49 results on '"Santos CX"'
Search Results
2. Evidence for the interaction between protein disulfide isomerase and NADPH oxidase as a regulatory mechanism for superoxide generation in phagocytic and vascular smooth muscle cells
- Author
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Costa, EP, Yamauchi, CH, Santos, CX, Laurindo, FRM, Lopes, LR, and Janiszewski, M
- Subjects
Meeting Abstract - Published
- 2003
3. P600 Endothelial Nox2 is protective against sepsis-induced severe hypotension and systemic inflammatory response.
- Author
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Trevelin, S C, Santos, CX, Zhang, M, Sag, C, Cunha, TM, Alves-Filho, JC, Lopes, LR, Cunha, FQ, Ivetic, A, and Shah, AM
- Subjects
- *
ENDOTHELIAL cells , *SEPSIS , *HYPOTENSION , *INFLAMMATION , *REGULATION of blood pressure , *PLETHYSMOGRAPHY - Abstract
Purpose: To investigate the role of endothelial Nox2 in sepsis-induced systemic inflammatory response and hypotension. Methods: All experiments were conduced in accordance with the Scientific Procedures Act. 1986 (UK Home Office). Mice with endothelial-target deletion of Nox2 were generated by crossing Nox2fl/fl mice with Tie2Cre transgenic animals. We compared Nox2fl/fl Tie2Cre+/− mice (Knockout,KO) with matched littermate Nox2fl/fl Tie2Cre-/- (Control) animals on a C57BL/6 background. Mice received an iv injection of lipopolysaccharide (LPS, 10mg/kg) or saline. Systolic and diastolic blood pressures were determined by tail-cuff plethysmography. A clinical severity score comprising signs of lethargy, piloerection, tremor, periorbital exudates, respiratory distress and diarrhoea was determined 2-hourly after LPS injection. After 12 hours sepsis, animals were euthanized and neutrophil sequestration in lungs was evaluated by myeloperoxidase assay. ROS production in mesenteric vessels was assessed by dihydroethidium- image assay and 3-nitrotyrosine staining was used as a readout of nitrosative stress. Levels of tumor necrosis factor alpha (TNF-alpha) were determined by enzymatic linked immunoabsorbent assay. Results: KO mice had lower ROS production in mesenteric vessels than controls after LPS injection. Despite similar basal values (94.46±2.1 vs 96.43±2.50mmHg for systolic blood pressure and 68.19±1.74 vs 69.26±2.56mmHg for diastolic pressure), KO mice had lower blood pressure than control mice, 6 hours after LPS injection (63.04±2.56 vs 76.70±1.38mmHg for systolic blood pressure and 48.24±2.77 vs 59.14±1.89 mmHg for diastolic pressure; p<0.05; n=9). The clinical severity scores were also more aggravate in KO mice compared to controls (p=0.0003, n=9). Mice with endothelial-target Nox2 deletion had higher number of neutrophils trapped in lungs (12480±207.4 vs 6489±75.09 cells/mg lung, p=0.0004, n=6), and increased levels of TNF-alpha in plasma (95.70±13.51 vs 33.69±11.59 pg/ml, p=0.0207, n=5) compared to controls. Interestingly, 3-nitrotyrosine staining in mesenteric vessels was higher in KO mice compared to controls. Conclusions: Endothelial cell Nox2 appears to be protective during sepsis by reducing the extent of hypotension, neutrophil sequestration in lungs and levels of TNF-alpha in plasma. The severe hypotension in KO mice during sepsis could be associated with evidence of greater nitrosative stress in mesenteric vessels than controls. The cellular mechanisms underlying the Nox2 protective effect are currently being investigated. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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4. Protein disulfide isomerase-mediated transcriptional upregulation of Nox1 contributes to vascular dysfunction in hypertension.
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Camargo LL, Trevelin SC, da Silva GHG, Dos Santos Dias AA, Oliveira MA, Mikhaylichenko O, Androwiki ACD, Dos Santos CX, Holbrook LM, Ceravolo GS, Denadai-Souza A, Ribeiro IMR, Sartoretto S, Laurindo FRM, Coltri PP, Antunes VR, Touyz R, Miller FJ Jr, Shah AM, and Lopes LR
- Subjects
- Animals, Rats, Male, Myocytes, Smooth Muscle metabolism, ErbB Receptors metabolism, ErbB Receptors genetics, Rats, Wistar, Transcription, Genetic, Protein Disulfide-Isomerases metabolism, Protein Disulfide-Isomerases genetics, NADPH Oxidase 1 metabolism, NADPH Oxidase 1 genetics, Hypertension physiopathology, Hypertension genetics, Hypertension metabolism, Rats, Inbred SHR, Up-Regulation, Muscle, Smooth, Vascular metabolism
- Abstract
Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)
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- 2024
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5. Cardiomyocyte protein O-GlcNAcylation is regulated by GFAT1 not GFAT2.
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Nabeebaccus AA, Verma S, Zoccarato A, Emanuelli G, Santos CX, Streckfuss-Bömeke K, and Shah AM
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- Animals, Fibroblasts metabolism, Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) genetics, Hexosamines biosynthesis, Hexosamines metabolism, Induced Pluripotent Stem Cells, Mice, Myocardium cytology, Protein Isoforms, Rats, Sprague-Dawley, Rats, Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) metabolism, Myocardium metabolism, Myocytes, Cardiac metabolism
- Abstract
In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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6. Nox4 regulates InsP 3 receptor-dependent Ca 2+ release into mitochondria to promote cell survival.
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Beretta M, Santos CX, Molenaar C, Hafstad AD, Miller CC, Revazian A, Betteridge K, Schröder K, Streckfuß-Bömeke K, Doroshow JH, Fleck RA, Su TP, Belousov VV, Parsons M, and Shah AM
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- Animals, Cell Survival, Inositol 1,4,5-Trisphosphate Receptors genetics, Myocardial Reperfusion Injury genetics, Myocardial Reperfusion Injury metabolism, NADPH Oxidase 4 genetics, Oxidative Stress, Rats, Calcium metabolism, Calcium Signaling, Inositol 1,4,5-Trisphosphate Receptors metabolism, Mitochondria, Heart metabolism, Myocytes, Cardiac metabolism, NADPH Oxidase 4 metabolism
- Abstract
Cells subjected to environmental stresses undergo regulated cell death (RCD) when homeostatic programs fail to maintain viability. A major mechanism of RCD is the excessive calcium loading of mitochondria and consequent triggering of the mitochondrial permeability transition (mPT), which is especially important in post-mitotic cells such as cardiomyocytes and neurons. Here, we show that stress-induced upregulation of the ROS-generating protein Nox4 at the ER-mitochondria contact sites (MAMs) is a pro-survival mechanism that inhibits calcium transfer through InsP
3 receptors (InsP3 R). Nox4 mediates redox signaling at the MAM of stressed cells to augment Akt-dependent phosphorylation of InsP3 R, thereby inhibiting calcium flux and mPT-dependent necrosis. In hearts subjected to ischemia-reperfusion, Nox4 limits infarct size through this mechanism. These results uncover a hitherto unrecognized stress pathway, whereby a ROS-generating protein mediates pro-survival effects through spatially confined signaling at the MAM to regulate ER to mitochondria calcium flux and triggering of the mPT., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)- Published
- 2020
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7. Static Stretching Intensity Does Not Influence Acute Range of Motion, Passive Torque, and Muscle Architecture.
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Santos CX, Beltrão NB, Pirauá ALT, Durigan JLQ, Behm D, and de Araújo RC
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- Humans, Male, Torque, Young Adult, Muscle Stretching Exercises physiology, Muscle Tonus physiology, Muscle, Skeletal physiology, Range of Motion, Articular physiology
- Abstract
Context: Although stretching exercises are commonly used in clinical and athletic practice, there is a lack of evidence regarding the methodological variables that guide the prescription of stretching programs, such as intensity., Objective: To investigate the acute effects of different stretching intensities on the range of motion (ROM), passive torque, and muscle architecture., Design: Two-group pretest-posttest design., Setting: Laboratory., Participants: Twenty untrained men were allocated into the low- or high-intensity group., Main Outcome Measures: Subjects were evaluated for initial (ROMinitial) and maximum (ROMmax) discomfort angle, stiffness, viscoelastic stress relaxation, muscle fascicle length, and pennation angle., Results: The ROM assessments showed significant changes, in both groups, in the preintervention and postintervention measures both for the ROMinitial (P < .01) and ROMmax angle (P = .02). There were no significant differences for stiffness and viscoelastic stress relaxation variables. The pennation angle and muscle fascicle length were different between the groups, but there was no significant interaction., Conclusion: Performing stretching exercises at high or low intensity acutely promotes similar gains in flexibility, that is, there are short-term/immediate gains in ROM but does not modify passive torque and muscle architecture.
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- 2020
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8. Viability of mobile applications for remote support of radiotherapy patients.
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Cunha CE, Fernandes R, Santos CX, Boccaletti KW, Pellizzon ACA, and Barbosa JHO
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- Brazil, Cell Phone instrumentation, Communication, Cross-Sectional Studies, Health Personnel, Humans, User-Computer Interface, Medical Oncology instrumentation, Mobile Applications, Radiotherapy instrumentation, Telemedicine instrumentation
- Abstract
Background: Technological advances of the 21st century have provided greater communication, regardless of socioeconomic class and age group. Actions to promote the development of health applications are emerging around the world., Objective: To provide a perspective on the viability and usability of mobile applications dedicated to radiotherapy patients for remote support to health professionals proposing solutions to encourage Brasil in the development of these digital tools., Methods: Cross-sectional exploratory study by systematic review and literature review. We searched the PubMed, BVS, IBGE, and WHO databases, from 2014 to 2018., Results: 6 articles were found with topics related to the use of mobile applications in the health area, two of which were published in Portuguese and four in the English, on oncology, from 2014 to 2018., Conclusions: We did not find an expressive number of works on this subject in Brasil. Mobile applications have the potential to assist in the remote support of radiotherapy patients. The latest studies suggest the need for a regulation of data protection protocols to be deployed.
