Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement

Downregulation of p22phox in Retinal Pigment Epithelial Cells Inhibits Choroidal Neovascularization in Mice

Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice

Background: Excess reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) promotes apoptotic cell death following ischemic/reperfusion injury. Effect of chlorpromazine and promethazine (C+P) on brain activity was reported to induce neuroprotection. The current study was designed to evaluate the inhibitory function of C+P on oxidative injury after stroke. Methods: Adult male Sprague-Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion. At the onset of reperfusion, rats received C+P, or apocynin (NOX inhibitor), or rottlerin [protein kinase C (PKC) δ inhibitor]. Brain damage was evaluated using infarct volumes and neurological deficits as well as apoptotic cell death (TUNEL). The enzymatic activity of NOX and ROS production as well as protein expressions of NOX subunits (gp91 phox , p67 phox , p47 phox , and p22 phox ), phosphorylation of PKC δ (p-PKC δ)/PKC δ and manganese superoxide dismutase (MnSOD) was examined. Neural SHSY5Y cells were used under 2 h of oxygen-glucose deprivation (OGD) followed by reoxygenation for 6 and 24 h with or without C+P treatment. ROS and protein levels of NOX subunits, p-PKC δ/PKC δ and MnSOD were detected. Moreover, measurement of PKC δ membrane translocation and detection of the interaction of p47 phox and PKC δ through co-immunoprecipitation were performed. Results: C+P reduced cerebral infarct volumes, neurological deficits, and apoptotic cell death in the ischemic rats, as well observed in the presence of NOX and PKC δ inhibitors. ROS production, NOX activity, expression of NOX subunits, p-PKC δ/PKC δ and MnSOD were significantly reduced by C+P. In ischemic rats administered with NOX and PKC δ inhibitors, ROS, activity of NOX and the NOX subunits protein levels were all decreased. In the OGD/R model, C+P decreased ROS and protein levels of NOX subunits, p-PKC δ/PKC δ and MnSOD. Furthermore, C+P reduced the PKC δ membrane translocation and the interaction of p47 phox and PKC δ. Conclusion: C+P treatments confers neuroprotection in severe stroke by suppressing oxidative stress and ROS production. PKC δ/NOX/MnSOD may be the vital regulators and the potential targets for an efficacious therapy following ischemic stroke.

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] A key risk factor for the development of atherosclerosis is familial hypercholesterolemia (FH), a genetic disease characterized by elevated levels of low density lipoprotein (LDL). Studies have shown that oxidative stress and vascular smooth muscle cell (VSMC) phenotypic modulation play critical roles in the development and stability of atherosclerotic plaques. The key source of reactive oxygen species (ROS) contributing to oxidative stress in the vasculature is the enzymatic complex nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Upregulation of intermediateconductance Ca[superscript 2 +]-activated K[superscript +] channels (K[subscript Ca]3.1) and modification of NADPH oxidase activity have been shown to mediate phenotypic modulation of coronary smooth muscle cells (CSMC). It remains unclear, however, whether K[subscript Ca]3.1 expression and activity are altered in atherosclerotic coronary smooth muscle of individuals with FH, and whether NADPH oxidase plays a role in atherosclerosis through regulation of K[subscript Ca]3.1 channels. Our objective was thus twofold, 1) to determine whether K[subscript Ca]3.1 expression and activity are increased in CSMCs isolated from FH swine, and 2) to determine if NADPH oxidase plays a role in growth factor-induced upregulation of K[subscript Ca]3.1. Right coronary artery (RCA) sections from 2 year old FH swine showed a [approximate sign]15 fold increase in artery stenosis accompanied by significantly elevated triglyceride and cholesterol values. In the media of the diseased FH coronaries, there was a trend for increased K[subscript Ca]3.1mRNA expression and K[subscript Ca]3.1 protein expression was elevated [approximate sign]20% compared to control coronaries. In addition, K[subscript Ca]3.1 channel activity increased almost 2- fold in coronary artery cells isolated from FH swine compared to control animals. In cultured CSMCs, basic fibroblast growth factor (bFGF) increased superoxide (O[subscript 2] [superscript .-]) production which was inhibited by treatment with the NADPH oxidase inhibitor apocynin (Apo). Treatment with bFGF increased K[subscript Ca]3.1 mRNA levels [aproximate sign]2.5 fold in both right coronary artery (RCA) sections and CSMCs, while addition of Apo prevented the increase. Furthermore, inhibition of NADPH oxidase abolished the bFGF-induced increase in coronary smooth muscle K[subscript Ca]3.1 protein expression and CSMC K[subscript Ca]3.1 channel activity. Treatment with bFGF significantly increased activator protein-1 (AP-1) promoter activity which was inhibited by addition of Apo. RCA and CSMC express all four cardiovascular Nox isoforms (Nox1, Nox2, Nox4, Nox5) with Nox4 being the predominant isoform. Treatment with bFGF decreased Nox1, Nox2, and Nox4 CSMC message, while treatment with Apo increased the mRNA expression of all four isoforms. Knock down of Nox2 and Nox4 did not affect the K[subscript Ca]3.1 message response to bFGF or Apo. Consistent with our earlier findings of increased medial K[subscript Ca]3.1 expression in FH coronaries; whole vessel K[subscript Ca]3.1 mRNA expression was increased in FH coronary smooth muscle and Nox2 rather than Nox4 was the predominant Nox isoform. Our findings demonstrate that K[subscript Ca]3.1 is upregulated in coronary smooth muscle of FH swine and support previous research indicating K[subscript Ca]3.1 plays a key role in the development and progression of atherosclerosis. Our findings also provide novel evidence that NADPH oxidase contributes to VSMC phenotypic modulation associated with atherosclerosis through AP-1 transcriptional upregulation of K[subscript Ca]3.1.

