bims-noxint Biomed News
on NADPH oxidases in tumorigenesis
Issue of 2022‒05‒15
eight papers selected by
Laia Caja Puigsubira
Uppsala University

  1. FASEB J. 2022 May;36 Suppl 1
      Previously, we have reported that exercise training enhances endothelium-dependent dilation in coronary arterioles distal to chronic occlusion, through increased contribution of the redox signaling molecule, hydrogen peroxide (H2 O2 ). In the present study, we tested the hypothesis that enhanced endothelium-dependent dilation in collateral-dependent arterioles after exercise training would be attributable to an increased contribution of NADPH oxidase (NOX) 4-derived reactive oxygen species. Yucatan miniature swine were surgically instrumented with an ameroid constrictor around the proximal left circumflex artery, which gradually induced occlusion of the artery and created a collateral-dependent vascular bed. Eight weeks postoperatively, swine were assigned to either a sedentary protocol consisting of normal pen activity or a progressive exercise-training regimen comprised of treadmill running 5 days a week, for 14 weeks. Coronary arterioles were isolated from the nonoccluded and collateral-dependent myocardial regions and bradykinin-mediated dilation via pressure myography was performed in the presence and absence of ML-171, gp91ds-tat and GKT136901, inhibiting NOX1, NOX2, and NOX1/4 isoforms, respectively. Additionally, using the superoxide probe, dihydroethidium, HPLC analysis was performed on isolated microvascular endothelial cells, pretreated with NOX inhibitors, to determine candidate sources of superoxide. Inhibition of NOX2 produced a significant rightward shift in EC50 values of arterioles from sedentary animals, independent of occlusion, as well as the nonoccluded arterioles of exercise-trained pigs. EC50 values of collateral-dependent arterioles from exercise-trained swine were not significantly altered by NOX2 inhibition. Contrastingly, collateral-dependent arterioles from exercise-trained swine exhibited a significant shift in EC50 values in the presence NOX1/4 inhibition, which was absent in other arteriole treatment groups. Importantly, NOX1 inhibition did not significantly shift EC50 values in any treatment group, suggesting responses to the NOX1/4 inhibitor, GKT136901, reflect the contribution of NOX4. Interestingly, microvascular endothelial cell superoxide levels were not different across arteriole treatment groups. Our present findings suggest that exercise training produces adaptations in collateral-dependent arterioles that appear to shift dependence from NOX2- to NOX4-derived reactive oxygen species for endothelium-dependent dilation. Taken together with our previous findings, our data support the assertion that enhanced dilation of collateral-dependent arterioles after exercise training is driven by enhanced NOX4-derived H2 O2 levels.
  2. FASEB J. 2022 May;36 Suppl 1
      NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to calcium binding. NOX5 has been identified as a key player for a variety of physiological and pathological conditions, yet a comprehensive understanding of its role in the cell has been lacking. Here we present a workflow to identify NOX5 protein partners as well as proteins which become oxidatively modified in a NOX5 dependent manner. These protein candidates can then be assessed for their role in mediating NOX5 dependent signaling in the cell which is involved in cellular differentiation, angiogenesis, and human disease. Investigating these results will allow us to understand how NOX5 carries out its physiological roles as well as provide indications for how dysregulation can lead to the initiation and progression of disease.
  3. BMC Nephrol. 2022 May 13. 23(1): 184
      BACKGROUND: Statins therapy has been primarily recommended for the prevention of cardiovascular risk in patients with chronic kidney diseases. Statins has also been proved some benefits in lipid-induced kidney diseases. The current study aims to investigate the protection and underlying mechanisms of statins on renal tubular injuries induced by cholesterol overloaded.METHODS: We used tubular suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys and mouse collecting duct cell line mpkCCD cells to investigate the effect of statins on reactive oxygen species (ROS) production induced by cholesterol. Protein and mRNA expression of NADPH oxidase 2 (NOX2) /NOX4 was examined by Western blot and RT-PCR in vitro studies and in rats with 5/6 nephrectomy and high-fat diet. Mitochondrial morphology and membrane potential was observed by Mito-tracker and JC-1.
    RESULTS: Statins treatment was associated with decreased NOX2 and NOX4 protein expression and mRNA levels in 5/6Nx rats with high-fat diet. Statins treatment markedly reduced the ROS production in IMCD suspensions and mpkCCD cells. Also, statins reduced NOX2 and NOX4 protein expression and mRNA levels in cholesterol overload mpkCCD cells and improved mitochondrial morphology and function.
    CONCLUSION: Statins prevented ROS production induced by cholesterol in the kidney, likely through inhibiting NOXs protein expression and improving mitochondrial function. Statins may be a therapeutic option in treating obesity-associated kidney diseases.
