NADPH oxidase inhibitors: a decade of discovery from Nox2ds to HTS

NADPH oxidases (Nox) are established as major sources of reactive oxygen species (ROS). Over the past two decades, Nox-derived ROS have emerged as pivotal in the development of myriad diseases involving oxidative stress. In contrast, Nox are also involved in signaling mechanisms necessary for normal cell function. The study of these enzymes in physiological and pathophysiological conditions is made considerably more complex by the discovery of 7 isoforms: Nox1 through 5 as well as Duox1 and 2, each with its own specific cytosolic components, regulatory control mechanisms, subcellular localization and/or tissue distribution. A clear understanding of the role individual isoforms play in a given system is hindered by the lack of isoform-specific inhibitors. In animal models, knockdown or knockout methodologies are providing definitive answers to perplexing questions of the complex interplay of multiple Nox isoforms in cell and tissue signaling. However, the complex structures and interactions of these heteromeric isozymes predict pleiotropic actions of the Nox subunits and thus suppression of these proteins is almost certain to have untoward effects. Thus, as both therapies and pharmacological tools, molecule-based inhibitors continue to prove extremely useful and rational in design. Unfortunately, many of the available inhibitors have proven non-specific, falling into the category of scavengers or inhibitors of more than one source of ROS. Here, we will review some of the efforts that have been undertaken to develop specific inhibitors of NADPH oxidase over the past decade, from the peptidic inhibitor Nox2ds-tat to more recent small molecule inhibitors that have emerged from high-throughput screening campaigns.     

The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.     

Nitric oxide synthases: targets for therapeutic strategies in neurological diseases

Glutamate excitotoxicity, oxidative stress, and mitochondrial dysfunctions are common features leading to neuronal death in cerebral ischemia, traumatic brain injury, Parkinson's disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Nitric oxide (NO) alone or in cooperation with superoxide anion and peroxynitrite is emerging as a predominant effector of neurodegeneration The use of NO synthase (NOS) inhibitors and mutant mice lacking each NOS isoform have provided evidence for the injurious effects of NO derived from neuronal or inducible isoforms. New neuroprotective strategies have been proposed with selective NOS inhibitors for the neuronal (ARL17477) or the inducible (1400 W) isoforms or with compounds combining in one molecule selective nNOS inhibition and antioxidant properties (BN 80933), in experimental ischemia-induced acute neuronal damage. The efficacy of these new strategies is well established in acute neuronal injury but remains to be determined in more chronic neurological diseases.     

NOX enzymes: potential target for the treatment of acute lung injury

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), is characterized by acute inflammation, disruption of the alveolar-capillary barrier, and in the organizing stage by alveolar pneumocytes hyperplasia and extensive lung fibrosis. The cellular and molecular mechanisms leading to the development of ALI/ARDS are not completely understood, but there is evidence that reactive oxygen species (ROS) generated by inflammatory cells as well as epithelial and endothelial cells are responsible for inflammatory response, lung damage, and abnormal repair. Among all ROS-producing enzymes, the members of NADPH oxidases (NOXs), which are widely expressed in different lung cell types, have been shown to participate in cellular processes involved in the maintenance of lung integrity. It is not surprising that change in NOXs' expression and function is involved in the development of ALI/ARDS. In this context, the use of NOX inhibitors could be a possible therapeutic perspective in the management of this syndrome. In this article, we summarize the current knowledge concerning some cellular aspects of NOXs localization and function in the lungs, consider their contribution in the development of ALI/ARDS and discuss the place of NOX inhibitors as potential therapeutical target.     

Targeting NADPH oxidases for the treatment of cancer and inflammation

NADPH oxidases are a family of oxidases that utilize molecular oxygen to generate hydrogen peroxide and superoxide, thus indicating physiological functions of these highly reactive and short-lived species. The regulation of these NADPH oxidases (nox) enzymes is complex, with many members of this family exhibiting complexity in terms of subunit composition, cellular location, and tissue-specific expression. While the complexity of the nox family (Nox1-5, Duox1, 2) is daunting, the complexity also allows for targeting of NADPH oxidases in disease states. In this review, we discuss which inflammatory and malignant disorders can be targeted by nox inhibitors, as well as clinical experience in the use of such inhibitors.     