- Published
- 2019
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9. Developing potential biomarkers through bedside-to-bench translation.
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Bromage DI, Santos CX, and Shah AM
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- Animals, Humans, Biomarkers, Molecular Diagnostic Techniques, Translational Research, Biomedical methods
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- 2019
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10. Paracrine Mechanisms of Redox Signalling for Postmitotic Cell and Tissue Regeneration.
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Hervera A, Santos CX, De Virgiliis F, Shah AM, and Di Giovanni S
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- Animals, Extracellular Vesicles metabolism, Humans, Myocytes, Cardiac cytology, Neurons cytology, Oxidation-Reduction, Mitosis, Myocytes, Cardiac metabolism, Neurons metabolism, Paracrine Communication, Signal Transduction
- Abstract
Adult postmitotic mammalian cells, including neurons and cardiomyocytes, have a limited capacity to regenerate after injury. Therefore, an understanding of the molecular mechanisms underlying their regenerative ability is critical to advance tissue repair therapies. Recent studies highlight how redox signalling via paracrine cell-to-cell communication may act as a central mechanism coupling tissue injury with regeneration. Post-injury redox paracrine signalling can act by diffusion to nearby cells, through mitochondria or within extracellular vesicles, affecting specific intracellular targets such as kinases, phosphatases, and transcription factors, which in turn trigger a regenerative response. Here, we review redox paracrine signalling mechanisms in postmitotic tissue regeneration and discuss current challenges and future directions., (Copyright © 2019. Published by Elsevier Ltd.)
- Published
- 2019
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11. Nox4 reprograms cardiac substrate metabolism via protein O-GlcNAcylation to enhance stress adaptation.
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Nabeebaccus AA, Zoccarato A, Hafstad AD, Santos CX, Aasum E, Brewer AC, Zhang M, Beretta M, Yin X, West JA, Schröder K, Griffin JL, Eykyn TR, Abel ED, Mayr M, and Shah AM
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- Adaptation, Physiological physiology, Animals, Cardiomegaly metabolism, Energy Metabolism physiology, Fatty Acids metabolism, Glucose metabolism, Glycolysis physiology, Hexosamines biosynthesis, Mice, Knockout, Mice, Transgenic, Myocytes, Cardiac metabolism, NADPH Oxidase 4 deficiency, NADPH Oxidase 4 genetics, Oxidation-Reduction, Proteome metabolism, Acetylglucosamine metabolism, Cardiomegaly physiopathology, Myocardium metabolism, NADPH Oxidase 4 physiology, Stress, Physiological physiology
- Abstract
Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4-dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial.
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- 2017
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12. Acute and Chronic Effects of Isometric Handgrip Exercise on Cardiovascular Variables in Hypertensive Patients: A Systematic Review.
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Farah BQ, Germano-Soares AH, Rodrigues SLC, Santos CX, Barbosa SS, Vianna LC, Cornelissen VA, and Ritti-Dias RM
- Abstract
The aim of this study was to describe, through a systematic review, the acute and chronic effects of isometric handgrip exercise on cardiovascular variables in hypertensive individuals. In this systematic review, we included studies that analyzed whether a single bout or a program with isometric exercises affect cardiovascular variables in hypertensive adults. The electronic database PubMed/Medline was searched for relevant studies published until May 2017. Of the 2927 studies initially identified, 2916 were excluded based on title and abstract and five on the basis of full-text assessment, leaving six studies remaining. In addition, one further study cited in the references of the included articles was included in this review, totaling seven studies included (five studies on the chronic effects of isometric handgrip exercise on cardiovascular parameters). None of the acute studies observed post-exercise hypotension. The majority of the chronic studies found decreases in office blood pressure after isometric handgrip training, with training ranging from 6 to 10 weeks, while heart rate variability parameters were improved in one study and did not change in another. Reduction in oxidative stress was observed; however, this variable was only analyzed in one study. In hypertensives, acute responses to isometric handgrip exercise are very limited due to the small number of studies, therefore more research is required. Furthermore, chronic isometric handgrip training reduces blood pressure; however, there is still a gap in the knowledge on the effects of this modality of exercise on other cardiovascular variables-such as endothelial function, oxidative stress, and cardiac autonomic modulation-which should be addressed in future studies., Competing Interests: The authors declare that there are no conflicts of interest.
- Published
- 2017
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13. Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop.
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Viswambharan H, Yuldasheva NY, Sengupta A, Imrie H, Gage MC, Haywood N, Walker AM, Skromna A, Makova N, Galloway S, Shah P, Sukumar P, Porter KE, Grant PJ, Shah AM, Santos CX, Li J, Beech DJ, Wheatcroft SB, Cubbon RM, and Kearney MT
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- Animals, Atherosclerosis pathology, Cells, Cultured, Endothelial Cells pathology, Endothelium, Vascular pathology, Human Umbilical Vein Endothelial Cells, Humans, Male, Mice, Mice, 129 Strain, Mice, Inbred C57BL, Mice, Transgenic, Organ Culture Techniques, Atherosclerosis metabolism, Endothelial Cells metabolism, Endothelium, Vascular metabolism, Insulin Resistance physiology, Signal Transduction physiology
- Abstract
Rationale: In the endothelium, insulin stimulates endothelial NO synthase (eNOS) to generate the antiatherosclerotic signaling radical NO. Insulin-resistant type 2 diabetes mellitus is associated with reduced NO availability and accelerated atherosclerosis. The effect of enhancing endothelial insulin sensitivity on NO availability is unclear., Objective: To answer this question, we generated a mouse with endothelial cell (EC)-specific overexpression of the human insulin receptor (hIRECO) using the Tie2 promoter-enhancer., Methods and Results: hIRECO demonstrated significant endothelial dysfunction measured by blunted endothelium-dependent vasorelaxation to acetylcholine, which was normalized by a specific Nox2 NADPH oxidase inhibitor. Insulin-stimulated phosphorylation of protein kinase B was increased in hIRECO EC as was Nox2 NADPH oxidase-dependent generation of superoxide, whereas insulin-stimulated and shear stress-stimulated eNOS activations were blunted. Phosphorylation at the inhibitory residue Y657 of eNOS and expression of proline-rich tyrosine kinase 2 that phosphorylates this residue were significantly higher in hIRECO EC. Inhibition of proline-rich tyrosine kinase 2 improved insulin-induced and shear stress-induced eNOS activation in hIRECO EC., Conclusions: Enhancing insulin sensitivity specifically in EC leads to a paradoxical decline in endothelial function, mediated by increased tyrosine phosphorylation of eNOS and excess Nox2-derived superoxide. Increased EC insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide. Inhibition of proline-rich tyrosine kinase 2 restores insulin-induced and shear stress-induced NO production. This study demonstrates for the first time that increased endothelial insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide., (© 2016 American Heart Association, Inc.)
- Published
- 2017
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14. Hypoxia induces heart regeneration in adult mice.
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Nakada Y, Canseco DC, Thet S, Abdisalaam S, Asaithamby A, Santos CX, Shah AM, Zhang H, Faber JE, Kinter MT, Szweda LI, Xing C, Hu Z, Deberardinis RJ, Schiattarella G, Hill JA, Oz O, Lu Z, Zhang CC, Kimura W, and Sadek HA
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- Animals, Cardiomyopathies metabolism, Cardiomyopathies pathology, Cell Proliferation, Cell Respiration, DNA Damage, Male, Mice, Mice, Inbred C57BL, Mitochondria metabolism, Mitosis, Myocardial Infarction metabolism, Myocardial Infarction pathology, Myocardium pathology, Myocytes, Cardiac cytology, Myocytes, Cardiac metabolism, Reactive Oxygen Species metabolism, Ventricular Function, Left, Heart growth & development, Hypoxia metabolism, Myocardium cytology, Myocardium metabolism, Regeneration, Regenerative Medicine methods
- Abstract
The adult mammalian heart is incapable of regeneration following cardiomyocyte loss, which underpins the lasting and severe effects of cardiomyopathy. Recently, it has become clear that the mammalian heart is not a post-mitotic organ. For example, the neonatal heart is capable of regenerating lost myocardium, and the adult heart is capable of modest self-renewal. In both of these scenarios, cardiomyocyte renewal occurs via the proliferation of pre-existing cardiomyocytes, and is regulated by aerobic-respiration-mediated oxidative DNA damage. Therefore, we reasoned that inhibiting aerobic respiration by inducing systemic hypoxaemia would alleviate oxidative DNA damage, thereby inducing cardiomyocyte proliferation in adult mammals. Here we report that, in mice, gradual exposure to severe systemic hypoxaemia, in which inspired oxygen is gradually decreased by 1% and maintained at 7% for 2 weeks, results in inhibition of oxidative metabolism, decreased reactive oxygen species production and oxidative DNA damage, and reactivation of cardiomyocyte mitosis. Notably, we find that exposure to hypoxaemia 1 week after induction of myocardial infarction induces a robust regenerative response with decreased myocardial fibrosis and improvement of left ventricular systolic function. Genetic fate-mapping analysis confirms that the newly formed myocardium is derived from pre-existing cardiomyocytes. These results demonstrate that the endogenous regenerative properties of the adult mammalian heart can be reactivated by exposure to gradual systemic hypoxaemia, and highlight the potential therapeutic role of hypoxia in regenerative medicine.
