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Inhibition of NOX4 reduced NOX4 expression and H2O2 generation in late EPCs. (A) Inhibition of NOX4 gene using NOX4 RNAi markedly reduced NOX4 mRNA expression in EPCs. (B) Lack of NOX4 decreased NOX4 protein level when compared to control in EPCs using a specific NOX4 antibody as shown in a representative Western Blot. (C) H2O2 generation was reduced after NOX4 inhibition using Amplex red. Values (means ± SEM; n = 3–5), *P < 0.05 from Ad-GFP control following a Student's paired t-test.
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Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. Howev...
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... Furthermore, our new data indicated that pro-angiogenic effects of NOX4 signalling in this setting were mediated via stabilisation of Nrf2 as an established downstream target ( Figure 3C-E). Indeed, Nrf2 KD was reported to inhibit endothelial cell migration and angiogenesis in hypoxia [34], functions which are promoted in endothelial progenitor cells by induction of NOX4-derived hydrogen peroxide and associated modulation of inflammation [35]. However, perhaps the most novel finding of the current study was identification of PLAC8 as a negative regulator of NOX4-mediated angiogenic signalling in hypoxic CB-ECFCs. ...
Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8–NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.
... NOX4-derived H 2 O 2 in part stimulates NOX2 to increase mitochondrial ROS via pSer36-p66Shc, thereby increasing VEGFR-2 signaling and angiogenesis in endothelial cells (19). NOX4-derived H 2 O 2 also stimulates endothelial progenitor cells (20). ...
... Expression of NOX4 mRNA was 1.6-fold (p = 0.02) and H 2 O 2 release 1.6-fold (p = 0.049) higher in samples of patients with asymptomatic CAS, when compared to symptomatic CAS ( Fig. 1A and B). As pointed out above, NOX4 mRNA expression is strongly correlated with NOX4 protein expression and H 2 O 2 release [16]. Surprisingly, here H 2 O 2 release was not directly correlated to NOX4 mRNA expression (Fig. 1D). ...
Carotid artery stenosis (CAS) develops from atherosclerotic lesions and plaques. Plaque rupture or stenosis may result in occlusion of the carotid artery. Accordingly, the asymptomatic disease becomes symptomatic, characterized by ischemic stroke or transient ischemic attacks, indicating an urgent need for better understanding of the underlying molecular mechanisms and eventually prevent symptomatic CAS. NOX4, a member of the NADPH oxidase family, has anti-atherosclerotic and anti-inflammatory properties in animal models of early atherosclerosis. We hypothesized that NOX4 mRNA expression is linked to protective mechanisms in CAS patients with advanced atherosclerotic lesions as well. Indeed, NOX4 mRNA expression is lower in patients with symptomatic CAS. A low NOX4 mRNA expression is associated with an increased risk of the development of clinical symptoms. In fact, NOX4 appears to be linked to plaque stability, apoptosis and plaque hemorrhage. This is supported by cleaved caspase-3 and glycophorin C and correlates inversely with plaque NOX4 mRNA expression. Even healing of a ruptured plaque appears to be connected to NOX4, as NOX4 mRNA expression correlates to fibrous cap collagen and is reciprocally related to MMP9 activity. In conclusion, low intra-plaque NOX4 mRNA expression is associated with an increased risk for symptomatic outcome and with reduced plaque stabilizing mechanisms suggesting protective effects of NOX4 in human advanced atherosclerosis.
... According to the data registered in Table 3, it can be observed that the low WSS generates the down-regulation in the expression of eNOS (endothelial nitric oxide synthase), the enzyme responsible for the NO release and the maintenance of the overall homeostasis in the vascular wall [52]. In addition, it generates the down-regulation of NOTCH1 (Neurogenic locus notch homolog protein 1), which is a mechanical sensor that maintains the junctional integrity of endothelium [53], and NOX 4 (NADPH oxidase 4), which produces H 2 O 2 as a signaling molecule for endothelial cell proliferation [54]. Without the expression of these genes, it is likely improbable that the endothelial lining can be regenerated over the TEVG surface. ...