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.

Endothelin (ET)-1 is a potent vasoconstrictor and mitogen, and because of these properties, it is thought to play a role in the development of hypertension.1,2 The vascular endothelium is a major source of ET-1 production, although a variety of other cell types also have been shown to synthesize and release ET-1. ET-1 is believed to act in a paracrine manner on ETA and ETB receptors on smooth muscle, which mediate contraction, cell proliferation, and hypertrophy. Activation of ETB receptors on endothelial cells stimulates the production of prostacyclin and nitric oxide to induce vasorelaxation and inhibition of sodium transport in renal tubules. Given these properties, considerable attention has been paid to the mechanisms of ET-1 action as it relates to the renal control of blood pressure and the pathogenesis of salt-dependent hypertension. Renal ET synthesis is increased in experimental animals maintained on a high-salt diet and ETA receptor antagonists lower arterial pressure primarily in salt-dependent models of hypertension.1,2 For the past 30 to 40 years, the actions of angiotensin (Ang) II has been arguably the most widely investigated factor in hypertension research. Although physiology textbooks agree on the major actions of Ang II, eg, vasoconstriction and release of aldosterone, recent attention has focused on its ability to stimulate the synthesis of ET-1,3–5 as well as reactive oxygen species.6 There are many reactive oxygen species such as superoxide, hydroxyl radical, and hydrogen peroxide that are produced by all cell types and can have profound effects on the vascular system to impact blood pressure regulation. Most recent attention has been paid to the role of superoxide. There are many enzymatic sources of superoxide including NADPH oxidase, xanthine oxidase, nitric oxide synthase, and cytochrome P450. The focus of the current review, however, is be on the …

Alzheimer’s disease (AD) is one of the main human dementias around the world which is constantly increasing every year due to several factors (age, genetics, environment, etc.) and there are no prevention or treatment options to cure it. AD is characterized by memory loss associated with oxidative stress (OS) in brain cells (neurons, astrocytes, microglia, etc.). OS can be produced by amyloid beta (Aβ) protein aggregation and its interaction with metals, mitochondrial damage and alterations between antioxidants and oxidant enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. NADPH oxidase produces reactive oxygen species (ROS) and it is overexpressed in AD, producing large amounts of superoxide anions and hydrogen peroxide which damage brain cells and the vasculature. In addition, it has been reported that NADPH oxidase causes an imbalance of pH which could also influence in the amyloid beta (Aβ) production. Therefore, NADPH oxidase had been proposed as a therapeutic target in AD. However, there are no drugs for AD treatment such as an NADPH oxidase inhibitor despite great efforts made to stabilize the ROS production using antioxidant molecules. So, in this work, we will focus our attention on NADPH oxidase (NOX2 and NOX4) in AD as well as in AD models and later discuss the use of NADPH oxidase inhibitor compounds in AD.

