Exp Neurobiol 2017; 26(4): 195-205
Published online August 31, 2017
© The Korean Society for Brain and Neural Sciences
Jong Youl Kim1, Joohyun Park1,2, Jong Eun Lee1,2* and Midori A. Yenari3*
1Department of Anatomy, Yensei University College of Medicine, 2BK21 Plus Project for Medical Sciences and Brain Research Institute, Yonsei University College of Medicine, Seoul 03722, Korea, 3Department of Neurology, University of California, San Francisco, and San Francisco Veterans Affairs Medical Center, San Francisco, California 94121, USA
Correspondence to: *To whom correspondence should be addressed.
Jong Eun Lee, TEL: 82-2-2228-1646, FAX: 82-2-365-0700
Midori A. Yenari, TEL: 1-415 750-2011, FAX: 1-415 750-2273
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.
Keywords: ischemic stroke, NADPH oxidase, superoxide, NOX inhibition
Stroke is caused by occlusion of or hemorrhage from a blood vessel supplying the brain. It is a cause of long-term disability and ranks as the third frequent cause of death following heart disease and cancer, yet despite high prevalence, the number of approved therapies remains low [1,2]. The major therapeutic strategy for treatment of acute ischemic stroke is rapid recanalization, either by pharmacological means through thrombolytic agents, or mechanical thrombectomy . However, the time window for intervention limits these therapies to a small number of patients, and their inappropriate use can actually worsen outcome.
Stroke, via occlusion of cerebral artery(ies) brings on energy depletion and subsequent death of cells in the vascular territory. Currently, understanding of the pathophysiology of ischemic stroke indicates that the mechanism of neuronal injury can be characterized according to infarct progression over time. The acute stage appears within minutes up to a few hours after ischemic stroke onset, during which ionic homeostasis is disrupted by the decrease in cerebral blood flow. This phenomenon leads to increased intracellular calcium concentrations and stimulation of glutamate release, producing excitotoxicity and a spreading depression throughout the ischemic region [4,5,6]. Water enters the intracellular space due to osmotic gradients, causing cells swell, and the resulting vasogenic edema can give rise to intracranial pressure, vascular compression and herniation . Furthermore, the generation of reactive oxygen species (ROS) can destroy membranes, mitochondria and DNA, leading to proteins misfolding and enzyme dysfunction. These factors are exacerbated in the event of reperfusion takes place. In the second-subacute phase occurs a few hours to a few days after ischemia. An inflammatory and cell death (apoptosis and necrosis) response develops as a result of the stimulatory influences of the acute phase [4,5,7]. In additional to these responses, high intracellular calcium concentrations built up during the acute phase lead to the overactivation of several proteolytic enzyme systems.
Recent studies have focused on the role of superoxide generating systems in immune cells and their consequences on reperfusion injury (RI), or that injury which results when the occluded vessel is reopened. Of the various systems by which cells can generate reactive oxygen species (ROS), the topic of this review will focus on superoxide produced by activation of the NADPH oxidase (NOX). Superoxide is soluble in solution and can combine with other ROS to form more toxic molecules after experimental ischemic stroke . The importance of NOX in production of ROS and subsequent neuronal cell death has been the subject of somewhat recent investigations. Several other enzymes found throughout the cell are involved in ROS generation, including xanthine oxidase, lipoxygenase, cylcoocxygenases (COX) and substrate coupled nitric oxide synthetase, but none of these can produce the large amount of ROS observed in nonphagocytic cells in both normal and pathological conditions . In the last decade, study into the NOX enzyme has made a major new turn with discovery of a large number of NOX homologs which may allow for a more targeted therapeutic approach. At present, no specific treatments exist for NOX induced ROS in central nervous system (CNS) diseases and most of these diseases have very few therapeutic options at all. This will be discussed in more detail in this review.
Oxidative stress leads to overload of ROS caused by excessive ROS production and/or impaired ROS reduction. Under conditions of oxidative stress, the overproduction of ROS becomes detrimental and possibly contributes to various acute and chronic diseases, such as myocardial infarction, hypertension, atherosclerosis, cancer, neurodegeneration, and inflammation. Consequently, oxidative stress has also been suggested as an important underlying mechanism of ischemic stroke .
Among many enzymes utilizing molecular oxygen as substrate, NOX is activated and can serve as a source of ROS in the CNS . Seven NOX family members have been identified and isoforms are denoted as NOX1 to NOX5, dual oxidase (Duox)-1 and -2. NOX1, 2 and 4 have been detected in the CNS, with cells such as neurons and intracranial vessels as well as microglia [12,13]. NOX3 is almost exclusively expressed in the inner ear but not in blood vessels. Gene deletion of NOX5 in the rodent genome is the least functionally characterized isoform compared to others . Nevertheless, several recent studies show that NOX5 is activated by calcium in human cells. Thus, it may be a key player in ROS generation in vascular cells [15,16]. Duox-1 and -2, also contain an oxidase domain, which appear to be expressed predominantly in epithelial cells . Recently,
NOX isoforms have been reported in the brain and its vasculature. Thus, cerebrovascular disease may benefit from NOX as a therapeutic target in the brain, as well as in its blood vessels. Recently, NOX may be a key player in the prevention of RI, an increasingly observed complication of delayed recanalization leading to brain edema or hemorrhage [23,24]. Thus, therapies to minimize RI might expand populations of stroke patients eligible for treatment. RI begins after ischemic stroke when the brain is quickly exposed to oxygenated blood, and ischemic mitochondria within the brain are rendered incapable of detoxifying reactive species .
A robust inflammatory reaction characterized by peripheral leukocyte influx into the cerebral parenchyma and activation of endogenous microglia follows ischemic stroke. While immune cells generate ROS through several enzyme systems, NOX is the major enzyme that generates superoxide in these cells. ROS elaborated by these cells can then activate other cells, even ischemic neurons, to secrete inflammatory factors. How NOX is activated in stroke is not entirely clear, but phosphorylation of the 47phox subunit appears key. This phosphorylation can occur through several kinases also upregulated and activated by brain ischemia, including several protein kinase C isoforms and the p38 and p21 been described , phagocytic NOX, also referred to as NOX2, is associated with immune cells. While NOX2 has been described in various cell types, NOX2 expression is greatest in myeloid cells , which, in the case of brain, are microglia . A previous study showed that microglial cells isolated from the human fetal brain expressed Nox2, p22phox, p40phox, p47phox, and p67phox transcripts . A large number of studies also demonstrated superoxide generating NOX systems in immune cells and their consequences on RI. Thus, inhibition of NOX can potentially reduce the amount of superoxide generated, and thus limit RI. This approach has the potential to not only treat acute ischemic stroke, but also reduce complications of recanalizing strategies by using it in combination with thrombolytics or mechanical thrombectomy devices.
NOX expression has been shown to increase in the brain after experimental stroke [29,30], and several groups have shown that mice deficient in the gp91phox subunit had smaller infarcts than wildtype mice [8,31,32,33,34]. Further, NOX appears to play a significant role in RI. While RI has been attributed to the restoration of blood oxygen leading to a surge in ROS, some studies indicate that the source of damaging ROS may, in fact, be due to the return of blood glucose, which then fuels NOX to generate more ROS. In this case, NOX serves as an electron donor to produce damaging levels of superoxide, and NOX activation is dependent on glucose . NOX also appears to be a primary source of ROS generated by NMDA receptor activation, thus suggesting another mechanism of ROS generation .
The targeting of NOX does not need to be restricted to the brain itself. While NOX and its isoforms are certainly present in microglia and other brain cell types, NOX present on circulating leukocytes contributes substantially to stroke pathology . In a prior study by our lab, the use of bone marrow chimeras allowed us to replace marrow derived cells lacking functional NOX2 into wild-type mice, and vice versa, replacing marrow with intact NOX2 into NOX2 deficient mice. After subjecting these chimeras to strokes, mice lacking circulating NOX2 but intact brain NOX2 had better outcomes compared to mice with intact circulating NOX2, but deficient brain NOX2. Vascular NOX is another relevant target. Aged mice have been found to have dysregulated cerebrovascular responses compared to similar aged mice deficient in NOX . Vascular NOX also appears to contribute to cerebrovascular dysfunction and behavioral deficits in models of Alzheimer's disease [38,39]. Thus, the development of NOX inhibiting compounds may not necessary have to penetrate the blood brain barrier (BBB) in order to be effective against stroke and related conditions.
Several pharmacological compounds have been studied to target NOX enzymes in ischemic stroke. However, these are generally very low in specificity and selectivity. There has not been much clinical development since many are associated with toxicities that could hinder their use in humans. Yet, safer and more specific inhibitors are under development.
DPI has been shown to protect the brain from stroke by blocking NOX . It impairs NOX via flavoprotein inhibition to prevent superoxide production. Intravenous DPI administration reduced magnetic resonance imaging detected ischemic lesion size in an experimental stroke model suggesting that it is capable of penetrating the BBB. Further, it was shown to decrease expression of extracellular matrix proteins MMP-2 and MMP-9, proteins known to contribute to BBB disruption . In a hypoxia model, DPI also improved the integrity of tight-junctions in the BBB by reducing ROS production . DPI treatment has also been associated with the inhibition of the inflammatory response accompanying stroke . However, DPI's usefulness is limited because it can also inhibit other flavoenzymes
Perhaps the most widely NOX inhibitor in experimental stroke is apocynin, also known as acetovanillone. Apocynin is a naturally occurring organic compound, originally derived from the root of Canadian hemp (
The effect of apocynin has been widely explored in experimental ischemic stroke. It has also been documented to penetrate into the brain following systemic delivery , and several groups have shown the salutary effects of treatment on lesion size, brain hemorrhage reduction and improvements in neurological deficits. Previous our lab reported that a dose of 2.5 mg/kg given just prior to reperfusion, or 1.5 hours after ischemic stroke, resulted in reduced infarct size and improved neurological outcome while markedly suppressing superoxide generation in the brain . However, at higher doses (3.75 and 5 mg/kg), apocynin showed no benefit, and actually increased the severity of brain hemorrhage. Thus, this rather narrow therapeutic dose range may limit its translation to the clinical level. However, other groups have shown salutary effects of apocynin at doses as high as 40~50 mg/kg [31,55], while another group found no effect of apocynin using a dose of about 4 mg/kg . In global cerebral ischemia, 5 mg/kg apocynin attenuated hippocampal injury when given prior to ischemia onset . In safety studies of uninjured mice, apocynin was well tolerated in single oral doses of up to 1000 mg/kg . Interestingly, apocynin failed to have any further beneficial effect in NOX2 deficient mice subjected to stroke, and might speak somewhat to its specificity or its dependence on NOX2 . In addition to apocynin, Altered NOX function has been linked to neurological disorders such as Alzheimer's (ALS) and Parkinson's diseases (PD) . Apocynin has been effective in ameliorating neuropathological damages in both
Other natural compounds of NOX inhibitor are honokiol and plumbagin. Honokiol was isolated from the herb Magnolia officinalis and has been of particular interest because this compound appears to inhibit superoxide production after the respiratory burst and not before the enzyme is activated as with other inhibitors . Honokiol has also been shown to lesion size in experimental ischemic stroke followed by reperfusion and this decrease was correlated to a reduction in neutrophil infiltration and activation, and decreased lipid peroxidation [65,66]. The only inhibitor to date that directly interacts with a specific NOX homolog seems to be a plant derived naphthoquinone called plumbagin . Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a natural yellow pigment that comes from the roots of the black walnut plant Plumbago zeylanica. Plumbagin inhibits non-phagocytic NOX activity in HEK293 and LN229, a cell line that only express NOX4 and in a cell line transfected for NOX4 . The regulation of NOX4 appears to be different from the other NOX homologs which require p47phox and p67phox, and it has been observed that NOX4 alone can produce superoxide activity [68,69,70]. The method by which it inhibits NOX4 is unknown but it is unlikely that it is due to cytotoxic effects as the cells were viable after 1 hour incubation with plumbagin . It has been shown to have significant anti-cancer activity and may work by blocking superoxide production as many cancers have been shown to produce ROS and specifically express NOX homologs [71,72,73,74]. NOX4 is the dominant NOX homolog in vascular smooth muscle cells and its inhibition by plumbagin may well explain its anti-atheroscerotic effect.
The most selective NOX inhibitor to date is a chimeric peptide gp91ds-tat . This peptide is constructed from the sequence of gp91phox that is known to be involved in the binding of gp91phox to p47phox and can inhibit the oxygen radical production in cell-free assays (gp91 docking sequence or gp91ds). In order to deliver this peptide into the cells, the gp91ds was linked to HIV coat peptide (HIVtat) that is known to be involved in internalization . This gp91ds-tat specifically binds to p47phox and prevents the formation of the NOX complex. While this is the most specific inhibitor for NOX, it cannot distinguish between the phagocytic or non-phagocytic enzyme and it has little oral bioavailability as it is a peptide.
One of a group of NOX inhibitors is the 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) which an irreversible serine protease inhibitor . AEBSF appears to have a direct effect on the plasma membrane components of NOX and interrupt with the binding of the cytosolic components p47phox and p67phox. AEBSF does not interfere with the electron transport and does not scavenge the oxygen radicals. Unfortunately, AEBSF modifies many proteins by covalent attachment preferentially on tyrosine, and to a lesser extent on lysine, histidine, and the amino-terminus. AEBSF is quite stable in aqueous solution and the extent to which the protein is modified continues to increase for several days and this significantly limits its use
Other NOX inhibitors include phenylarsine oxide and gliotoxin . Phenylarsine oxide, via its interaction with cysteine residues, appears to prevent NADPH enzyme assembly. However, this inhibitor is quite non-specific. Due to its interactions with the cysteine groups it will also interact with other enzymes and proteins [79,80]. Gliotoxin is extracted from Aspergillus and it appears to inhibit the enzyme by blocking the phosphorylation of p47phox [53,81]. It also seems to react with the thiol groups, thus limiting its specificity. To date, there do not appear to be any studies examining the therapeutic value of these compounds in stroke or stroke models.