- Published
- 2017
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15. Apocynin and Nox2 regulate NF-κB by modifying thioredoxin-1 redox-state.
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Trevelin SC, Dos Santos CX, Ferreira RG, de Sá Lima L, Silva RL, Scavone C, Curi R, Alves-Filho JC, Cunha TM, Roxo-Júnior P, Cervi MC, Laurindo FR, Hothersall JS, Cobb AM, Zhang M, Ivetic A, Shah AM, Lopes LR, and Cunha FQ
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- Animals, Granulomatous Disease, Chronic chemically induced, Granulomatous Disease, Chronic genetics, Granulomatous Disease, Chronic metabolism, Granulomatous Disease, Chronic pathology, Lipopolysaccharides toxicity, Male, Mice, Mice, Knockout, NADPH Oxidase 2 genetics, NF-kappa B genetics, Oxidation-Reduction drug effects, Sepsis chemically induced, Sepsis genetics, Sepsis metabolism, Sepsis pathology, Thioredoxins genetics, Acetophenones pharmacology, NADPH Oxidase 2 metabolism, NF-kappa B metabolism, Signal Transduction drug effects, Thioredoxins metabolism
- Abstract
The reactive-oxygen-species-(ROS)-generating-enzyme Nox2 is essential for leukocyte anti-microbial activity. However its role in cellular redox homeostasis and, consequently, in modulating intracellular signaling pathways remains unclear. Herein, we show Nox2 activation favors thioredoxin-1 (TRX-1)/p40phox interaction, which leads to exclusion of TRX-1 from the nucleus. In contrast, the genetic deficiency of Nox2 or its pharmacological inhibition with apocynin (APO) results in reductive stress after lipopolysaccharide-(LPS)-cell stimulation, which causes nuclear accumulation of TRX-1 and enhanced transcription of inflammatory mediators through nuclear-factor-(NF)-κB. The NF-κB overactivation is prevented by TRX-1 oxidation using inhibitors of thioredoxin reductase-1 (TrxR-1). The Nox2/TRX-1/NF-κB intracellular signaling pathway is involved in the pathophysiology of chronic granulomatous disease (CGD) and sepsis. In fact, TrxR-1 inhibition prevents nuclear accumulation of TRX-1 and LPS-stimulated hyperproduction of tumor-necrosis-factor-(TNF)-α by monocytes and neutrophils purified from blood of CGD patients, who have deficient Nox2 activity. TrxR-1 inhibitors, either lanthanum chloride (LaCl
3 ) or auranofin (AUR), also increase survival rates of mice undergoing cecal-ligation-and-puncture-(CLP). Therefore, our results identify a hitherto unrecognized Nox2-mediated intracellular signaling pathway that contributes to hyperinflammation in CGD and in septic patients. Additionally, we suggest that TrxR-1 inhibitors could be potential drugs to treat patients with sepsis, particularly in those with CGD.- Published
- 2016
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16. Cell-specific effects of Nox2 on the acute and chronic response to myocardial infarction.
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Sirker A, Murdoch CE, Protti A, Sawyer GJ, Santos CX, Martin D, Zhang X, Brewer AC, Zhang M, and Shah AM
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- Animals, Apoptosis genetics, Disease Models, Animal, Echocardiography, Female, Fibrosis, Heart Ventricles metabolism, Heart Ventricles pathology, Heart Ventricles physiopathology, Hemodynamics, Mice, Mice, Transgenic, Myocardial Infarction diagnosis, Myocardial Infarction physiopathology, Myocytes, Cardiac metabolism, NADPH Oxidase 2, Organ Specificity genetics, Reactive Oxygen Species metabolism, Ventricular Dysfunction, Left, Ventricular Remodeling, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism, Myocardial Infarction etiology, Myocardial Infarction metabolism, NADPH Oxidases genetics, NADPH Oxidases metabolism
- Abstract
Background: Increased reactive oxygen species (ROS) production is involved in the process of adverse cardiac remodeling and development of heart failure after myocardial infarction (MI). NADPH oxidase-2 (Nox2) is a major ROS source within the heart and its activity increases after MI. Furthermore, genetic deletion of Nox2 is protective against post-MI cardiac remodeling. Nox2 levels may increase both in cardiomyocytes and endothelial cells and recent studies indicate cell-specific effects of Nox2, but it is not known which of these cell types is important in post-MI remodeling., Methods and Results: We have generated transgenic mouse models in which Nox2 expression is targeted either to cardiomyocytes (cardio-Nox2TG) or endothelial cells (endo-Nox2TG). We here studied the response of cardio-Nox2TG mice, endo-Nox2TG mice and matched wild-type littermates (WT) to MI induced by permanent left coronary artery ligation up to 4weeks. Initial infarct size assessed by magnetic resonance imaging (MRI) and cardiac dysfunction were similar among groups. Cardiomyocyte hypertrophy and interstitial fibrosis were augmented in cardio-Nox2TG compared to WT after MI and post-MI survival tended to be worse whereas endo-Nox2TG mice showed no significant difference compared to WT., Conclusions: These results indicate that cardiomyocyte rather than endothelial cell Nox2 may have the more important role in post-MI remodeling., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2016
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17. Redox signaling in the cardiomyocyte: From physiology to failure.
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Santos CX, Raza S, and Shah AM
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- Heart Failure physiopathology, Humans, Myocytes, Cardiac pathology, Protein Processing, Post-Translational, Reactive Oxygen Species, Signal Transduction, Myocytes, Cardiac physiology, Oxidation-Reduction
- Abstract
The specific effect of oxygen and reactive oxygen species (ROS) in mediating post-translational modification of protein targets has emerged as a key mechanism regulating signaling components, a process termed redox signaling. ROS act in the post-translational modification of multiple target proteins including receptors, kinases, phosphatases, ion channels and transcription factors. Both O2 and ROS are major source of electrons in redox reactions in aerobic organisms. Because the heart has the highest O2 consumption among body organs, it is not surprising that redox signaling is central to heart function and pathophysiology. In this article, we review some of the main cardiac redox signaling pathways and their roles in the cardiomyocyte and in heart failure, with particular focus on the specific molecular targets of ROS in the heart., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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18. Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling.
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Santos CX, Hafstad AD, Beretta M, Zhang M, Molenaar C, Kopec J, Fotinou D, Murray TV, Cobb AM, Martin D, Zeh Silva M, Anilkumar N, Schröder K, Shanahan CM, Brewer AC, Brandes RP, Blanc E, Parsons M, Belousov V, Cammack R, Hider RC, Steiner RA, and Shah AM
- Subjects
- Animals, Cell Line, Humans, NADPH Oxidase 4, Oxidation-Reduction, Eukaryotic Initiation Factor-2 metabolism, NADPH Oxidases metabolism, Protein Phosphatase 1 metabolism, Receptors, Neuropeptide Y antagonists & inhibitors, Signal Transduction
- Abstract
Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine-threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species-generating NADPH oxidase-4 (Nox4) is induced downstream of ATF4, binds to a PP1-targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4-regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia-reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4-GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2016
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19. Contractile Function During Angiotensin-II Activation: Increased Nox2 Activity Modulates Cardiac Calcium Handling via Phospholamban Phosphorylation.
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Zhang M, Prosser BL, Bamboye MA, Gondim ANS, Santos CX, Martin D, Ghigo A, Perino A, Brewer AC, Ward CW, Hirsch E, Lederer WJ, and Shah AM
- Subjects
- Animals, Disease Models, Animal, Mice, Mice, Transgenic, Models, Cardiovascular, NADPH Oxidase 2, Phosphorylation physiology, Reactive Oxygen Species metabolism, Renin-Angiotensin System physiology, Sarcoplasmic Reticulum metabolism, Angiotensin II metabolism, Calcium metabolism, Calcium-Binding Proteins metabolism, Cardiovascular Diseases metabolism, Membrane Glycoproteins metabolism, Myocardial Contraction physiology, Myocytes, Cardiac metabolism, NADPH Oxidases metabolism
- Abstract
Background: Renin-angiotensin system activation is a feature of many cardiovascular conditions. Activity of myocardial reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (NADPH oxidase 2 or Nox2) is enhanced by angiotensin II (Ang II) and contributes to increased hypertrophy, fibrosis, and adverse remodeling. Recent studies found that Nox2-mediated reactive oxygen species production modulates physiological cardiomyocyte function., Objectives: This study sought to investigate the effects of cardiomyocyte Nox2 on contractile function during increased Ang II activation., Methods: We generated a cardiomyocyte-targeted Nox2-transgenic mouse model and studied the effects of in vivo and ex vivo Ang II stimulation, as well as chronic aortic banding., Results: Chronic subpressor Ang II infusion induced greater cardiac hypertrophy in transgenic than wild-type mice but unexpectedly enhanced contractile function. Acute Ang II treatment also enhanced contractile function in transgenic hearts in vivo and transgenic cardiomyocytes ex vivo. Ang II-stimulated Nox2 activity increased sarcoplasmic reticulum (SR) Ca(2+) uptake in transgenic mice, increased the Ca(2+) transient and contractile amplitude, and accelerated cardiomyocyte contraction and relaxation. Elevated Nox2 activity increased phospholamban phosphorylation in both hearts and cardiomyocytes, related to inhibition of protein phosphatase 1 activity. In a model of aortic banding-induced chronic pressure overload, heart function was similarly depressed in transgenic and wild-type mice., Conclusions: We identified a novel mechanism in which Nox2 modulates cardiomyocyte SR Ca(2+) uptake and contractile function through redox-regulated changes in phospholamban phosphorylation. This mechanism can drive increased contractility in the short term in disease states characterized by enhanced renin-angiotensin system activation., (Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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20. Redox regulation of cardiac hypertrophy.