Reactive oxygen species (ROS) generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)1 mediate cellular signalings involved in normal physiological processes, and aberrant control of Nox1 has been implicated in the pathogenesis of various diseases. Therefore, Nox1 could have great potential as a therapeutic target. Here, we identified a novel Nox1 inhibitor, NOS31 secreted from Stretomyces sp. and analyzed its chemical structure. Furthermore, NOS31 was found to selectively inhibit Nox1-mediated ROS generation, with only a marginal effect on other Nox isoforms (Nox2-5) and no ROS scavenging activity. This compound blocked both Nox organizer 1 (NOXO1)/Nox activator 1 (NOXA1)-dependent and phorbol 12-myristate 13-acetate-stimulated Nox1-mediated ROS production in colon cancer cells. NOS31 inhibited the proliferation of several colon carcinoma and gastric cancer cell lines that upregulate the Nox1 system, whereas it had no appreciable effect on normal cells with low levels of Nox1. The finding suggests that NOS31 is a unique, potent Nox1 inhibitor of microbial origin and raises its possibility as a therapeutic agent for inhibiting gastrointestinal cancer cell growth.

Procyanidins have been associated with a reduced risk of cardiovascular diseases such as atherosclerosis. However, the molecular mechanisms underlying this benefit are not fully understood. Increased reactive oxygen species (ROS) production generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a common problem in different cardiovascular diseases. Our objective was to evaluate the effects of procyanidin-rich fractions from distilled grape pomace on NADPH oxidase activity in human umbilical vein endothelial cells (HUVEC). Three differently polymerized and galloylated procyanidin fractions were analyzed for their NADPH oxidase inhibitory activity in cell lysates and in HUVEC cultures. All of the three fractions, up to 1 µg/ml, equally inhibited isolated NADPH oxidase in HUVEC lysates in a concentration-dependent manner and independently of any superoxide anion scavenging activities. The procyanidin fractions even blocked NADPH oxidase activity in intact HUVEC, inhibiting ROS production at both extra- and intracellular levels. The fractions achieved the same effects that known NADPH oxidase inhibitors, such as diphenylene iodonium and apocynin, but they presented better hydrosolubility. Our results demonstrated that procyanidin from grape pomace inhibit human endothelial NADPH oxidase regardless of their polymerization degree and galloylation percentage. Therefore, procyanidins are suitable NADPH oxidase inhibitors which could serve as models for therapeutic alternatives for cardiovascular diseases.

Reactive oxygen species (ROS) produced in the neuronal, renal, and vascular systems not only influence cardiovascular physiology but are also strongly implicated in pathological signaling leading to hypertension. Different sources of ROS have been identified, ranging from xanthine-xanthine oxidase and mitochondria to NADPH oxidase (Nox) enzymes. Of 7 Nox family members, Nox1, Nox2, and Nox4 (and Nox5 in humans) influence the cardiovascular system. Their activation processes and cell and tissue distribution vary widely, adding complexity to understanding their functional roles. Whether these systems act collectively or independently in disease conditions is unclear, but recently feed forward mechanisms have been established between ROS sources. Studies published in Hypertension over the last few years are the focus of this review, and they provide a framework with which to consider the roles of Nox enzymes in neuronal, renal, and vascular hypertensive mechanisms, as well as cardiac remodeling, and their relationships with other ROS-generating systems. ### Neuronal ROS in Hypertension Redox signaling in the central nervous system is well recognized in neuronal control of blood pressure (BP), as well as in response to angiotensin II (Ang II) and aldosterone, which are linked to ROS-dependent hypertension. Recently, new roles for ROS have been described in the hypothalamus and brain stem, nucleus tractus solitarius (NTS), subfornical organ (SFO), rostral ventrolateral medulla, and area postrema (Figure 1). Figure 1. Neuronal NADPH oxidase–dependent ROS involved in central regulation of hypertension. NADPH oxidase homologues, mainly Nox2 and Nox4, are found in different regions of the neuronal system and are reported to have a role in the neuropathogenesis of hypertension by enhancing the sympathetic nerve activity. Nox-induced ROS initiate a forward loop in cross-activation of different receptors and between Nox and mitochondrial ROS. OVLT indicates organum vasculosum of the lamina terminalis; PVN, paraventricular nucleus; PP, posterior pituitary; AP, area postrema; RVLM, rostral ventrolateral medulla. Several …

Apocyanin, a microglial NADPH oxidase inhibitor prevents dopaminergic neuronal degeneration in lipopolysaccharide induced Parkinson's disease model