Vascular grafts (VGs) are medical devices intended to replace the function of a blood vessel. Available VGs in the market present low patency rates for small diameter applications setting the VG failure. This event arises from the inadequate response of the cells interacting with the biomaterial in the context of operative conditions generating chronic inflammation and a lack of regenerative signals where stenosis or aneurysms can occur. Tissue Engineered Vascular grafts (TEVGs) aim to induce the regeneration of the native vessel to overcome these limitations. Besides the biochemical stimuli, the biomaterial and the particular micro and macrostructure of the graft will determine the specific behavior under pulsatile pressure. The TEVG must support blood flow withstanding the exerted pressure, allowing the proper compliance required for the biomechanical stimulation needed for regeneration. Although the international standards outline the specific requirements to evaluate vascular grafts, the challenge remains in choosing the proper biomaterial and manufacturing TEVGs with good quality features to perform satisfactorily. In this review, we aim to recognize the best strategies to reach suitable mechanical properties in cell-free TEVGs according to the reported success of different approaches in clinical trials and pre-clinical trials.
... Furthermore, induction of hindlimb ischaemia in mice lacking Nox2 has been associated with a substantial reduction in the number of circulating EPCs as well as with an impaired vascular regeneration (Urao et al. 2008;Schröder et al. 2009). Inhibition of other NADPH oxidase isoforms that have relevance to vascular events, notably that of Nox4, is also implicated in increased rate of cellular death induced by pro-inflammatory cytokine, TNF-α and diminished proliferative and migratory capacity of EPCs (Hakami et al. 2017). Despite suppression of overall NADPH oxidase activity by relatively specific wide-spectrum inhibitors, including VAS2870 or DPI, has largely been linked with diminished hypoxia-mediated EPCs dysfunctions (Liu et al. 2017), reports showing increases in migratory and tube-forming capacity of OECs upon stimulation of PKC signalling pathway by phorbol myristate acetate (PMA, 10 nM) also exist. ...
... These findings imply that OECs may cope better with the oxidative stress induced by the ischaemia-reperfusion injury and therefore may make effective therapeutics to mitigate post-stroke barrier damage to maintain neurovascular homeostasis. Although suppression of NADPH oxidase by specific targeting of Nox2 or Nox4 subunit has been correlated with significant decreases in ROS level in EPCs (Urao et al. 2008;Schröder et al. 2009;Hakami et al. 2017), the inhibition of PKC-β in the current study led to dramatic increases in NADPH oxidase activity and superoxide anion release. It is possible that the inhibition of PKC-β may completely neutralise the minimal basal oxidant activity and superoxide anion generation in OECs and may, as a consequence, trigger a negative feedback mechanism to compensate these changes in OECs. ...
Outgrowth endothelial cells (OECs) provide an endogenous repair mechanism and thus maintain endothelial barrier integrity. As inhibition of protein kinase C-β (PKC-β) activity has been shown to attenuate endothelial damage in various pathological conditions including hyperglycaemia and ischaemic injury, the present study comparatively assessed the effect of LY333531, a PKC-β inhibitor, on the cerebral barrier integrity formed by OECs or human brain microvascular endothelial cells (HBMECs). To this end, an in vitro model of human BBB established by co-culture of astrocytes and pericytes with either OECs or HBMECs was exposed to 4 h of oxygen-glucose deprivation with/out LY333531 (0.05 µM). The inhibition of PKC-β protected the integrity and function of the BBB formed by HBMECs, as evidenced by increases in transendothelial electrical resistance and decreases in sodium fluorescein flux. It also attenuated ischaemia-evoked actin cytoskeleton remodelling, oxidative stress, and apoptosis in HBMECs. In contrast, treatments with LY333531 exacerbated the deleterious effect of ischaemia on the integrity and function of BBB formed by OECs while augmenting the levels of oxidative stress, apoptosis, and cytoskeletal reorganisation in OECs. Interestingly, the magnitude of damage in all aforementioned parameters, notably oxidative stress, was lower with low dose of LY333531 (0.01 µM). It is therefore possible that the therapeutic concentration of LY333531 (0.05 µM) may neutralise the activity of NADPH oxidase and thus trigger a negative feedback mechanism which in turn exacerbate the detrimental effects of ischaemic injury. In conclusion, targeting PKC-β signalling pathway in ischaemic settings requires close attention while using OECs as cellular therapeutic.
... Nonconditioned starvation medium (DMEM, 1 g l À1 glucose, 0.1% FBS) and endothelial growth media (EGM, Lonza) were used as negative and positive controls, respectively. Since endothelial cells are sensitive to NOX4-inhibition [37], data with CM collected from TREiNOX treatments were analyzed separately from that of medium from untreated cells, and plain EGM supplemented with TREiNOX was used as the experimental control. Assay quantification was performed using the Angiogenesis Analyzer Plugin of the ImageJ Software (NIH, Bethesda, MD, USA) on randomly captured images with a 4Âobjective on a Nikon Eclipse TI inverted microscope (Nikon, Tokyo, Japan). ...