- Author
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Sag CM, Santos CX, and Shah AM
- Subjects
- Animals, Humans, Oxidation-Reduction, Reactive Oxygen Species metabolism, Signal Transduction physiology, Cardiomegaly metabolism, NADPH Oxidases metabolism
- Abstract
It is increasingly evident that redox-dependent modifications in cellular proteins and signaling pathways (or redox signaling) play important roles in many aspects of cardiac hypertrophy. Indeed, these redox modifications may be intricately linked with the process of hypertrophy wherein there is not only a significant increase in myocardial O2 consumption but also important alterations in metabolic processes and in the local generation of O2-derived reactive species (ROS) that modulate and/or amplify cell signaling pathways. This article reviews our current knowledge of redox signaling pathways and their roles in cardiac hypertrophy. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System"., (Copyright © 2014. Published by Elsevier Ltd.)
- Published
- 2014
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21. The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.
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Puente BN, Kimura W, Muralidhar SA, Moon J, Amatruda JF, Phelps KL, Grinsfelder D, Rothermel BA, Chen R, Garcia JA, Santos CX, Thet S, Mori E, Kinter MT, Rindler PM, Zacchigna S, Mukherjee S, Chen DJ, Mahmoud AI, Giacca M, Rabinovitch PS, Aroumougame A, Shah AM, Szweda LI, and Sadek HA
- Subjects
- Acetylcysteine pharmacology, Animals, Cell Proliferation drug effects, DNA Damage, Free Radical Scavengers pharmacology, Mice, Mitochondria metabolism, Myocytes, Cardiac metabolism, Zebrafish, Cell Cycle Checkpoints, Myocytes, Cardiac cytology, Reactive Oxygen Species metabolism
- Abstract
The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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22. Endoplasmic reticulum stress and Nox-mediated reactive oxygen species signaling in the peripheral vasculature: potential role in hypertension.
- Author
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Santos CX, Nabeebaccus AA, Shah AM, Camargo LL, Filho SV, and Lopes LR
- Subjects
- Animals, Diabetes Mellitus metabolism, Humans, Hypertension metabolism, Insulin Resistance, Blood Vessels metabolism, Endoplasmic Reticulum Stress, NADPH Oxidases metabolism, Reactive Oxygen Species metabolism, Signal Transduction
- Abstract
Significance: Reactive oxygen species (ROS) are produced during normal endoplasmic reticulum (ER) metabolism. There is accumulating evidence showing that under stress conditions such as ER stress, ROS production is increased via enzymes of the NADPH oxidase (Nox) family, especially via the Nox2 and Nox4 isoforms, which are involved in the regulation of blood pressure. Hypertension is a major contributor to cardiovascular and renal disease, and it has a complex pathophysiology involving the heart, kidney, brain, vessels, and immune system. ER stress activates the unfolded protein response (UPR) signaling pathway that has prosurvival and proapoptotic components., Recent Advances: Here, we summarize the evidence regarding the association of Nox enzymes and ER stress, and its potential contribution in the setting of hypertension, including the role of other conditions that can lead to hypertension (e.g., insulin resistance and diabetes)., Critical Issues: A better understanding of this association is currently of great interest, as it will provide further insights into the cellular mechanisms that can drive the ER stress-induced adaptive versus maladaptive pathways linked to hypertension and other cardiovascular conditions. More needs to be learnt about the precise signaling regulation of Nox(es) and ER stress in the cardiovascular system., Future Directions: The development of specific approaches that target individual Nox isoforms and the UPR signaling pathway may be important for the achievement of therapeutic efficacy in hypertension.
- Published
- 2014
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23. Nox2 NADPH oxidase has a critical role in insulin resistance-related endothelial cell dysfunction.
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Sukumar P, Viswambharan H, Imrie H, Cubbon RM, Yuldasheva N, Gage M, Galloway S, Skromna A, Kandavelu P, Santos CX, Gatenby VK, Smith J, Beech DJ, Wheatcroft SB, Channon KM, Shah AM, and Kearney MT
- Subjects
- Acetylcholine pharmacology, Animals, Cells, Cultured, Chromatography, High Pressure Liquid, Immunoblotting, Insulin Resistance genetics, Male, Membrane Glycoproteins genetics, Mice, Mice, Knockout, NADPH Oxidase 2, NADPH Oxidases genetics, Polymerase Chain Reaction, Vasodilator Agents pharmacology, Endothelial Cells drug effects, Insulin Resistance physiology, Membrane Glycoproteins metabolism, NADPH Oxidases metabolism
- Abstract
Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance-induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance-mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance-related vascular disease.
- Published
- 2013
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24. A 28-kDa splice variant of NADPH oxidase-4 is nuclear-localized and involved in redox signaling in vascular cells.
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Anilkumar N, San Jose G, Sawyer I, Santos CX, Sand C, Brewer AC, Warren D, and Shah AM
- Subjects
- Animals, DNA Damage, Enzyme Activation, Fluorescent Antibody Technique, HEK293 Cells, Histones metabolism, Humans, Microscopy, Confocal, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Molecular Weight, Mutagenesis, Site-Directed, NADPH Oxidase 4, NADPH Oxidases genetics, Oxidation-Reduction, Phosphorylation, Primary Cell Culture, Protein Isoforms, RNA Interference, Rats, Reactive Oxygen Species metabolism, Signal Transduction, Transfection, Cell Nucleus enzymology, Fibroblasts enzymology, Human Umbilical Vein Endothelial Cells enzymology, Muscle, Smooth, Vascular enzymology, Myocytes, Cardiac enzymology, Myocytes, Smooth Muscle enzymology, NADPH Oxidases metabolism
- Abstract
Objective: Reactive oxygen species-generating nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase proteins (Noxs) are involved in cell differentiation, migration, and apoptosis. Nox4 is unique among Noxs in being constitutively active, and its subcellular localization may therefore be particularly important. In this study, we identified and characterized a novel nuclear-localized 28-kDa splice variant of Nox4 in vascular cells., Approach and Results: Nox4 immunoreactivity was noted in the nucleus and nucleolus of vascular smooth muscle cells and multiple other cell types by confocal microscopy. Cell fractionation, sequence analyses, and siRNA studies indicated that the nuclear-localized Nox4 is a 28-kDa splice variant, Nox4D, which lacks putative transmembrane domains. Nox4D overexpression resulted in significant NADPH-dependent reactive oxygen species production as detected by several different methods and caused increased phosphorylation of extracellular-signal-regulated kinase1/2 and the nuclear transcription factor Elk-1. Overexpression of Nox4D could also induce DNA damage as assessed by γ-H2AX phosphorylation. These effects were inhibited by a single amino acid substitution in the Nox4D NADPH-binding region., Conclusions: Nox4D is a nuclear-localized and functionally active splice variant of Nox4 that may have important pathophysiologic effects through modulation of nuclear signaling and DNA damage.
- Published
- 2013
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25. Pathological cardiac hypertrophy alters intracellular targeting of phosphodiesterase type 5 from nitric oxide synthase-3 to natriuretic peptide signaling.
- Author
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Zhang M, Takimoto E, Lee DI, Santos CX, Nakamura T, Hsu S, Jiang A, Nagayama T, Bedja D, Yuan Y, Eaton P, Shah AM, and Kass DA
- Subjects
- Animals, Cardiomegaly pathology, Cyclic GMP metabolism, Cyclic Nucleotide Phosphodiesterases, Type 5 genetics, Disease Models, Animal, Female, Heart Failure metabolism, Heart Failure pathology, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Myocardium enzymology, Myocardium pathology, Myocytes, Cardiac enzymology, Myocytes, Cardiac pathology, Nitric Oxide Synthase Type III genetics, Oxidative Stress physiology, Reactive Oxygen Species metabolism, Ventricular Remodeling physiology, Atrial Natriuretic Factor metabolism, Cardiomegaly metabolism, Cyclic Nucleotide Phosphodiesterases, Type 5 metabolism, Nitric Oxide Synthase Type III metabolism, Signal Transduction physiology
- Abstract
Background: In the normal heart, phosphodiesterase type 5 (PDE5) hydrolyzes cGMP coupled to nitric oxide- (specifically from nitric oxide synthase 3) but not natriuretic peptide (NP)-stimulated guanylyl cyclase. PDE5 is upregulated in hypertrophied and failing hearts and is thought to contribute to their pathophysiology. Because nitric oxide signaling declines whereas NP-derived cGMP rises in such diseases, we hypothesized that PDE5 substrate selectivity is retargeted to blunt NP-derived signaling., Methods and Results: Mice with cardiac myocyte inducible PDE5 overexpression (P5(+)) were crossed to those lacking nitric oxide synthase 3 (N3(-)), and each model, the double cross, and controls were subjected to transaortic constriction. P5(+) mice developed worse dysfunction and hypertrophy and enhanced NP stimulation, whereas N3(-) mice were protected. However, P5(+)/N3(-) mice behaved similarly to P5(+) mice despite the lack of nitric oxide synthase 3-coupled cGMP generation, with protein kinase G activity suppressed in both models. PDE5 inhibition did not alter atrial natriuretic peptide-stimulated cGMP in the resting heart but augmented it in the transaortic constriction heart. This functional retargeting was associated with PDE5 translocation from sarcomeres to a dispersed distribution. P5(+) hearts exhibited higher oxidative stress, whereas P5(+)/N3(-) hearts had low levels (likely owing to the absence of nitric oxide synthase 3 uncoupling). This highlights the importance of myocyte protein kinase G activity as a protection for pathological remodeling., Conclusions: These data provide the first evidence for functional retargeting of PDE5 from one compartment to another, revealing a role for natriuretic peptide-derived cGMP hydrolysis by this esterase in diseased heart myocardium. Retargeting likely affects the pathophysiological consequence and the therapeutic impact of PDE5 modulation in heart disease.