A significant role for alveolar macrophages (AM) in the pathophysiology of sepsis-induced acute lung injury (ALI) has been shown; however, the mechanisms behind AM-related lung injury remain relatively uncertain. We examined the role of AM nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in pulmonary endothelial cell septic injury. NADPH oxidase is one of the major sources of cellular reactive oxygen species and has been implicated in endothelial injury in ALI. Pulmonary microvascular endothelial cells (PMVEC) monolayers were grown on Transwell inserts and incubated with wild-type and NADPH oxidase-deficient AM in the presence or absence of cytomix (equimolar TNF-alpha, IL-1beta, and IFN-gamma). Injury to the monolayers was assessed by trans-PMVEC Evans blue (EB)-labeled albumin flux. We found AM under cytomix stimulation caused significant EB-albumin flux across the PMVEC monolayers, and this effect was attenuated by the genetic deletion of AM NADPH oxidase. The pharmacological inhibition of AM NADPH oxidase with apocynin and PR-39 also significantly reduced AM-dependent PMVEC injury. In the AM-PMVEC cocultures, we also assessed PMVEC injury through measurement of protein oxidation and lipid peroxidation. AM were shown to cause a significant increase in these markers of PMVEC injury, which was also attenuated by the inhibition of NADPH oxidase or through the use of NADPH oxidase-deficient AM. PMVEC NADPH oxidase was shown not to significantly contribute to PMVEC injury in our studies. From our findings we have concluded that AM NADPH oxidase is crucial for the septic increase in pulmonary vascular permeability.

An elevated level of tumor necrosis factor (TNF)-α is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-α activates nuclear factor (NF)-kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-α and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-α-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-α with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-α-induced ROS production was measured using 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate assay. TNF-α-induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-α-induced upregulation of interleukin (IL)-1β and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-α-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-α/β and p65, degradation of IkappaBα, binding of NF-kappaB to its binding motif, and upregulation of IL-1β and VCAM-1 induced by TNF-α were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-α-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1β and VCAM-1, in human cardiomyocytes.

An accumulating body of evidence suggests that transient or physiological reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases act as a redox signal to re-establish homeostasis. The capacity to re-establish homeostasis progressively declines during aging but is maintained in long-lived animals to promote healthy aging. In the model organism Caenorhabditis elegans, ROS generated by dual oxidases (Duox) are important for extracellular matrix integrity, pathogen defense, oxidative stress resistance, and longevity. The Duox enzymatic activity is tightly regulated and under cellular control. Developmental molting cycles, pathogen infections, toxins, mitochondrial-derived ROS, drugs, and small GTPases (e.g., RHO-1) can activate Duox (BLI-3) to generate ROS, whereas NADPH oxidase inhibitors and negative regulators, such as MEMO-1, can inhibit Duox from generating ROS. Three mechanisms-of-action have been discovered for the Duox/BLI-3-generated ROS: (1) enzymatic activity to catalyze crosslinking of free tyrosine ethyl ester in collagen bundles to stabilize extracellular matrices, (2) high ROS bursts/levels to kill pathogens, and (3) redox signaling activating downstream kinase cascades to transcription factors orchestrating oxidative stress and immunity responses to re-establish homeostasis. Although Duox function at the cell surface is well established, recent genetic and biochemical data also suggests a novel role for Duoxs at the endoplasmic reticulum membrane to control redox signaling. Evidence underlying these mechanisms initiated by ROS from NADPH oxidases, and their relevance for human aging, are discussed in this review. Appropriately controlling NADPH oxidase activity for local and physiological redox signaling to maintain cellular homeostasis might be a therapeutic strategy to promote healthy aging.

Hyperhydricity is a physiological disorder associated with oxidative stress. Reactive oxygen species (ROS) generation in plants is initiated by various enzymatic sources, including plasma membrane-localized nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, cell wall-bound peroxidase (POD), and apoplastic polyamine oxidase (PAO). The origin of the oxidative burst associated with hyperhydricity remains unknown. To investigate the role of NADPH oxidases, POD, and PAO in ROS production and hyperhydricity, exogenous hydrogen peroxide (H2O2) and inhibitors of each ROS-producing enzyme were applied to explore the mechanism of oxidative stress induction in garlic plantlets in vitro. A concentration of 1.5mM H2O2 increased endogenous ROS production and hyperhydricity occurrence and enhanced the activities of NADPH oxidases, POD, and PAO. During the entire treatment period, NADPH oxidase activity increased continuously, whereas POD and PAO activities exhibited a transient increase and subsequently declined. Histochemical and cytochemical visualization demonstrated that specific inhibitors of each enzyme effectively suppressed ROS accumulation. Moreover, superoxide anion generation, H2O2 content, and hyperhydric shoot frequency in H2O2-stressed plantlets decreased significantly. The NADPH oxidase inhibitor was the most effective at suppressing superoxide anion production. The results suggested that NADPH oxidases, POD, and PAO were responsible for endogenous ROS induction. NADPH oxidase activation might play a pivotal role in the oxidative burst in garlic plantlets in vitro during hyperhydricity.