Daniel N. Meijles

The Quest for Selective Nox Inhibitors and Therapeutics: Challenges, Triumphs and Pitfalls

SIGNIFICANCE Numerous studies in animal models and human subjects corroborate that elevated levels of reactive oxygen species (ROS) play a pivotal role in the progression of multiple diseases. As a major source of ROS in many organ systems, the NADPH oxidase (Nox) has become a prime target for therapeutic development. RECENT ADVANCES In recent years, intense efforts have been dedicated to the development of pan- and isoform-specific Nox inhibitors as opposed to antioxidants that proved ineffective in clinical trials. Over the past decade, an array of compounds has been proposed in an attempt to fill this void. CRITICAL ISSUES Although many of these compounds have proven effective as Nox enzyme family inhibitors, isoform specificity has posed a formidable challenge to the scientific community. This review surveys the most prominent Nox inhibitors, and discusses potential isoform specificity, known mechanisms of action, and shortcomings. Some of these inhibitors hold substantial promise as targeted therapeutics. FUTURE DIRECTIONS Increased insight into the mechanisms of action and regulation of this family of enzymes as well as atomic structures of key Nox subunits are expected to give way to a broader spectrum of more potent, efficacious, and specific molecules. These lead molecules will assuredly serve as a basis for drug development aimed at treating a wide array of diseases associated with increased Nox activity.

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production

Background: p47phox is a regulatory subunit of NADPH oxidase, which produces superoxide. Results: We propose a molecular dynamics model of p47phox phosphorylation and assembly with p22phox in NADPH oxidase activation supported by biochemical experiments. Conclusion: Ser-379 phosphorylation in the C-terminal tail is a molecular switch in p47phox activation. Significance: This report provides novel structural and mechanistic information of p47phox activation. 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 p47phox cDNA transfected 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-derived ROS in PPARγ signaling and cell-cycle progression of lung alveolar epithelial cells.

Reactive oxygen species (ROS) play important roles in peroxisome proliferator-activated receptor γ (PPARγ) signaling and cell-cycle regulation. However, the PPARγ redox-signaling pathways in lung alveolar epithelial cells remain unclear. In this study, we investigated the in vivo and in vitro effects of PPARγ activation on the levels of lung ROS production and cell-cycle progression using C57BL/6J wild-type and Nox2 knockout mice (n=10) after intraperitoneal injection of a selective PPARγ agonist (GW1929, 5 mg/kg body wt, daily) for 14 days. Compared to vehicle-treated mice, GW1929 increased significantly the levels of ROS production in wild-type lungs, and this was accompanied by significant up-regulation of PPARγ, Nox2, PCNA, and cyclin D1 and phosphorylation of ERK1/2 and p38MAPK. These effects were absent in Nox2 knockout mice. In cultured alveolar epithelial cells, GW1929 (5 μM for 24 h) increased ROS production and promoted cell-cycle progression from G0/G1 into S and G2/M phases, and these effects were abolished by (1) adding a PPARγ antagonist (BADGE, 1 μM), (2) knockdown of PPARγ using siRNA, or (3) knockout of Nox2. In conclusion, PPARγ activation through Nox2-derived ROS promotes cell-cycle progression in normal mouse lungs and in cultured normal alveolar epithelial cells.

Divergent Effects of p47phox Phosphorylation at S303-4 or S379 on Tumor Necrosis Factor-α Signaling via TRAF4 and MAPK in Endothelial Cells

Objectives—To define the mechanism of p47phox phosphorylation in regulating endothelial cell response to tumor necrosis factor-&agr; (TNF&agr;) stimulation. Methods and Results—We replaced 11 serines (303-4, 310, 315, 320, 328, 345, 348, 359, 370, and 379) with alanines and investigated their effects on TNF&agr; (100 U/mL, 30 minutes)–induced acute O2.− production and mitogen-activated protein kinase phosphorylation in endothelial cells. Seven constructs, S303-4A (double), S310A, S315A, S328A, S345A, S370A, and S379A, significantly reduced the O2.− production, and 4 of them (S328A, S345A, S370A, and S379A) also inhibited TNF&agr;-induced extracellular-signal–regulated kinase (ERK) 1/2 phosphorylation. Blocking the phosphorylation of S303-4 and S379 inhibited most effectively TNF&agr;-induced O2.− production. However, phosphorylation of S303-4 was not required for TNF&agr;-induced p47phox membrane translocation and binding to TNF receptor–associated factor 4, ERK1/2 activation, and subsequent vascular cell adhesion molecule-1 expression. Knockout of p47phox or knockdown of TNF receptor–associated factor 4 using siRNA abolished TNF&agr;-induced ERK1/2 phosphorylation, and inhibition of ERK1/2 activation significantly reduced the TNF&agr;-induced vascular cell adhesion molecule-1 expression. Conclusion—Phosphorylation of p47phox at different serine sites plays distinct roles in endothelial cell response to TNF&agr; stimulation. Double serine (S303-4) phosphorylation is crucial for acute O2.− production, but is not involved in TNF&agr; signaling through TNF receptor–associated factor 4 and ERK1/2. p47phox requires serine phosphorylation at distinct sites to support specific signaling events in response to TNF&agr;.