- Published
- 2012
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26. ER stress is associated with reduced ABCA-1 protein levels in macrophages treated with advanced glycated albumin - reversal by a chemical chaperone.
- Author
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Castilho G, Okuda LS, Pinto RS, Iborra RT, Nakandakare ER, Santos CX, Laurindo FR, and Passarelli M
- Subjects
- ATP Binding Cassette Transporter 1, ATP-Binding Cassette Transporters genetics, Animals, Cell Line, Cell Line, Tumor, Endoplasmic Reticulum Stress drug effects, Glycation End Products, Advanced, Immunoblotting, Macrophages, Peritoneal drug effects, Mice, Glycated Serum Albumin, ATP-Binding Cassette Transporters metabolism, Endoplasmic Reticulum Stress physiology, Macrophages, Peritoneal metabolism, Molecular Chaperones pharmacology, Serum Albumin pharmacology
- Abstract
ATP-binding cassette transporter A1 mediates the export of excess cholesterol from macrophages, contributing to the prevention of atherosclerosis. Advanced glycated albumin (AGE-alb) is prevalent in diabetes mellitus and is associated with the development of atherosclerosis. Independently of changes in ABCA-1 mRNA levels, AGE-alb induces oxidative stress and reduces ABCA-1 protein levels, which leads to macrophage lipid accumulation. These metabolic conditions are known to elicit endoplasmic reticulum (ER) stress. We sought to determine if AGE-alb induces ER stress and unfolded protein response (UPR) in macrophages and how disturbances to the ER could affect ABCA-1 content and cholesterol efflux in macrophages. AGE-alb induced a time-dependent increase in ER stress and UPR markers. ABCA-1 content and cellular cholesterol efflux were reduced by 33% and 47%, respectively, in macrophages treated with AGE-alb, and both were restored by treatment with 4-phenyl butyric acid (a chemical chaperone that alleviates ER stress), but not MG132 (a proteasome inhibitor). Tunicamycin, a classical ER stress inductor, also impaired ABCA-1 expression and cholesterol efflux (showing a decrease of 61% and 82%, respectively), confirming the deleterious effect of ER stress in macrophage cholesterol accumulation. Glycoxidation induces macrophage ER stress, which relates to the reduction in ABCA-1 and in reverse cholesterol transport, endorsing the adverse effect of macrophage ER stress in atherosclerosis. Thus, chemical chaperones that alleviate ER stress may represent a useful tool for the prevention and treatment of atherosclerosis in diabetes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
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27. Protection against lethal leptospirosis after vaccination with LipL32 coupled or coadministered with the B subunit of Escherichia coli heat-labile enterotoxin.
- Author
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Grassmann AA, Félix SR, dos Santos CX, Amaral MG, Seixas Neto AC, Fagundes MQ, Seixas FK, da Silva EF, Conceição FR, and Dellagostin OA
- Subjects
- Animals, Bacterial Outer Membrane Proteins administration & dosage, Bacterial Toxins administration & dosage, Bacterial Vaccines administration & dosage, Cricetinae, Disease Models, Animal, Enterotoxins administration & dosage, Escherichia coli Proteins administration & dosage, Female, Leptospira interrogans immunology, Leptospirosis immunology, Leptospirosis mortality, Lipoproteins administration & dosage, Survival Analysis, Vaccination methods, Vaccines, Subunit administration & dosage, Vaccines, Subunit immunology, Vaccines, Synthetic administration & dosage, Vaccines, Synthetic immunology, Bacterial Outer Membrane Proteins immunology, Bacterial Toxins immunology, Bacterial Vaccines immunology, Enterotoxins immunology, Escherichia coli Proteins immunology, Leptospirosis prevention & control, Lipoproteins immunology
- Abstract
Leptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species of Leptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD(50)) of Leptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P < 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.
- Published
- 2012
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28. Protein disulfide isomerase redox-dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation.
- Author
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de A Paes AM, Veríssimo-Filho S, Guimarães LL, Silva AC, Takiuti JT, Santos CX, Janiszewski M, Laurindo FR, and Lopes LR
- Subjects
- Amino Acid Substitution, Anti-Bacterial Agents pharmacology, Bacitracin pharmacology, Cell Membrane genetics, Enzyme Activation drug effects, Enzyme Activation physiology, Enzyme Inhibitors pharmacology, Humans, Mutation, Missense, NADPH Oxidases genetics, Oxidation-Reduction drug effects, Protein Disulfide-Isomerases genetics, Protein Transport drug effects, Protein Transport physiology, Cell Membrane enzymology, Cytosol enzymology, NADPH Oxidases metabolism, Neutrophils enzymology, Protein Disulfide-Isomerases metabolism, Superoxides metabolism
- Abstract
Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI-mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell-free system, PDI inhibitors scrRNase (100 μg/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by ~40%), whereas PDIred diminished (by ~60%) superoxide generation. No change occurred after incubation with PDI serine-mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by ~70%. Thus, oxidized PDI supports, whereas reduced PDI down-regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol-mediated interaction with p47(phox) in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47(phox). PDI-p47(phox) association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47(phox) in cytosol. Incubation of purified PDI (>80% reduced) and p47(phox) in vitro promoted their arachidonate-dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re)cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox-dependent enzyme complex organizer.
- Published
- 2011
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29. Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo.
- Author
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Ray R, Murdoch CE, Wang M, Santos CX, Zhang M, Alom-Ruiz S, Anilkumar N, Ouattara A, Cave AC, Walker SJ, Grieve DJ, Charles RL, Eaton P, Brewer AC, and Shah AM
- Subjects
- Angiotensin II pharmacology, Animals, Endothelium, Vascular physiology, Hydrogen Peroxide metabolism, Male, Mice, Mice, Transgenic, NADPH Oxidase 4, Nitric Oxide physiology, Reactive Oxygen Species metabolism, Blood Pressure, Endothelium, Vascular enzymology, NADPH Oxidases physiology, Vasodilation
- Abstract
Objective: Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H(2)O(2)), but its role in vivo remains unclear., Methods and Results: We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H(2)O(2) production and H(2)O(2)-induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants., Conclusion: Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H(2)O(2) production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms.
- Published
- 2011
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30. Redox signaling in cardiac myocytes.
- Author
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Santos CX, Anilkumar N, Zhang M, Brewer AC, and Shah AM
- Subjects
- Animals, Cell Differentiation physiology, Cell Proliferation, Heart Diseases metabolism, Heart Diseases physiopathology, Humans, Hypertrophy metabolism, Hypertrophy physiopathology, Mice, Mitochondria metabolism, Myocardial Ischemia metabolism, Myocardial Ischemia physiopathology, NADPH Oxidases metabolism, Oxidative Stress, Oxygen metabolism, Rabbits, Rats, Thioredoxins metabolism, Excitation Contraction Coupling physiology, Myocytes, Cardiac physiology, Oxidation-Reduction, Reactive Oxygen Species metabolism, Stress, Physiological
- Abstract
The heart has complex mechanisms that facilitate the maintenance of an oxygen supply-demand balance necessary for its contractile function in response to physiological fluctuations in workload as well as in response to chronic stresses such as hypoxia, ischemia, and overload. Redox-sensitive signaling pathways are centrally involved in many of these homeostatic and stress-response mechanisms. Here, we review the main redox-regulated pathways that are involved in cardiac myocyte excitation-contraction coupling, differentiation, hypertrophy, and stress responses. We discuss specific sources of endogenously generated reactive oxygen species (e.g., mitochondria and NADPH oxidases of the Nox family), the particular pathways and processes that they affect, the role of modulators such as thioredoxin, and the specific molecular mechanisms that are involved-where this knowledge is available. A better understanding of this complex regulatory system may allow the development of more specific therapeutic strategies for heart diseases., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2011
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31. Evaluation of mild hyperhomocysteinemia during the development of atherosclerosis in apolipoprotein E-deficient and normal mice.
- Author
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Aléssio AC, Santos CX, Debbas V, Oliveira LC, Haddad R, and Annichino-Bizzacchi JM
- Subjects
- Animals, Apolipoproteins E genetics, Atherosclerosis pathology, Atherosclerosis prevention & control, Diet, Endoplasmic Reticulum drug effects, Endoplasmic Reticulum pathology, Folic Acid pharmacology, Hyperhomocysteinemia chemically induced, Hyperhomocysteinemia physiopathology, Male, Methionine pharmacology, Mice, Mice, Inbred C57BL, Mice, Knockout, Time Factors, Apolipoproteins E deficiency, Atherosclerosis complications, Hyperhomocysteinemia complications
- Abstract
We focused on the effect of mild hyperhomocysteinemia (HHcy) on the development of atherosclerosis, using apolipoprotein E-deficient (apoE(-/-)) and normal mice. Mice received diets enriched in methionine with low or high levels of folate, B(12) and B(6) (diets B and C, respectively), and diet only with low levels of folate, B(12) and B(6) (diets D), to induce mild HHcy. Normal mice fed on diets B, C and D presented mild HHcy, but they did not develop atherosclerotic lesions after 24 weeks of diet. In addition, increased endoplasmic reticulum stress was present in normal mice fed on diet B, compared to others groups. ApoE(-/-) mice fed on diet B for 20 weeks presented the greatest atherosclerotic lesion area at the aortic sinus than other groups. These results suggest that the methionine may have a toxic effect on endothelium, and the B-vitamins addition on diet may have a protective effect in the long term, despite the increase on homocysteine levels. Mild HHcy accelerated the development of atherosclerosis in apoE(-/-) mice, and supplementation with B-vitamins is important for prevention of vascular disease, principally in the long term., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2011
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32. Protein disulfide isomerase and host-pathogen interaction.