Towards specific NADPH oxidase inhibition by small synthetic peptides

Reactive oxygen species (ROS) production by the phagocyte NADPH oxidase is essential for host defenses against pathogens. ROS are very reactive with biological molecules such as lipids, proteins and DNA, potentially resulting in cell dysfunction and tissue insult. Excessive NADPH oxidase activation and ROS overproduction are believed to participate in disorders such as joint, lung, vascular and intestinal inflammation. NADPH oxidase is a complex enzyme composed of six proteins: gp91phox (renamed NOX2), p22phox, p47phox, p67phox, p40phox and Rac1/2. Inhibitors of this enzyme could be beneficial, by limiting ROS production and inappropriate inflammation. A few small non-peptide inhibitors of NADPH oxidase are currently used to inhibit ROS production, but they lack specificity as they inhibit NADPH oxidase homologues or other unrelated enzymes. Peptide inhibitors that target a specific sequence of NADPH oxidase components could be more specific than small molecules. Here we review peptide-based inhibitors, with particular focus on a molecule derived from gp91phox/NOX2 and p47phox, and discuss their possible use as specific phagocyte NADPH oxidase inhibitors.     

Targeting NOX enzymes in the central nervous system: therapeutic opportunities

Among the pathogenic mechanisms underlying central nervous system (CNS) diseases, oxidative stress is almost invariably described. For this reason, numerous attempts have been made to decrease reactive oxygen species (ROS) with the administration of antioxidants as potential therapies for CNS disorders. However, such treatments have always failed in clinical trials. Targeting specific sources of reactive oxygen species in the CNS (e.g. NOX enzymes) represents an alternative promising option. Indeed, NOX enzymes are major generators of ROS, which regulate progression of CNS disorders as diverse as amyotrophic lateral sclerosis, schizophrenia, Alzheimer disease, Parkinson disease, and stroke. On the other hand, in autoimmune demyelinating diseases, ROS generated by NOX enzymes are protective, presumably by dampening the specific immune response. In this review, we discuss the possibility of developing therapeutics targeting NADPH oxidase (NOX) enzymes for the treatment of different CNS pathologies. Specific compounds able to modulate the activation of NOX enzymes, and the consequent production of ROS, could fill the need for disease-modifying drugs for many incurable CNS pathologies.     

Targeting NOX enzymes in pulmonary fibrosis

Oxidative stress has been associated with a number of human fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Oxidative stress is most often defined as an imbalance between the generation of reactive oxygen species (ROS) in excess of the capacity of cells/tissues to detoxify or scavenge them. Additionally, the regulated production of ROS participates in cellular signaling. Therapeutic strategies to treat IPF have, thus far, focused on augmenting anti-oxidant capacity. Recent studies have demonstrated a critical role for ROS-generating enzymatic systems, specifically, NADPH oxidase (NOX) family oxidoreductases in fibrotic processes. In this review, we examine the evidence for NOX isoforms in the generation and perpetuation of fibrosis, and the potential to target this gene family for the treatment of IPF and related fibrotic disorders.     

Cerebral ischemia revisited: new insights as revealed using in vitro brain slice preparations

The elucidation of the pathophysiological mechanisms of cerebral ischemia/hypoxia dictates the use of experimental models which mimic this disabling brain condition. In vivo experimental models have been available for many decades and are responsible for the bulk of, though incomplete, knowledge we have about these mechanisms. Since study in isolation of each postulated mechanism is impossible in vivo, the need for an in vitro experimental model has intensified in recent years. Consequently, rat and guinea pig hippocampal slice preparations have emerged as the models of choice. This review attempts to highlight some of the results obtained using brain slices in the study of cerebral ischemia/hypoxia and compare them to those obtained in vivo. Both the biochemical and the physiological correlates of energy metabolism, ion homeostasis, neurotransmission and neuromodulation of this brain condition are reviewed. The agreements, and especially the disagreements, between the in vivo and in vitro findings are emphasized. Details are given of the possible roles of both lactic acid, Ca2+ and excitotoxins in the neuronal damage inflicted by cerebral ischemia/hypoxia. Recent attempts to protect brain slices against experimental cerebral ischemic/hypoxic damage are also reviewed here briefly.     

Cerebral ischemia revisited: New insights as revealed using in vitro brain slice preparations

Summary The elucidation of the pathophysiological mechanisms of cerebral ischemia/hypoxia dictates the use of experimental models which mimic this disabling brain condition. In vivo experimental models have been available for many decades and are responsible for the bulk of, though incomplete, knowledge we have about these mechanisms. Since study in isolation of each postulated mechanism is impossible in vivo, the need for an in vitro experimental model has intensified in recent years. Consequently, rat and guinea pig hippocampal slice preparations have emerged as the models of choice. This review attempts to highlight some of the results obtained using brain slices in the study of cerebral ischemia/hypoxia and compare them to those obtained in vivo. Both the biochemical and the physiological correlates of energy metabolism, ion homeostasis, neurotransmission and neuromodulation of this brain condition are reviewed. The agreements, and especially the disagreements, between the in vivo and in vitro findings are emphasized. Details are given of the possible roles of both lactic acid, Ca²⁺ and excitotoxins in the neuronal damage inflicted by cerebral ischemia/hypoxia. Recent attempts to protect brain slices against experimental cerebral ischemic/hypoxic damage are also reviewed here briefly.     

siRNA-mediated knock-down of NOX3: therapy for hearing loss?