Cardiac stromal cells (CSCs) are the main players in fibrosis. Dysmetabolic conditions (metabolic syndrome ‐ MetS, and type 2 diabetes ‐ DM2) are strong pathogenetic contributors to cardiac fibrosis. Moreover, modulation of the oxidative state (OxSt) and autophagy is a fundamental function affecting the fibrotic commitment of CSCs, that are adversely modulated in MetS/DM2. We aimed to characterize CSCs from dysmetabolic patients, and to obtain a beneficial phenotypic setback from such fibrotic commitment by modulation of OxSt and autophagy. CSCs were isolated from 38 patients, stratified as MetS, DM2, or controls. Pharmacological modulation of OxSt and autophagy was obtained by treatment with trehalose and NOX4/NOX5 inhibitors (TREiNOX). Flow‐cytometry and RT‐qPCR analyses showed significantly increased expression of myofibroblasts markers in MetS‐CSCs at baseline (GATA4, ACTA2, THY1/CD90) and after starvation (COL1A1, COL3A1). MetS‐ and DM2‐CSCs displayed a paracrine profile distinct from control cells, as evidenced by heatmap analysis of 30 secreted cytokines, with significant reduction in VEGF and endoglin confirmed by ELISA. DM2‐CSCs showed significantly reduced support to endothelial cells in angiogenic assays, and significantly increased H2O2 release and NOX4/5 expression levels. Autophagy impairment after starvation (reduced ATG7 and LC3‐II proteins) was also detectable in DM2‐CSCs. TREiNOX treatment significantly reduced ACTA2, COL1A1, COL3A1, and NOX4 expression in both DM2‐ and MetS‐CSCs, as well as GATA4 and THY1/CD90 in DM2, all versus control cells. Moreover, TREiNOX significantly increased VEGF release by DM2‐CSCs, and VEGF and endoglin release by both MetS‐ and DM2‐CSCs, also recovering the angiogenic support to endothelial cells by DM2‐CSCs. In conclusion, DM2 and MetS worsen microenvironmental conditioning by CSCs. Appropriate modulation of autophagy and OxSt in human CSCs appears to restore these features, mostly in DM2‐CSCs, suggesting a novel strategy against cardiac fibrosis in dysmetabolic patients. This article is protected by copyright. All rights reserved.
... Indeed, an experimental model of RA revealed that NADPH oxidase is responsible for increased endothelial oxidative stress, and an in vitro administration of diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase activity, reduced the production of superoxide anions [41]. Furthermore, recent findings report that NOX4-type NADPH oxidase is important for proliferation, migration and apoptosis of EPCs [42], while a positive correlation between the NOX-mediated oxidative stress and the dysfunctions of circulating EPCs in dyslipidemia has been described, suggesting that a suppression of NOX might offer a novel strategy through which to improve EPC functions [42,43]. ...
... Indeed, an experimental model of RA revealed that NADPH oxidase is responsible for increased endothelial oxidative stress, and an in vitro administration of diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase activity, reduced the production of superoxide anions [41]. Furthermore, recent findings report that NOX4-type NADPH oxidase is important for proliferation, migration and apoptosis of EPCs [42], while a positive correlation between the NOX-mediated oxidative stress and the dysfunctions of circulating EPCs in dyslipidemia has been described, suggesting that a suppression of NOX might offer a novel strategy through which to improve EPC functions [42,43]. ...
Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disease characterized by the swelling of multiple joints, pain and stiffness, and accelerated atherosclerosis. Sustained immune response and chronic inflammation, which characterize RA, may induce endothelial activation, damage and dysfunction. An equilibrium between endothelial damage and repair, together with the preservation of endothelial integrity, is of crucial importance for the homeostasis of endothelium. Endothelial Progenitor Cells (EPCs) represent a heterogenous cell population, characterized by the ability to differentiate into mature endothelial cells (ECs), which contribute to vascular homeostasis, neovascularization and endothelial repair. A modification of the number and function of EPCs has been described in numerous chronic inflammatory and auto-immune conditions; however, reports that focus on the number and functions of EPCs in RA are characterized by conflicting results, and discrepancies exist among different studies. In the present review, the authors describe EPCs’ role and response to RA-related endothelial modification, with the aim of illustrating current evidence regarding the level of EPCs and their function in this disease, to summarize EPCs’ role as a biomarker in cardiovascular comorbidities related to RA, and finally, to discuss the modulation of EPCs secondary to RA therapy.
... Moreover, data from KO mice suggest that Nox4 might offer further protective benefits within the endothelium by promoting angiogenesis [144,145]. EC migration and proliferation is inhibited when the NOX4 gene is silenced; whilst EC with overexpression of NOX4 have increased angiogenesis in vitro [146,147]. Deletion of NOX4 in mice reduces H 2 O 2 production and inhibits EC tube formation. Moreover, the addition of low concentrations of H 2 O 2 restores EC tube formation-highlighting the important role of Nox4 derived H 2 O 2 in endothelial function [145]. ...
Oxidative stress within the vascular endothelium, due to excess generation of reactive oxygen species (ROS), is thought to be fundamental to the initiation and progression of the cardiovascular complications of type 2 diabetes mellitus. The term ROS encompasses a variety of chemical species including superoxide anion (O2•−), hydroxyl radical (OH−) and hydrogen peroxide (H2O2). While constitutive generation of low concentrations of ROS are indispensable for normal cellular function, excess O2•− can result in irreversible tissue damage. Excess ROS generation is catalysed by xanthine oxidase, uncoupled nitric oxide synthases, the mitochondrial electron transport chain and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Amongst enzymatic sources of O2•− the Nox2 isoform of NADPH oxidase is thought to be critical to the oxidative stress found in type 2 diabetes mellitus. In contrast, the transcriptionally regulated Nox4 isoform, which generates H2O2, may fulfil a protective role and contribute to normal glucose homeostasis. This review describes the key roles of Nox2 and Nox4, as well as Nox1 and Nox5, in glucose homeostasis, endothelial function and oxidative stress, with a key focus on how they are regulated in health, and dysregulated in type 2 diabetes mellitus.
... Results show that NOX4-derived ROS are peculiar for proliferation and migration functions of EPCs and counteract the detrimental effect induced by pro-inflammatory cytokine in EPCs. The authors suggested also that NOX4 could facilitate the efficient function of EPCs, leading to successful neovascularization [159]. NADPH oxidase, thanks to different localization and to the possibility of tuning the amount of ROS generated, represents a peculiar source of ROS for redox signaling related to endothelial cell differentiation. ...
One of the major sources of reactive oxygen species (ROS) generated within stem cells is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOXs), which are critical determinants of the redox state beside antioxidant defense mechanisms. This balance is involved in another one that regulates stem cell fate: indeed, self-renewal, proliferation, and differentiation are decisive steps for stem cells during embryo development, adult tissue renovation, and cell therapy application. Ex vivo culture-expanded stem cells are being investigated for tissue repair and immune modulation, but events such as aging, senescence, and oxidative stress reduce their ex vivo proliferation, which is crucial for their clinical applications. Here, we review the role of NOX-derived ROS in stem cell biology and functions, focusing on positive and negative effects triggered by the activity of different NOX isoforms. We report recent findings on downstream molecular targets of NOX-ROS signaling that can modulate stem cell homeostasis and lineage commitment and discuss the implications in ex vivo expansion and in vivo engraftment, function, and longevity. This review highlights the role of NOX as a pivotal regulator of several stem cell populations, and we conclude that these aspects have important implications in the clinical utility of stem cells, but further studies on the effects of pharmacological modulation of NOX in human stem cells are imperative.
... The transplantation of integrin β1overexpressing ECFCs restored the local vascular network by physically engrafting within neovessels and rescued local blood flow [230]. A recent investigation focused on NADPH oxidase 4 (NOX4) [233], which is constitutively active and promotes H2O2 production, thereby stimulating ECFC proliferation and migration [234]. NOX4 overexpression stimulated ECFC migration in vitro, albeit this effect was mediated by enhanced anion superoxide, rather than H2O2, production. ...
Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. In turn, circulating endothelial colony-forming cells (ECFCs) represent truly endothelial precursors, which display high clonogenic potential and have the documented ability to originate de novo blood vessels in vivo. Therefore, ECFCs are regarded as the most promising cellular candidate to promote therapeutic angiogenesis in patients suffering from CVD. The current briefly summarizes the available information about the origin and characterization of ECFCs and then widely illustrates the preclinical studies that assessed their regenerative efficacy in a variety of ischemic disorders, including acute myocardial infarction, peripheral artery disease, ischemic brain disease, and retinopathy. Then, we describe the most common pharmacological, genetic, and epigenetic strategies employed to enhance the vasoreparative potential of autologous ECFCs by manipulating crucial pro-angiogenic signaling pathways, e.g., extracellular-signal regulated kinase/Akt, phosphoinositide 3-kinase, and Ca 2+ signaling. We conclude by discussing the possibility of targeting circulating ECFCs to rescue their dysfunctional phenotype and promote neovascularization in the presence of CVD.