- Author
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Stolf BS, Smyrnias I, Lopes LR, Vendramin A, Goto H, Laurindo FR, Shah AM, and Santos CX
- Subjects
- Animals, Cytosol metabolism, Endoplasmic Reticulum metabolism, Humans, NADPH Oxidases metabolism, Oxidation-Reduction, Protein Transport, Host-Pathogen Interactions, Protein Disulfide-Isomerases metabolism
- Abstract
Reactive oxygen species (ROS) production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i) pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation) and (ii) phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER) and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.
- Published
- 2011
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33. NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis.
- Author
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Zhang M, Brewer AC, Schröder K, Santos CX, Grieve DJ, Wang M, Anilkumar N, Yu B, Dong X, Walker SJ, Brandes RP, and Shah AM
- Subjects
- Animals, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Mice, Mice, Transgenic, Myocardium metabolism, NADPH Oxidase 4, NADPH Oxidases genetics, Vascular Endothelial Growth Factor A metabolism, Heart physiopathology, NADPH Oxidases metabolism, Neovascularization, Physiologic, Stress, Physiological
- Abstract
Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses, but the role of different ROS sources remains unclear. Here we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level, which increases in cardiomyocytes under stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models, but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy, and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte hypoxia inducible factor 1 and the release of vascular endothelial growth factor, resulting in increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a unique inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of nonspecific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.
- Published
- 2010
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34. Mechanisms and implications of reactive oxygen species generation during the unfolded protein response: roles of endoplasmic reticulum oxidoreductases, mitochondrial electron transport, and NADPH oxidase.
- Author
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Santos CX, Tanaka LY, Wosniak J, and Laurindo FR
- Subjects
- Apoptosis physiology, Enzyme Activation, Homeostasis, Isoenzymes metabolism, Oxidation-Reduction, Protein Disulfide-Isomerases metabolism, Signal Transduction physiology, Stress, Physiological, Vascular Diseases metabolism, Electron Transport physiology, Endoplasmic Reticulum enzymology, Mitochondria metabolism, NADPH Oxidases metabolism, Oxidoreductases metabolism, Reactive Oxygen Species metabolism
- Abstract
Cellular mechanisms governing redox homeostasis likely involve their integration with other stresses. Endoplasmic reticulum (ER) stress triggers complex adaptive or proapoptotic signaling defined as the unfolded protein response (UPR), involved in several pathophysiological processes. Since protein folding is highly redox-dependent, convergence between ER stress and oxidative stress has attracted interest. Evidence suggests that ROS production and oxidative stress are not only coincidental to ER stress, but are integral UPR components, being triggered by distinct types of ER stressors and contributing to support proapoptotic, as well as proadaptive UPR signaling. Thus, ROS generation can be upstream or downstream UPR targets and may display a UPR-specific plus a nonspecific component. Enzymatic mechanisms of ROS generation during UPR include: (a) Multiple thiol-disulfide exchanges involving ER oxidoreductases including flavooxidase Ero1 and protein disulfide isomerase (PDI); (b) Mitochondrial electron transport; (c) Nox4 NADPH oxidase complex, particularly Nox4. Understanding the roles of such mechanisms and how they interconnect with the UPR requires more investigation. Integration among such ROS sources may depend on Ca(2+) levels, ROS themselves, and PDI, which associates with NADPH oxidase and regulates its function. Oxidative stress may frequently integrate with a background of ER stress/UPR in several diseases; here we discuss a focus in the vascular system.
- Published
- 2009
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35. Protein disulfide isomerase (PDI) associates with NADPH oxidase and is required for phagocytosis of Leishmania chagasi promastigotes by macrophages.
- Author
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Santos CX, Stolf BS, Takemoto PV, Amanso AM, Lopes LR, Souza EB, Goto H, and Laurindo FR
- Subjects
- Animals, Anti-Bacterial Agents, Brefeldin A pharmacology, Cricetinae, Enzyme Inhibitors pharmacology, Male, Mice, Protein Disulfide-Isomerases antagonists & inhibitors, Golgi Apparatus enzymology, Leishmania, Leishmaniasis enzymology, Multienzyme Complexes metabolism, NADH, NADPH Oxidoreductases metabolism, Phagocytosis, Protein Disulfide-Isomerases metabolism
- Abstract
PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref-A, an ER-Golgi-disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild-type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss- or gain-of-function experiments indicated that PMA-driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi.
- Published
- 2009
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- View/download PDF
36. A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation.
- Author
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de Souza Malaspina TS, Zambuzzi WF, dos Santos CX, Campanelli AP, Laurindo FR, Sogayar MC, and Granjeiro JM
- Subjects
- Alkaline Phosphatase analysis, Biomarkers analysis, Blotting, Western, Calcification, Physiologic physiology, Calcium Phosphates analysis, Cell Count, Cell Differentiation physiology, Cell Line, Cell Proliferation, Chromatography, High Pressure Liquid, Dinitrobenzenes, Glutathione analysis, Haptens, Humans, Nitrophenols, Organophosphorus Compounds, Oxidation-Reduction, Oxidative Stress physiology, Phenotype, Protein Tyrosine Phosphatases analysis, Proto-Oncogene Proteins analysis, Time Factors, Glutathione metabolism, Osteoblasts physiology, Protein Tyrosine Phosphatases metabolism, Proto-Oncogene Proteins metabolism
- Abstract
Objective: Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels., Design: hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays., Results: LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter., Conclusions: Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. In this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation.
- Published
- 2009
- Full Text
- View/download PDF
37. Cross-talk between mitochondria and NADPH oxidase: effects of mild mitochondrial dysfunction on angiotensin II-mediated increase in Nox isoform expression and activity in vascular smooth muscle cells.
- Author
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Wosniak J Jr, Santos CX, Kowaltowski AJ, and Laurindo FR
- Subjects
- Angiotensin II pharmacology, Animals, Base Sequence, Blotting, Western, Cell Line, Comet Assay, DNA Damage drug effects, DNA, Mitochondrial drug effects, Gene Expression Regulation, Enzymologic drug effects, Glutathione metabolism, Humans, Isoenzymes genetics, Mitochondria drug effects, Molecular Sequence Data, Myocytes, Smooth Muscle drug effects, NADPH Oxidases genetics, Nitrogen Oxides metabolism, Oxygen Consumption genetics, Oxygen Consumption physiology, Polymerase Chain Reaction, Rabbits, Reactive Oxygen Species metabolism, Vasoconstrictor Agents pharmacology, Isoenzymes metabolism, Mitochondria metabolism, Muscle, Smooth, Vascular cytology, Myocytes, Smooth Muscle enzymology, Myocytes, Smooth Muscle metabolism, NADPH Oxidases metabolism
- Abstract
Mitochondria and NADPH oxidase activation are concomitantly involved in pathogenesis of many vascular diseases. However, possible cross-talk between those ROS-generating systems is unclear. We induced mild mitochondrial dysfunction due to mitochondrial DNA damage after 24 h incubation of rabbit aortic smooth muscle (VSMC) with 250 ng/mL ethidium bromide (EtBr). VSMC remained viable and had 29% less oxygen consumption, 16% greater baseline hydrogen peroxide, and unchanged glutathione levels. Serum-stimulated proliferation was unaltered at 24 h. Although PCR amplification of several mtDNA sequences was preserved, D-Loop mtDNA region showed distinct amplification of shorter products after EtBr. Such evidence for DNA damage was further enhanced after angiotensin-II (AngII) incubation. Remarkably, the normally observed increase in VSMC membrane fraction NADPH oxidase activity after AngII was completely abrogated after EtBr, together with failure to upregulate Nox1 mRNA expression. Conversely, basal Nox4 mRNA expression increased 1.6-fold, while being unresponsive to AngII. Similar loss in AngII redox response occurred after 24 h antimycin-A incubation. Enhanced Nox4 expression was unassociated with endoplasmic reticulum stress markers. Protein disulfide isomerase, an NADPH oxidase regulator, exhibited increased expression and inverted pattern of migration to membrane fraction after EtBr. These results unravel functionally relevant cross-talk between mitochondria and NADPH oxidase, which markedly affects redox responses to AngII.
- Published
- 2009
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- View/download PDF
38. Tartrate-resistant acid phosphatase activity and glutathione levels are modulated during hFOB 1.19 osteoblastic differentiation.
- Author
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de Souza Malaspina TS, dos Santos CX, Campanelli AP, Laurindo FR, Sogayar MC, and Granjeiro JM
- Subjects
- Animals, Calcification, Physiologic, Cell Line, Cell Proliferation, Cell Shape, Extracellular Matrix chemistry, Extracellular Matrix metabolism, Humans, Osteoblasts cytology, Oxidative Stress, Tartrate-Resistant Acid Phosphatase, Acid Phosphatase metabolism, Cell Differentiation physiology, Glutathione metabolism, Isoenzymes metabolism, Osteoblasts physiology
- Abstract
Tartrate-resistant acid phosphatase (TRAP) is a well-known marker of osteoclasts and bone resorption. Here we have investigated whether osteoblast-like cells (hFOB 1.19) present TRAP activity and how would be its pattern of expression during osteoblastic differentiation. We also observed how the osteoblastic differentiation affected the reduced glutathione levels. TRAP activity was measured using the p-nitrophenylphosphate substrate. The osteogenic potential of hFOB 1.19 cells was studied by measuring alkaline phosphatase activity and mineralized nodule formation. Oxidative stress was determined by HPLC and DNTB assays. TRAP activity and the reduced glutathione-dependent microenvironment were modulated during osteoblastic differentiation. During this phase, TRAP activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day, decreasing thereafter. We demonstrate that TRAP activity is modulated during osteoblastic differentiation, possibly in response to the redox state of the cell, since it seemed to depend on suitable levels of reduced glutathione.