Cisplatin is a widely used chemotherapeutic agent that causes significant hearing loss. Previous studies have shown that cisplatin exposure is associated with increase in reactive oxygen species (ROS) in the cochlea. The inner ear expresses a unique isoform of NADPH oxidase, NOX3. This enzyme may be the primary source of ROS generation in the cochlea. The knockdown of NOX3 by pretreatment with siRNA prevented cisplatin ototoxicity, as demonstrated by preservation of hearing thresholds and inner ear sensory cells. Trans-tympanic NOX3 siRNA reduced the expression of NOX3 and biomarkers of cochlear damage, including transient receptor vanilloid 1 (TRPV1) channel and kidney injury molecule-1 (KIM-1) in cochlear tissues. In addition, siRNA against NOX3 reduced apoptosis as demonstrated by TUNEL staining, and prevented the increased expression of Bax and abrogated the decrease in Bcl2 expression following cisplatin administration. Trans-tympanic administration of siRNA directed against NOX3 may provide a useful method of attenuating cisplatin ototoxicity. In this paper, we review recent publications dealing with the role of NOX3 in ototoxicity and the effects of siRNA against cisplatin-induced hearing loss.     

急性脑梗死超早期静脉溶栓治疗24例疗效报告

急性缺血性脑血管病起病6h内,脑血管造影发现70～80％的病人,在出现症状的区域内,脑血管有血栓性或狭窄性病变^[1]。所以在急性脑梗死（ACI）超早期,用能溶解血栓及栓子的药物,使阻塞的血管再通,从而挽救缺血的脑组织,是近年达成的共识。本文对2005年10月～2007年12月,对发病6h内的（ACI）患者予以尿激酶（UK）静脉溶栓治疗进行了研究。     

Heat shock response in the central nervous system

The heat shock response is induced in nervous tissue in a variety of clinically significant experimental models including ischemic brain injury (stroke), trauma, thermal stress and status epilepticus. Excessive excitatory neurotransmission or the inability to metabolically support normal levels of excitatory neurotransmission may contribute to neuronal death in the nervous system in many of the same pathophysiologic circumstances. We demonstrated that in vitro glutamate-neurotransmitter induced excitotoxicity is attenuated by the prior induction of the heat shock response. A short thermal stress induced a pattern of protein synthesis characteristic of the highly conserved heat shock response and increased the expression of heat shock protein (HSP) mRNA. Protein synthesis was necessary for the neuroprotective effect. The study of the mechanisms of heat shock mediated protection may lead to important clues as to the basic mechanisms underlying the molecular actions of the HSP and the factors important for excitotoxic neuronal injury. The clinical relevance of these findings in vitro is suggested by experiments performed by others in vivo demonstrating that pretreatment of animals with a submaximal thermal or ischemis stress confers protection from a subsequent ischemic insult.     

Regulation and termination of NADPH oxidase activity

NADPH oxidase of phagocytes plays a crucial role in host defense by producing reactive oxygen species (ROS) that are intended to kill invading microbes. Many other cells produce ROS for signaling purposes. The respiratory burst oxidase in human neutrophils is the main but not exclusive subject of this review, because it is archetypical and has been studied most extensively. The activity of this enzyme must be controlled in phagocytes to prevent collateral damage, and in non-phagocytic cells to perform its signaling role. With many stimuli, NADPH oxidase activity is transient. Various forms of evidence indicate that sustained NADPH oxidase activity requires continuous renewal of the enzyme complex, without which rapid deactivation occurs. This review considers mechanisms that have been proposed to terminate the phagocyte respiratory burst. Changes in the phosphorylation state of p47(phox) and in the species of nucleotide bound to Rac seem to be the dominant factors in deactivation.     

Vascular endothelial growth factors (VEGFs) and stroke

Vascular endothelial growth factors (VEGFs) have been shown to participate in atherosclerosis, arteriogenesis, cerebral edema, neuroprotection, neurogenesis, angiogenesis, postischemic brain and vessel repair, and the effects of transplanted stem cells in experimental stroke. Most of these actions involve VEGF-A and the VEGFR-2 receptor, but VEGF-B, placental growth factor, and VEGFR-1 have been implicated in some cases as well. VEGF signaling pathways represent important potential targets for the acute and chronic treatment of stroke.     

Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or “All that you did and did not want to know about Nox inhibitory peptides”

Phagocytes utilize reactive oxygen species (ROS) to kill pathogenic microorganisms. The source of ROS is an enzymatic complex (the NADPH oxidase), comprising a membrane-associated heterodimer (flavocytochrome b (558)), consisting of subunits Nox2 and p22(phox), and four cytosolic components (p47(phox), p67(phox), p40(phox), and Rac). The primordial ROS (superoxide) is generated by the reduction of molecular oxygen by NADPH via redox centers located on Nox2. This process is activated by the translocation of the cytosolic components to the membrane and their assembly with Nox2. Membrane translocation is preceded by interactions among cytosolic components. A number of proteins structurally and functionally related to Nox2 have been discovered in many cells (the Nox family) and these have pleiotropic functions related to the production of ROS. An intense search is underway to design therapeutic means to modulate Nox-dependent overproduction of ROS, associated with diseases. Among drug candidates, a central position is held by synthetic peptides reflecting domains in oxidase components involved in NADPH oxidase assembly. Peptides, corresponding to domains in Nox2, p22(phox), p47(phox), and Rac, found to be oxidase activation inhibitory in vitro, are reviewed. Usually, peptides are inhibitory only when added preceding assembly of the complex. Although competition with intact components seems most likely, less obvious mechanisms are, sometimes, at work. The use of peptides as inhibitory drugs in vivo requires the development of methods to assure cell penetration, resistance to degradation, and avoidance of toxicity, and modest successes have been achieved. The greatest challenge remains the discovery of peptide inhibitors acting specifically on individual Nox isoforms.     

Neuroprotection against hypoxia/ischemia: δ-opioid receptor-mediated cellular/molecular events

Hypoxic/ischemic injury remains the most dreaded cause of neurological disability and mortality. Despite the humbling experiences due to lack of promising therapy, our understanding of the complex cascades underlying the neuronal insult has led to advances in basic science research. One of the most noteworthy has been the effect of opioid receptors, especially the delta-opioid receptor (DOR), on hypoxic/ischemic neurons. Our recent studies, and those of others worldwide, present strong evidence that sheds light on DOR-mediated neuroprotection in the brain, especially in the cortex. The mechanisms of DOR neuroprotection are broadly categorized as: (1) stabilization of the ionic homeostasis, (2) inhibition of excitatory transmitter release, (3) attenuation of disrupted neuronal transmission, (4) increase in antioxidant capacity, (5) regulation of intracellular pathways—inhibition of apoptotic signals and activation of pro-survival signaling, (6) regulation of specific gene and protein expression, and (7) up-regulation of endogenous opioid release and/or DOR expression. Depending upon the severity and duration of hypoxic/ischemic insult, the release of endogenous opioids and DOR expression are regulated in response to the stress, and DOR signaling acts at multiple levels to confer neuronal tolerance to harmful insult. The phenomenon of DOR neuroprotection offers a potential clue for a promising target that may have significant clinical implications in our quest for neurotherapeutics.     

Hh signaling in regeneration of the ischemic heart

Myocardial infarction (MI) is caused by the occlusion of a coronary artery due to underlying atherosclerosis complicated by localized thrombosis. The blockage of blood flow leads to cardiomyocyte (CM) death in the infarcted area. Adult mammalian cardiomyocytes have little capacity to proliferate in response to injury; however, some pathways active during embryogenesis and silent during adult life are recruited in response to tissue injury. One such example is hedgehog (Hh) signaling. Hh is involved in the embryonic development of the heart and coronary vascular system. Pathological conditions including ischemia activate Hh signaling in adult tissues. This review highlights the involvement of Hh signaling in ischemic tissue regeneration with a particular emphasis on heart regeneration and discusses its potential role as a therapeutic agent.     

Cerebral ischemia is the main cause for the onset of heat stroke syndrome in rabbits.

During the onset of heat stroke, rabbits displayed hyperthermia (42.8 degrees C), and decreased cerebral perfusion pressure and decreased cerebral blood flow (as reflected by a prolonged cerebral circulation time) compared to those of normothermic rabbits. On the other hand febrile rabbits, during the fever plateau did not show the above responses, although they had a similar level of hyperthermia (42.4 degrees C). The data support the concept that cerebral ischemia is the main cause for the onset of the heat stroke syndrome.