmented O-GlcNAc levels. Aortic segments incubated with PUGNAc also exhibited increased levels of ROS, assessed by dihydroethidium fluorescence, and augmented •O2- production, determined by lucigenin-enhanced chemiluminescence. Additionally, PUGNAc treatment increased Nox1 and Nox4 protein expression in aorta and VSMCs. Translocation of p47phox subunit from the cytosol to the membrane was greater in aortas incubated with PUGNAc. VSMCs displayed increased p22phox protein expression after PUGNAc incubation, suggesting that NADPH oxidase is activated in conditions where O-GlcNAc protein levels are increased. In conclusion, O-GlcNAc levels reduce endothelium-dependent relaxation by overproduction of •O2- via activation of NADPH oxidase. This may represent an additional mechanism by which augmented O-GlcNAc levels impair vascular function

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

IKKα inactivation promotes Kras-initiated lung adenocarcinoma development through disrupting major redox regulatory pathways.

Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two distinct and predominant types of human lung cancer. IκB kinase α (IKKα) has been shown to suppress lung SCC development, but its role in ADC is unknown. We found inactivating mutations and homologous or hemizygous deletions in the CHUK locus, which encodes IKKα, in human lung ADCs. The CHUK deletions significantly reduced the survival time of patients with lung ADCs harboring KRAS mutations. In mice, lung-specific Ikkα ablation (IkkαΔLu ) induces spontaneous ADCs and promotes KrasG12D-initiated ADC development, accompanied by increased cell proliferation, decreased cell senescence, and reactive oxygen species (ROS) accumulation. IKKα deletion up-regulates NOX2 and down-regulates NRF2, leading to ROS accumulation and blockade of cell senescence induction, which together accelerate ADC development. Pharmacologic inhibition of NADPH oxidase or ROS impairs KrasG12D-mediated ADC development in IkkαΔLu mice. Therefore, IKKα modulates lung ADC development by controlling redox regulatory pathways. This study demonstrates that IKKα functions as a suppressor of lung ADC in human and mice through a unique mechanism that regulates tumor cell-associated ROS metabolism

Siglec-E promotes β2-integrin-dependent NADPH oxidase activation to suppress neutrophil recruitment to the lung

Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of β2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b β2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy

Expression and modulation of an NADPH oxidase in mammalian astrocytes

Amyloid β peptides generate oxidative stress in hippocampal astrocytes through a mechanism sensitive to inhibitors of the NADPH oxidase [diphenylene iodonium (DPI) and apocynin]. Seeking evidence for the expression and function of the enzyme in primary hippocampal astrocytes, we confirmed the expression of the subunits of the phagocyte NADPH oxidase by Western blot analysis and by immunofluorescence and coexpression with the astrocyte-specific marker glial fibrillary acidic protein both in cultures and in vivo. Functional assays using lucigenin luminescence, dihydroethidine, or dicarboxyfluorescein fluorescence to measure the production of reactive oxygen species (ROS) demonstrated DPI and apocynin-sensitive ROS generation in response to the phorbol ester PMA and to raised [Ca2+]c after application of ionomycin or P2u receptor activation. Stimulation by PMA but not Ca2+ was inhibited by the protein kinase C (PKC) inhibitors staurosporine and hispidin. Responses were absent in transgenic mice lacking gp91phox. Expression of gp91phox and p67phox was increased in reactive astrocytes, which showed increased rates of both resting and stimulated ROS generation. NADPH oxidase activity was modulated by intracellular pH, suppressed by intracellular alkalinization, and enhanced by acidification. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone suppressed basal ROS generation but markedly increased PMA-stimulated ROS generation. This was independent of mitochondrial ROS production, because it was unaffected by mitochondrial depolarization with rotenone and oligomycin. Thus, the NADPH oxidase is expressed in astrocytes and is functional, activated by PKC and intracellular calcium, modulated by pHi, and upregulated by astrocyte activation. The astrocytic NADPH oxidase is likely to play important roles in CNS physiology and pathology