Selective inactivation of NADPH oxidase 2 causes regression of vascularization and the size and stability of atherosclerotic plaques

BACKGROUND A variety of NADPH oxidase (Nox) isoforms including Noxs 1, 2, 4 and 5 catalyze the formation of reactive oxygen species (ROS) in the vascular wall. The Nox2 isoform complex has arguably received the greatest attention in the progression of atherogenesis in animal models. Thus, in the current study we postulated that specific Nox2 oxidase inhibition could reverse or attenuate atherosclerosis in mice fed a high-fat diet. METHODS We evaluated the effect of isoform-selective Nox2 assembly inhibitor on the progression and vascularization of atheromatous plaques. Apolipoprotein E-deficient mice (ApoE-/-) were fed a high fat diet for two months and treated over 15 days with Nox2ds-tat or control sequence (scrambled); 10 mg/kg/day, i.p. Mice were sacrificed and superoxide production in arterial tissue was detected by cytochrome C reduction assay and dihydroethidium staining. Plaque development was evaluated and the angiogenic markers VEGF, HIF1-α and visfatin were quantified by real time qRT-PCR. MMP-9 protein release and gelatinolytic activity was determined as a marker for vascularization. RESULTS Nox2ds-tat inhibited Nox-derived superoxide determined by cytochrome C in carotid arteries (2.3 ± 0.1 vs 1.7 ± 0.1 O2(•-) nmol/min*mg protein; P < 0.01) and caused a significant regression in atherosclerotic plaques in aorta (66 ± 6 μm(2) vs 37 ± 1 μm(2); scrmb vs. Nox2ds-tat; P < 0.001). Increased VEGF, HIF-1α, MMP-9 and visfatin expression in arterial tissue in response to high-fat diet were significantly attenuated by Nox2ds-tat which in turn impaired both MMP-9 protein expression and activity. CONCLUSION Given these results, it is quite evident that selective Nox inhibitors can reverse vascular pathology arising with atherosclerosis.

Nox and Inflammation in the Vascular Adventitia

For nearly half a century, strong associations have been made linking early adventitial activation in disease with endothelial dysfunction,1,2 thus challenging the notion that the luminal endothelium is the initial sensor and propagator of cardiovascular disease. Over the past 2 decades, perturbational studies have established a causal role of adventitial factors in this regard. This review-based update acts as a springboard for a revitalized debate of the adventitia’s role in vascular disease. From a pathological perspective, the adventitia reportedly plays a deleterious role via production of large amounts of nicotinamide adenine dinucleotide phosphate oxidase (Nox)-derived reactive oxygen species (ROS) in response to vascular injury and disease.3,4 A combined adventitial fibroblast and inflammatory cell infiltration facilitated by the vasa vasorum (VV) is expected to synergize and propagate vessel inflammation.5 In fact, Noxes are highly activated by inflammatory cytokines, hormones, lipids, and angiotensin II (Ang II).6 In the adventitia, Nox-derived ROS is the dominant culprit for intracellular redox signaling cascade activation, either in an autocrine or in a paracrine manner, leading to subsequent vascular cell activation, proliferation, hypertrophy, or apoptosis and thereby promoting vascular diseases including hypertension and atherosclerosis.7 Despite these discoveries, studies delving into the role of the adventitia in disease propagation or progression remain limited. This gap in knowledge presents a relatively untapped opportunity to study vascular disease pathogenesis in a renewed light. This focused mini-review addresses the role of adventitial Nox-derived ROS from a perivascular viewpoint looking inwards to promoting vascular inflammation and disease. Forming the outermost layer of the vessel wall, the adventitia is emerging as a prominent channel for the progression of vascular remodeling. Within the adventitial milieu, adventitial fibroblasts/myofibroblasts, lymphocytes, stem cell–like vascular and hematopoietic lineage progenitors, and endothelial cells reside.8–10 To date, the …

Consensus in silico computational modelling of the p22phox subunit of the NADPH oxidase