There are NOX inhibitors that have either been specifically developed by the pharmaceutical industry or are in clinical trials. Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, a mimic of glutathione peroxidase which also reacts with peroxynitrite, inhibits a variety of enzymes such as lipoxygenases, nitric oxide synthases, NOX, protein kinase C, and Hz/Kz-ATPase . Ebselen is therefore quite non-specific, but is being used at some centers for the treatment of stroke in Japan. Ebselen has shown efficacy if the treatment is started within 24 hours of the stroke [83,84]. Currently, a multicenter phase 3 ebselen trial is ongoing (www.strokecenter.org).
A few other inhibitors have been developed by the pharmaceutical industry including VAS2870 (Vasopharm) [56,85]. The VAS2870 has been shown to be neuroprotective in preclinical stroke experiments . It appears to a more specific to inhibitor of all NOX isoforms, and is neither general flavoprotein inhibitor nor ROS scavengers [85,86]. VAS2879 appears to be a pan-NOX inhibitor, as it prevents assembly or conformational change to active NOX complexes [87,88]. When VAS2870 was admitted before complex assembly induction, it inhibited a NOX2 activity . Therefore, the order by which a NOX complex assembly activator and a NOX inhibitor are added in cell-free assays is crucial. A recent study showed that VAS2870 has potential off-target effects, suggesting that the compound alkylates cysteine thiol residues . To date, there has been only one study examining this compound in a stroke model . In this study, VAS2870 treatment led to superior lesion size reduction and improvement in neurological deficits compared to no treatment.
tensin receptor-1 blockers (ARBs), and the HMGCoA reductase inhibitor (statins) drugs can all indirectly inhibit NOX activity [86,90]. Statins, ACE inhibitors and ARBs have all been shown to protect the brain from experimental stroke for various reasons. In particular, statins have been shown to improve outcome from experimental stroke via their ability to upregulate endothelial nitric oxide synthase . All of these compounds have been shown to correlate to an anti-inflammatory effect, although whether this is directly due to NOX inhibition seems less likely, with these compounds acting as a related immune target.
In conclusion, NOX appear to an important player in ischemic stroke, particularly as it relates to reperfusion injury due to its generation of superoxide. Superoxide is well known to contribute to the progression of stroke and related cerebrovascular diseases as well as neurodegenerative conditions. By inhibiting NOX, several preclinical studies have reported improved neurological outcome, and reduced severity of brain injury including brain hemorrhage. While available inhibitors may be non-specific or have a relatively narrow therapeutic window, we suggest that it is still worthwhile to investigate NOX inhibitors, and to carry out studies to develop safe and selective drugs for treatment of clinical stroke, its complications and for related conditions as well. Further, there is rapidly increasing knowledge of the role of the various NOX isoforms. Whether one form versus another is relevant to one type of pathology compared to another is still unknown. Such knowledge could also drive the development of appropriate therapies as well. Clearly, several laboratory and preclinical animal model studies have shown the salutary effects of several NOX isoforms as viable targets for treatment of ischemic stroke. While limiting elaboration of damaging ROS into the ischemic brain during reperfusion, its inhibition leads to the inhibition of the local inflammatory response and, as a result, may prevent secondary complications of stroke such as BBB disruption and brain hemorrhage. Since reperfusion injury, including hemorrhagic transformation and the development of a frank parenchymal hematoma, are significant complications of thrombolysis, NOX inhibitors have the potential to be used in conjunction with recombinant tissue plasminogen activator (rt-PA), in addition to being used as a stand alone neuroprotectant. However, pharmacological development of a specific and safe inhibitor is still lacking. Several inhibitors have been studied by various labs and, while shown to be effective in laboratory models, are either non-specific in their inhibition, or may have off target toxicities that could limit translational development. The issue of specificity may not be as critical, since several NOX isoforms have been shown to contribute to stroke pathophysiology, and off target effects, such as non-specific anti-oxidant properties, may contribute to neuroprotection. More importantly, it will be important to develop therapies with less toxicity. Finally, the issue of whether such compounds may pass the BBB may be less important compared to other therapies, since NOX is present on circulating leukoctyes, which could be targeted without requiring a NOX inhibitor to enter the brain.