- Published
- 2008
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39. Novel role of protein disulfide isomerase in the regulation of NADPH oxidase activity: pathophysiological implications in vascular diseases.
- Author
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Laurindo FR, Fernandes DC, Amanso AM, Lopes LR, and Santos CX
- Subjects
- Endothelium, Vascular enzymology, Humans, Muscle, Smooth, Vascular cytology, Oxidation-Reduction, Protein Disulfide-Isomerases chemistry, Protein Disulfide-Isomerases metabolism, Signal Transduction, Sulfhydryl Compounds chemistry, Muscle, Smooth, Vascular enzymology, NADPH Oxidases metabolism, Protein Disulfide-Isomerases physiology, Vascular Diseases etiology
- Abstract
Vascular cell NADPH oxidase complexes are key sources of signaling reactive oxygen species (ROS) and contribute to disease pathophysiology. However, mechanisms that fine-tune oxidase-mediated ROS generation are incompletely understood. Besides known regulatory subunits, upstream mediators and scaffold platforms reportedly control and localize ROS generation. Some evidence suggest that thiol redox processes may coordinate oxidase regulation. We hypothesized that thiol oxidoreductases are involved in this process. We focused on protein disulfide isomerase (PDI), a ubiquitous dithiol disulfide oxidoreductase chaperone from the endoplasmic reticulum, given PDI's unique versatile role as oxidase/isomerase. PDI is also involved in protein traffic and can translocate to the cell surface, where it participates in cell adhesion and nitric oxide internalization. We recently provided evidence that PDI exerts functionally relevant regulation of NADPH oxidase activity in vascular smooth muscle and endothelial cells, in a thiol redox-dependent manner. Loss-of-function experiments indicate that PDI supports angiotensin II-mediated ROS generation and Akt phosphorylation. In addition, PDI displays confocal co-localization and co-immunoprecipitates with oxidase subunits, indicating close association. The mechanisms of such interaction are yet obscure, but may involve subunit assembling stabilization, assistance with traffic, and subunit disposal. These data may clarify an integrative view of oxidase activation in disease conditions, including stress responses.
- Published
- 2008
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- View/download PDF
40. Vitamin C improves the effect of a new nitric oxide donor on the vascular smooth muscle from renal hypertensive rats.
- Author
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Rodrigues GJ, Lunardi CN, Lima RG, Santos CX, Laurindo FR, da Silva RS, and Bendhack LM
- Subjects
- Animals, Aorta pathology, Calcium analysis, Calcium metabolism, Disease Models, Animal, Dose-Response Relationship, Drug, Drug Synergism, Kidney blood supply, Male, Muscle, Smooth, Vascular pathology, Muscle, Smooth, Vascular physiopathology, Nitric Oxide analysis, Nitric Oxide metabolism, Organ Culture Techniques, Phenylephrine pharmacology, Rats, Rats, Wistar, Ruthenium chemistry, Superoxides analysis, Superoxides metabolism, Time Factors, Vasodilation drug effects, Aorta drug effects, Ascorbic Acid pharmacology, Hypertension, Renal physiopathology, Muscle, Smooth, Vascular drug effects, Nitric Oxide Donors pharmacology, Organometallic Compounds pharmacology
- Abstract
Impaired relaxation induced by the new nitric oxide (NO) donor [Ru(NH.NHq)(terpy)NO(+)](3+) (TERPY) has been observed in the aortic rings from renal hypertensive rats (2K-1C). An increased production of reactive oxygen species (ROS) in the aortas from 2K-1C rats are capable of reducing NO bioavailability. Therefore, this study aimed at investigating the effects of an antioxidant (vitamin C) on the relaxant effect of NO released from TERPY on the 2K-1C rat aorta. As for vascular reactivity, the potency of TERPY is greater in the control rats (2K) than in 2K-1C whereas the maximum relaxation (ME) is not significantly different between the 2K and 2K-1C rat aortas. The relaxation of TERPY is potentiated only in the 2K-1C aortic ring treated with vitamin C. TERPY has a lower effect in decreasing cytosolic Ca(2+) concentration ([Ca(2+)]c) in vascular smooth muscle cells (VSMCs) from 2K-1C rats. This effect is also potentiated in 2K-1C aortic cells treated with vitamin C, but it is not altered in 2K cells. The basal cytosolic NO concentration ([NO]c) is lower in 2K-1C than in 2K cells, and the bioavailability of the NO released from TERPY is larger in 2K than in 2K-1C VSMCs. The superoxide radical concentration ([O(2)(*-)]) is higher in the 2K-1C aorta, and vitamin C reduces the [O(2)(*-)] in the 2K-1C aorta. Taken together, these results show that in the aortas of renal hypertensive 2K-1C rats, released NO from the new NO donor is not available to produce a similar effect in 2K aorta due to increased [O(2)(*-)].
- Published
- 2008
- Full Text
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41. Assessment of superoxide production and NADPH oxidase activity by HPLC analysis of dihydroethidium oxidation products.
- Author
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Laurindo FR, Fernandes DC, and Santos CX
- Subjects
- Animals, Cells, Cultured, Chromatography, High Pressure Liquid methods, Ethidium metabolism, Humans, Oxidation-Reduction, Chemistry Techniques, Analytical methods, Ethidium analogs & derivatives, NADPH Oxidases physiology, Superoxides metabolism
- Abstract
Assessment of low-level superoxide in nonphagocytic cells is crucial for assessing redox-dependent signaling pathways and the role of enzymes such as the NADPH oxidase complex. However, most superoxide probes present inherent limitations. Particularly, assessment of dihydroethidium (DHE) fluorescence is limited regarding a lack of possible quantification and simultaneous detection of its two main products: 2-hydroxyethidium, more specific for superoxide, and ethidium, which reflects H2O2-dependent pathways involving metal proteins. HPLC separation and analysis of those two main products have been described. This chapter reports procedures used for the validation of superoxide measurements in vascular system. Superoxide assessment was performed for cultured cells and tissue fragments incubated with DHE, followed by acetonitrile extraction and HPLC run, with simultaneous fluorescence detection of 2-hydroxyethidium and ethidium and ultraviolet detection of remaining DHE. It also describes procedures for DHE-based NADPH oxidase activity assays using HPLC or fluorometry. Such methods can enhance accuracy and allow better quantitation of vascular superoxide measurements.
- Published
- 2008
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42. Isoflavone and the heme oxygenase system in ischemic acute kidney injury in rats.
- Author
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Watanabe M, de Moura Neiva LB, da Costa Santos CX, Martins Laurindo FR, and de Fátima Fernandes Vattimo M
- Subjects
- Animals, Catalase metabolism, Isoflavones administration & dosage, Isoflavones pharmacology, Isoflavones therapeutic use, Kidney drug effects, Kidney metabolism, Kidney Diseases prevention & control, Male, Malondialdehyde metabolism, Oxidative Stress drug effects, Plant Extracts administration & dosage, Plant Extracts therapeutic use, Protective Agents administration & dosage, Protective Agents therapeutic use, Rats, Rats, Wistar, Renal Circulation drug effects, Reperfusion Injury prevention & control, Sulfhydryl Compounds metabolism, Heme Oxygenase-1 drug effects, Phytotherapy, Plant Extracts pharmacology, Protective Agents pharmacology, Glycine max
- Abstract
Ischemic acute kidney injury (AKI) is mediated by reactive oxygen species and inflammatory infiltration, among others. The present study was performed to evaluate the interference of isoflavone in ischemic AKI in rats and to determine whether it was mediated by the heme oxygenase (HO) system. The treatment with dry extract of isoflavone, 8 mg/kg/day, resulted in improved renal function reducing oxidative damage. The use of HO inhibitor zinc protoporphyrin (ZnPP), 50 micromol/kg, exacerbated the rates of oxidative damage, urinary peroxides, malondialdehyde (MDA), thiols and catalyse activity, whereas an HO inducer, Hemin, 1 mg/100 g body weight, demonstrated a protective effect on renal function, with a reduction in the rates of oxidative damage. Isoflavone showed a promising role in the protection against oxidative renal dysfunction and its role is probably mediated by heme-oxygenase.
- Published
- 2007
- Full Text
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43. Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems.
- Author
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Fernandes DC, Wosniak J Jr, Pescatore LA, Bertoline MA, Liberman M, Laurindo FR, and Santos CX
- Subjects
- Animals, Blood Vessels enzymology, Catheterization, Cell Line, Transformed, Chromatography, High Pressure Liquid, Ethidium metabolism, Fluorescence, Iliac Artery injuries, Male, Muscle, Smooth, Vascular enzymology, Muscle, Smooth, Vascular metabolism, Myocytes, Smooth Muscle enzymology, Myocytes, Smooth Muscle metabolism, Oxidation-Reduction, Peroxidase metabolism, Rabbits, Wounds and Injuries enzymology, Wounds and Injuries etiology, Blood Vessels metabolism, Ethidium analogs & derivatives, NADPH Oxidases metabolism, Superoxides metabolism
- Abstract
Dihydroethidium (DHE) is a widely used sensitive superoxide (O2(*-)) probe. However, DHE oxidation yields at least two fluorescent products, 2-hydroxyethidium (EOH), known to be more specific for O2(*-), and the less-specific product ethidium. We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree peroxynitrite/carbon dioxide system led to EOH and ethidium formation. Peroxidase/H2O2 but not H2O2 alone yielded ethidium as the main product. In vascular smooth muscle cells incubated with ANG II (100 nM, 4 h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in vascular smooth muscle cell membrane fractions, showing that EOH was uniquely increased after ANG II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was approximately 1.5- to 2-fold increase in EOH/DHE and ethidium/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of ethidium product and EOH/ethidium ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in ethidium generation. Thus HPLC analysis of DHE-derived oxidation products can improve assessment of O2(*-) production or NADPH oxidase activity in many vascular experimental studies.