The p22(phox) protein is an essential subunit of the cytochrome b(558) of the NADPH oxidase (Nox) complex which by generating reactive oxygen species (ROS) plays important role in regulating cellular function. p22(phox) stabilises the Nox enzyme, assists in catalytic core maturation and in the meantime provides an anchoring site for cytosolic regulatory subunits to bind. However, the protein structure of the p22(phox) is still uncertain. In this study we use an in silico computational bioinformatic approach to produce a consensus 3-dimensional model of the p22(phox). Based on published protein sequence data of human p22(phox) and by using transmembrane specific protein prediction algorithms, we found that p22(phox) consists of two domains: an N-terminal transmembrane domain (124 a.a.) and a C-terminal cytoplasmic domain (71 a.a.). In its predicted most stable form, p22(phox) contains three transmembrane helices leading to an extracellular N-terminus and an extensive (39 a.a.) extracellular loop between helices 2 and 3. Furthermore, we locate the cytosolic domain phosphorylation site at threonine(147) which literature shows is capable of priming the p22(phox), in order to accept its binding partners. Our results are consistent with the biological characterisation of p22(phox) derived from experiments using specific antibody or genetic manipulation. Our 3-D protein model provides insights into the biological function of p22(phox) and cytochrome b(558), and can be used as tool to investigate the regulatory mechanism of Nox isoforms.

Endothelial Nox1 oxidase assembly in human pulmonary arterial hypertension; driver of Gremlin1-mediated proliferation

Pulmonary arterial hypertension (PAH) is a rapidly degenerating and devastating disease of increased pulmonary vessel resistance leading to right heart failure. Palliative modalities remain limited despite recent endeavors to investigate the mechanisms underlying increased pulmonary vascular resistance (PVR), i.e. aberrant vascular remodeling and occlusion. However, little is known of the molecular mechanisms responsible for endothelial proliferation, a root cause of PAH-associated vascular remodeling. Lung tissue specimens from PAH and non-PAH patients and hypoxia-exposed human pulmonary artery endothelial cells (ECs) (HPAEC) were assessed for mRNA and protein expression. Reactive oxygen species (ROS) were measured using cytochrome c and Amplex Red assays. Findings demonstrate for the first time an up-regulation of NADPH oxidase 1 (Nox1) at the transcript and protein level in resistance vessels from PAH compared with non-PAH patients. This coincided with an increase in ROS production and expression of bone morphogenetic protein (BMP) antagonist Gremlin1 (Grem1). In HPAEC, hypoxia induced Nox1 subunit expression, assembly, and oxidase activity leading to elevation in sonic hedgehog (SHH) and Grem1 expression. Nox1 gene silencing abrogated this cascade. Moreover, loss of either Nox1, SHH or Grem1 attenuated hypoxia-induced EC proliferation. Together, these data support a Nox1-SHH-Grem1 signaling axis in pulmonary vascular endothelium that is likely to contribute to pathophysiological endothelial proliferation and the progression of PAH. These findings also support targeting of Nox1 as a viable therapeutic option to combat PAH.

p22phox C242T Single-Nucleotide Polymorphism Inhibits Inflammatory Oxidative Damage to Endothelial Cells and Vessels.

Background— The NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A single-nucleotide polymorphism, C242T of the p22phox subunit of NADPH oxidase, has been reported to be negatively associated with coronary heart disease and may predict disease prevalence. However, the underlying mechanisms remain unknown. Methods and Results— With the use of computer molecular modeling, we discovered that C242T single-nucleotide polymorphism causes significant structural changes in the extracellular loop of p22phox and reduces its interaction stability with Nox2 subunit. Gene transfection of human pulmonary microvascular endothelial cells showed that C242T p22phox significantly reduced Nox2 expression but had no significant effect on basal endothelial O2 .– production or the expression of Nox1 and Nox4. When cells were stimulated with tumor necrosis factor-&agr; (or high glucose), C242T p22phox significantly inhibited tumor necrosis factor-&agr;–induced Nox2 maturation, O2 .– production, mitogen-activated protein kinases and nuclear factor &kgr;B activation, and inflammation (all P<0.05). These C242T effects were further confirmed using p22phox short-hairpin RNA–engineered HeLa cells and Nox2–/– coronary microvascular endothelial cells. Clinical significance was investigated by using saphenous vein segments from non–coronary heart disease subjects after phlebotomies. TT (C242T) allele was common (prevalence of ≈22%) and, in comparison with CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O2 .– generation in response to high-glucose challenge. Conclusions— C242T single-nucleotide polymorphism causes p22phox structural changes that inhibit endothelial Nox2 activation and oxidative response to tumor necrosis factor-&agr; or high-glucose stimulation. C242T single-nucleotide polymorphism may represent a natural protective mechanism against inflammatory cardiovascular diseases.

MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension

Background Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis. Methods and Results In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis. Conclusions Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH.