Abstract P342: Mitochondria-Targeted Antioxidant Therapy Prevents Angiotensin II--Mediated Connexin43 Remodeling and Sudden Arrhythmic Death

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Ali A Sovari ◽  
Shadi Zandieh ◽  
Shahriar Iravanian ◽  
Lianzhi Gu ◽  
Euy-Myong Jeong ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Protein Carbonyl ◽  
Oxidase Inhibitor ◽  
Control Treatment ◽  
Arrhythmic Death ◽  
Protein Levels ◽  
Mitochondrial Superoxide ◽  
Remodeling Suppression ◽  
The Right

Introduction: Angiotensin II activation and associated elevation in ROS have been implicated in pathogenesis of arrhythmia. Nevertheless commonly used antioxidants have been ineffective in clinical trials. We created a transgenic mouse model of cardiac restricted overexpression of ACE (ACE8/8). These mice show spontaneous VT/ VF, SCD, and a reduction in Cx43 level, which impairs conduction and predisposes to arrhythmia. We sought to determine the role and the major source of ROS by angiotensin II in VT/ VF and Cx43 remodeling. Method: Wild type and ACE8/8 mice with and without 2 weeks of treatment with a NOS inhibitor (L-NIO, 25mg/Kg IP injections daily), a mitochondria-targeted antioxidant (Mito-TEMPO, 0.7mg/Kg IP injections daily), a NADPH oxidase inhibitor (Apocynin 80mg/L in drinking water), and ACE8/8 crossed with P67DN were studied. Western blotting (with derivatization to dinitrophenylhydrozone to detect oxidized protein levels), detection of superoxide production in mitochondria by red mitochondrial superoxide indicator and immunohistochemistry staining for Cx43 were performed. EP study was performed by a 1.1F octapolar catheter through pacing the right ventricle using a burst pacing protocol. Results: Proteins were more oxidized (increased protein-carbonyl detection), and Cx43 was reduced in ACE8/8 to 33% of control. Treatment with Mito-TEMPO prevented SCD and improved survival in ACE8/8 mice (p=0.0005, hazard ratio 4.76 with 95% CI of 1.96 to 11.53). Inducibility of VT/VF was higher in ACE8/8 mice compare to WT (87.5% vs. 2.3%) and VT inducibility was reduced with Mito-TEMPO treatment (50% in treatment group). Cx43 level was increased by 1.7 fold with Mito-TEMPO treatment. Treatments with L-NIO, Apocynin and crossing with P67DN mice did not prevent VT/VF and SCD in ACE8/8 mice. Conclusion: In a model of angiotensin II activation mitochondria-targeted antioxidant, prevents VT/VF/SCD and Cx43 remodeling. Suppression of NADPH oxidase activity by Apocynin and crossing the ACE8/8 mice with P67DN or inhibition of NOS by L-NIO did not prevent the arrhythmic deaths in ACE8/8 mice. This result suggests that mitochondria are the major source of ROS by angiotensin II and mitochondria-targeted antioxidants may be effective antiarrhythmic drugs.

Apoptotic cascade initiated by angiotensin II in neonatal cardiomyocytes: role of DNA damage

2003 ◽  
Vol 285 (6) ◽  
pp. H2364-H2372 ◽  
Author(s):  
Valentina Grishko ◽  
Viktor Pastukh ◽  
Viktoriya Solodushko ◽  
Mark Gillespie ◽  
Junichi Azuma ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Dna Damage ◽  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Ventricular Remodeling ◽  
Oxidase Inhibitor ◽  
Neonatal Cardiomyocytes ◽  
Diphenylene Iodonium ◽  
Nitric Oxide Inhibitor