- Published
- 2007
- Full Text
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44. Regulation of NAD(P)H oxidase by associated protein disulfide isomerase in vascular smooth muscle cells.
- Author
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Janiszewski M, Lopes LR, Carmo AO, Pedro MA, Brandes RP, Santos CX, and Laurindo FR
- Subjects
- Acridines, Angiotensin II pharmacology, Animals, Aorta, Bacitracin pharmacology, Binding Sites, Blotting, Western, Cell Line, Cell Line, Transformed, Cell Membrane enzymology, Enzyme Activation drug effects, Enzyme Inhibitors pharmacology, Gene Expression, Green Fluorescent Proteins genetics, Homeostasis, Humans, Luminescent Measurements, Microscopy, Confocal, NADPH Oxidases analysis, Oligonucleotides, Antisense genetics, Oxidation-Reduction, Protein Disulfide-Isomerases antagonists & inhibitors, Protein Disulfide-Isomerases genetics, Protein Folding, Protein Subunits metabolism, Rabbits, Recombinant Fusion Proteins, Superoxides metabolism, Transfection, Muscle, Smooth, Vascular enzymology, NADPH Oxidases metabolism, Protein Disulfide-Isomerases physiology
- Abstract
NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22(phox), Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22(phox), Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.
- Published
- 2005
- Full Text
- View/download PDF
45. Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation.
- Author
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Linares E, Giorgio S, Mortara RA, Santos CX, Yamada AT, and Augusto O
- Subjects
- Animals, Carbon Dioxide metabolism, Chromatography, High Pressure Liquid, Dihydroxyphenylalanine metabolism, Electron Spin Resonance Spectroscopy, Hydroxylation, Leishmaniasis pathology, Macrophage Activation, Macrophages drug effects, Macrophages metabolism, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Microscopy, Confocal, Microscopy, Fluorescence, Nitric Oxide metabolism, Nitric Oxide Synthase Type II, Peroxidase metabolism, Tyrosine metabolism, Carbonates metabolism, Free Radicals metabolism, Leishmania infantum pathogenicity, Leishmaniasis metabolism, Macrophages parasitology, Nitric Oxide biosynthesis, Nitric Oxide Synthase metabolism, Peroxynitrous Acid metabolism, Tyrosine analogs & derivatives
- Abstract
The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.
- Published
- 2001
- Full Text
- View/download PDF
46. Direct evidence for apo B-100-mediated copper reduction: studies with purified apo B-100 and detection of tryptophanyl radicals.
- Author
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Batthyány C, Santos CX, Botti H, Cerveñansky C, Radi R, Augusto O, and Rubbo H
- Subjects
- Apolipoprotein B-100, Apolipoproteins B isolation & purification, Chromatography, High Pressure Liquid, Electron Spin Resonance Spectroscopy, Free Radicals metabolism, Humans, Kinetics, Lipoproteins, LDL isolation & purification, Lipoproteins, LDL metabolism, Nitroso Compounds metabolism, Oxidation-Reduction, Spin Trapping, Tryptophan metabolism, Apolipoproteins B metabolism, Copper metabolism
- Abstract
Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely alpha-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of alpha-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.
- Published
- 2000
- Full Text
- View/download PDF
47. Role of the carbonate radical anion in tyrosine nitration and hydroxylation by peroxynitrite.
- Author
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Santos CX, Bonini MG, and Augusto O
- Subjects
- Anions metabolism, Bicarbonates metabolism, Carbon Dioxide metabolism, Chromatography, High Pressure Liquid, Electron Spin Resonance Spectroscopy, Free Radicals analysis, Hydrogen-Ion Concentration, Hydroxyl Radical metabolism, Hydroxylation, Kinetics, Nitric Oxide metabolism, Spectrophotometry, Ultraviolet, Tyrosine analysis, Carbonates metabolism, Free Radicals metabolism, Nitrates metabolism, Tyrosine analogs & derivatives, Tyrosine metabolism
- Abstract
Peroxynitrite has been receiving increasing attention as the pathogenic mediator of nitric oxide cytotoxicity. In most cases, the contribution of peroxynitrite to diseases has been inferred from detection of 3-nitrotyrosine in injured tissues. However, presently it is known that other nitric oxide-derived species can also promote protein nitration. Mechanistic details of protein nitration remain under discussion even in the case of peroxynitrite, although recent literature data strongly suggest a free radical mechanism. Here, we confirm the free radical mechanism of tyrosine modification by peroxynitrite in the presence and in the absence of the bicarbonate-carbon dioxide pair by analyzing the stable tyrosine products and the formation of the tyrosyl radical at pH 5.4 and 7.4. Stable products, 3-nitrotyrosine, 3-hydroxytyrosine, and 3, 3-dityrosine, were identified by high performance liquid chromatography and UV spectroscopy. The tyrosyl radical was detected by continuous-flow and spin-trapping electron paramagnetic resonance (EPR). 3-Hydroxytyrosine was detected at pH 5.4 and its yield decreased in the presence of the bicarbonate-carbon dioxide pair. In contrast, the yields of the tyrosyl radical increased in the presence of the bicarbonate-carbon dioxide pair and correlated with the yields of 3-nitrotyrosine under all tested experimental conditions. Taken together, the results demonstrate that the promoting effects of carbon dioxide on peroxynitrite-mediated tyrosine nitration is due to the selective reactivity of the carbonate radical anion as compared with that of the hydroxyl radical. Colocalization of 3-hydroxytyrosine and 3-nitrotyrosine residues in proteins may be useful to discriminate between peroxynitrite and other nitrating species., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
48. Uric acid oxidation by peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation.
- Author
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Santos CX, Anjos EI, and Augusto O
- Subjects
- Allantoin metabolism, Alloxan metabolism, Cyclic N-Oxides metabolism, Electron Spin Resonance Spectroscopy, Free Radical Scavengers metabolism, Horseradish Peroxidase metabolism, Humans, Hydrogen Peroxide metabolism, Hydrogen-Ion Concentration, Kinetics, Lipid Peroxides metabolism, Lipoproteins, LDL metabolism, Liposomes metabolism, Nitrous Acid metabolism, Oxidation-Reduction, Oxygen metabolism, Peroxynitrous Acid, Thiobarbituric Acid Reactive Substances metabolism, Free Radicals metabolism, Lipid Metabolism, Nitrates metabolism, Oxidants metabolism, Uric Acid metabolism
- Abstract
Uric acid has been considered to be an efficient scavenger of peroxynitrite but the reaction between urate and peroxynitrite has been only partially characterized. Also, previous studies have indicated that urate may increase peroxynitrite-mediated oxidation of low density lipoprotein (LDL). Here, we examined the reaction between urate and peroxynitrite by combining kinetic, oxygen consumption, spin trapping, and product identification studies; in parallel, we tested the effect of urate upon peroxynitrite-mediated lipid oxidation. Our results demonstrated that urate reacts with peroxynitrite with an apparent second order rate constant of 4.8 x 10(2) M(-1). s(-1) in a complex process, which is accompanied by oxygen consumption and formation of allantoin, alloxan, and urate-derived radicals. The main radical was identified as the aminocarbonyl radical by the electrospray mass spectra of its 5, 5-dimethyl-l-pyrroline N-oxide adduct. Mechanistic studies suggested that urate reacts with peroxynitrous acid and with the radicals generated from its decomposition to form products that can further react with peroxynitrite anion. These many reactions may explain the reported efficiency of urate in inhibiting some peroxynitrite-mediated processes. Production of the aminocarbonyl radical, however, may propagate oxidative reactions. We demonstrated that this radical is likely to be the species responsible for the effects of urate in amplifying peroxynitrite-mediated oxidation of liposomes and LDL, which was monitored by the formation of lipid peroxides and thiobarbituric acid-reactive substances. The aminocarbonyl radical was not detectable during urate attack by other oxidants and consequently it is unlikely to be responsible for all previously described prooxidant effects of uric acid., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
49. Toxic effects of alcohol intake on prostate of rats.
- Author
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Novelli EL, Rodrigues NL, Santos CX, Martinez FE, and Novelli JL
- Subjects
- Alanine Transaminase blood, Alkaline Phosphatase blood, Animals, Body Weight drug effects, Male, Prostate metabolism, Rats, Rats, Wistar, Superoxide Dismutase blood, Alcohol Drinking blood, Ethanol adverse effects, Prostate drug effects
- Abstract
Background: The present report was carried out to determine whether alcohol intake could induce prostate lesions., Methods: We tested male rats for 300 days. Animals were divided into three groups: controls received only tap water as liquid diet; the chronic alcohol intake group received only ethanol solution in semivoluntary research; and the withdrawal group received the same treatment as chronic alcohol intake until 240 days, after which they reverted to drinking water., Results: Chronic alcohol intake increased lipoperoxide concentrations and acid phosphatase activities. Cu-Zn superoxide dismutase (SOD) was decreased at 60 days, but approached controls values at 300 days following treatment. The serum increased alkaline phosphatase, and alanine transaminase activities reflected the chronic toxic effect of ethanol., Conclusions: Since SOD activity was unable to scavenge superoxide radical and lipoperoxide formation, we can conclude that superoxide is an important intermediate in prostate damage of chronic alcohol intake.
- Published
- 1997
- Full Text
- View/download PDF
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