    Keywords:  Cholesterol; Collecting ducts; ROS; Statins
  4. FASEB J. 2022 May;36 Suppl 1
      Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA). Long term IH (LT-IH) triggers epigenetic reprogramming of the redox state involving DNA hypermethylation leading to persistent sympathetic activation and hypertension. Present study examined whether IH also activates epigenetic mechanism(s) other than DNA methylation. Lysine modifications of histones is another major epigenetic mechanism associated with gene regulation. Interplay between lysine deacetylases (HDACs) and demethylases (KDMs) differentially regulate gene expression. Lysine acetylation of histone opens up the chromatin thereby facilitating binding of transcription factors. On the other hand, KDMs regulate transcription by demethylating histone lysines which activate gene expression. Here we report that exposure of rat pheochromocytoma (PC)-12 cells to IH in vitro exhibit reduced HDAC5 and increased KDM6B enzyme activity leading to increased HIF-1α stability and transcriptional activity. Treatment of rats with Trichostatin A (TSA), an inhibitor of HDACs increased HIF-1 dependent NADPH oxidase (NOX)-4 transcription, in adrenal medullae (AM), resulting in elevated plasma catecholamines and blood pressure. Treating IH exposed rats with GSKJ4 an inhibitor of KDMs blocked HIF-1 dependent transcriptional activation of NOX4, as well as absence of elevated plasma catecholamines and hypertension. These findings indicate a hitherto uncharacterized role of HDACs and KDMs as early epigenetic regulators in IH-augmented sympathetic nerve activation and hypertension in rodent models of IH.
  5. FASEB J. 2022 May;36 Suppl 1
      The vasoactive peptide angiotensin II (Ang II) and hyperglycemia contribute to the initiation and the progression of glomerular fibrosis via activation of glomerular mesangial cells (MCs) and subsequent extracellular matrix expansion. We have previously shown that oxidative stress is critical for MC fibrotic response to Ang II or high concentrations of glucose (HG). Here, we demonstrate that Dual oxidase 2 (Duox2), a member of the Nox/Duox family of NADPH oxidases, is present in MCs and that its protein expression, along with its associated maturation factor, DuoxA2, are upregulated by Ang II and HG. Small interfering RNA (siRNA)-mediated downregulation of Duox2 significantly reduces Ang II-induced increase in reactive oxygen (ROS) generation and prevents the stimulatory effect of Ang II on MC fibrotic injury (as assessed by measuring a-smooth muscle actin and fibronectin expression). To demonstrate that Duox2 activation is Ca2+ -dependent, we show that the extracellular Ca2+ chelator BAPTA prevented Ang II-induced ROS generation and the stimulation of MC fibrotic injury by Ang II. Moreover, treatment of MCs with ionomycin resulted in increased ROS production and enhanced MC fibrotic injury. These effects were abrogated by siRNA targeting Duox2. Fura-2 fluorescence was utilized as well to show calcium mobilization in response to Ang II and this effect was abrogated with siDuox2 transfection. These data indicate that Ang II-stimulated Duox2 activation and ROS generation subsequently lead to MC fibrotic injury. In summary, we have identified a novel role for Duox2 and its cognate maturation factor, DuoxA2, as a major source of ROS in response to Ang II and HG and established the significance of Duox2 in Ang II-mediated MC activation and fibrotic injury. Therapeutic targeting of this pathway may prevent or reverse pathophysiologic manifestations of renal fibrotic diseases.
  6. J Appl Physiol (1985). 2022 May 12.
      Angiotensin-converting enzyme 2 (ACE2) or exercise training (ExT) is beneficial to hypertension, but their combined effects remain unknown. In this study, lentivirus containing enhanced green fluorescent protein (eGFP) and ACE2 were microinjected into the paraventricular nucleus (PVN) of young male spontaneous hypertensive rats (SHRs), and SHRs were assigned into 5 groups: sedentary (SHR), SHR-ExT, SHR-eGFP, ACE2 gene (SHR-ACE2), ACE2 gene combined with ExT (SHR-ACE2-ExT). Wistar-Kyoto (WKY) rats were used as a control. ACE2 gene or ExT significantly delayed the elevation of blood pressure, the combined effect prevented the development and progression of prehypertension. Either ACE2 over-expression or ExT improved arterial baroreflex sensitivity (BRS), while combined effect normalized BRS in SHR. Compared with SHR, SHR-ACE2 and SHR-ExT displayed a significantly higher level in ACE2 protein, but had lower plasma norepinephrine (NE) and angiotensin II (AngII) as well as angiotensin II type 1 receptor (AT1) protein expression in the PVN. SHR-ACE2-ExT showed the largest decrease in AngII and AT1 protein expression. Reactive oxygen species (ROS) level and NADPH oxidase (NOX2 and NOX4) protein expression in PVN were also decreased in SHR-ACE2-ExT group than in SHR-ACE2 and SHR-ExT groups. It was concluded that the combined effect has effectively prevented prehypertension progression and baroreflex dysfunction in SHR, which is associated with the reduction in AngII/AT1 axis function and oxidative stress in the PVN.
    Keywords:  baroreflex; exercise training; hypertension; paraventricular nucleus; renin-angiotensin system
  7. FASEB J. 2022 May;36 Suppl 1
      The circulating level of endothelin (ET), a potent vasoactive peptide, decreases with postnatal age. ET is also of pathophysiological significance in the kidney. ET is generated via endothelin converting enzyme (ECE)-induced proteolytic processing of pro-molecule big ET to biologically active peptides. Hydrogen peroxide (H2 O2 ), a reactive oxygen species (ROS), transactivates the promoter of ECE1. Oxyradicals contribute to kidney pathophysiology, but whether ROS regulates ET production in the kidneys remains unresolved. The present study shows that ECE1 is the predominant isoform in neonatal pig kidneys. We also demonstrated that H2 O2 induces ECE1-dependent ET1-3 production in the neonatal pig renal proximal tubules (PT) and vascular endothelial cells (EC). A uniaxial stretch of neonatal pig PT and EC increased NADPH oxidase (NOX) 2 and 4 levels and stimulated H2 O2 production. Mechanical stretch increased cellular ECE1 production in the PC and EC cells, which catalase mimetic EUK 134 and NOX inhibitor apocynin reversed. Mechanical stretch also triggered cellular ET1-3 generation in PT and EC. Mechanical stretch-induced ET1-3 production was attenuated by EUK 134, apocynin, and a selective ECE1 inhibitor CGS 35066. Short-term unilateral urinary tract obstruction (UUO), an inducer of the mechanical stretch of kidney cells and oxyradical production, increased ECE1-dependent ET1-3 production in neonatal pig kidneys. Despite removing the obstruction, acute UUO generated ROS, reduced kidney perfusion, and increased renal vascular resistance (RVR). UUO also elevated early biomarkers of acute kidney injury (AKI). Inhibition of ECE1 and ET receptors protected against UUO-induced increase in RVR and early AKI. These data suggest that mechanical stretch-driven ROS generation stimulates ET production in neonatal pig renal epithelial and vascular endothelial cells. ET then promotes AKI by causing renal hypoperfusion. This signaling pathway may underlie UUO-induced renal insufficiency in infants.
  8. FASEB J. 2022 May;36 Suppl 1
      The regulation of cellular signaling across plasma membranes is a well-organized process through cross-talk of biomolecules such as proteins and lipids. Peripheral membrane proteins (PMPs) are a class of water soluble, membrane associated, proteins that play a critical role in transducing these signals. Due to the dynamic nature of their membrane interactions, relatively smooth surfaces, and lack of deep ligand binding pockets, PMPs are commonly associated with the ~85% of the human proteome that is classified as 'undruggable' by current drug discovery methodologies. One such PMP is p47phox that acts as an organizer/scaffold subunit essential for activation of transmembrane enzyme NADPH oxidase 2 (NOX2). The NOX2 complex is activated by translocation of the PX domain of p47phox (p47phox -PX) through recognition of phospholipids (PIPs) localized in the plasma membrane. NOX2 is implicated in diseases such as cardiovascular, cancers, and neurodegenerative disorders, however, despite its importance, past efforts at inhibiting NOX2 activation have failed. The challenge lies in a lack of specificity among the NOX family of enzymes due to a highly conserved catalytic pocket. Inhibition of the complex prior to the activation event by targeting p47phox -PX presents a novel alternative strategy for reviving NOX2 inhibition. Fragment-based drug design (FBDD) was developed over the past twenty years and has already shown promise at targeting 'undruggable' proteins. Here, we initiate a FBDD study against p47phox -PX. We discovered a small molecule binder, myo-inositol-1-phosphate (I1P), that mimics the headgroup of p47phox -PX's natural PIP ligand. I1P has an ~800uM affinity towards the PX domain, as displayed by nuclear magnetic resonance spectroscopy (NMR) titrations, which will act as a scaffold for inhibitor design. Through a NMR based fragment screen, we identified 13 additional fragments that bind to p47phox -PX. Initial characterization of fragment hits displayed a number of high uM to low mM affinities within the lipid headgroup recognition pocket, which is promising for fragment advancement towards inhibitors. Future endeavors to probe the interactions between fragments and the PX domain of p47phox will further optimize the affinity of the inhibitor to p47phox -PX and increase selectivity against other PX domains. An array of biochemical, biophysical, and cellular assays will establish and optimize our inhibitor as a novel p47phox -PX small molecule drug and deactivator of the NOX2 complex.