Angiotensin II contributes to ventricular remodeling by promoting both cardiac hypertrophy and apoptosis; however, the mechanism underlying the latter phenomenon is poorly understood. One possibility that has been advanced is that angiotensin II activates NADPH oxidase, generating free radicals that trigger apoptosis. In apparent support of this notion, it was found that angiotensin II-mediated apoptosis in the cardiomyocyte is blocked by the NADPH oxidase inhibitor diphenylene iodonium. However, three lines of evidence suggest that peroxynitrite, rather than superoxide, is responsible for angiotensin II-mediated DNA damage and apoptosis. First, the inducible nitric oxide inhibitor aminoguanidine prevents angiotensin II-induced DNA damage and apoptosis. Second, based on ligation-mediated PCR, the pattern of angiotensin II-induced DNA damage resembles peroxynitritemediated damage rather than damage caused by either superoxide or nitric oxide. Third, angiotensin II activates p53 through the phosphorylation of Ser15 and Ser20, residues that are commonly phosphorylated in response to DNA damage. It is proposed that angiotensin II promotes the oxidation of DNA, which in turn activates p53 to mediate apoptosis.

scholarly journals Heat Shock Protein 70 and CHIP Promote Nox4 Ubiquitination and Degradation within the Losartan Antioxidative Effect in Proximal Tubule Cells

2015 ◽  
Vol 36 (6) ◽  
pp. 2183-2197 ◽  
Author(s):  
Andrea F. Gil Lorenzo ◽  
Valeria V. Costantino ◽  
Martin López Appiolaza ◽  
Valeria Cacciamani ◽  
Maria E. Benardon ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Proximal Tubule ◽  
Ros Generation ◽  
Antioxidative Effect ◽  
Proximal Tubule Cells ◽  
Caveolin 1 ◽  
Protein Levels ◽  
Hsp70 Protein

Background: Angiotensin II/Angiotensin II type 1 receptor (AT1R) effects are dependent on ROS production stimulated by NADPH oxidase activation. Hsp70 regulates a diverse set of signaling pathways through their interactions with proteins. CHIP is a E3 ubiquitin ligase that targets proteins for polyubiquitination and degradation. Aim: We study whether Hsp70/CHIP contribute to the negative regulation of Nox4 after AT1R blockage. Methods/Results: Primary culture of proximal tubule epithelial cells (PTCs) from SHR and WKY were stimulated with Angiotensin II (AII) or treated with Losartan (L) or Losartan plus Angiotensin II (L+AII). Losartan decreased AT1R and Nox4 while enhancing caveolin-1 and Hsp70 protein expression in SHR PTCs. Immunoprecipitation and immunofluorescence proved interaction and colocalization of increased Hsp70/CHIP with decreased Nox4 in SHR PTCs (L) vs (All). Hsp72 knockdown resulted in enhanced Nox4 protein levels, NADPH oxidase activity and ROS generation in (L+AII) revealing that Losartan was unable to abrogate AII effects on Nox4 expression and oxidative activity. Moreover, MG132 exposed PTCs (L) demostrated blocked ubiquitinated Nox4 degradation and increased colocalization of Nox4/Ubiquitin by inmunofluorescence. Conversely, Hsp72 depletion reduced Nox4/Ubiquitin colocalization causing Nox4 upregulation due to proteosomal degradation inhibition, although Losartan treatment. Conclusion: Our study demonstrates that Hsp70 and CHIP mediates the ubiquitination and proteasomal degradation of Nox4 as part of the antioxidative effect of Losartan in SHR.

scholarly journals NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa

2010 ◽  
Vol 298 (3) ◽  
pp. R707-R712 ◽  
Author(s):  
Yiling Fu ◽  
Rui Zhang ◽  
Deyin Lu ◽  
Haifeng Liu ◽  
Kiran Chandrashekar ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Primary Source ◽  
Oxidase Inhibitor ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
Ang Ii ◽  
Xanthine Oxidase Inhibitor ◽  
Si Rna ◽  
Full Activation

Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O2−) and angiotensin II (ANG II). We have reported that NaCl-induced O2−in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O2−in MD is unknown. Thus we determined the pathways by which ANG II increased O2−in the MD by measuring O2−in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O2−levels to increase by more than twofold ( P < 0.01). This increase was blocked by losartan (AT1receptor blocker) but not PD-123319 (AT2receptor antagonist). Apocynin (a NOX inhibitor) decreased O2−by 86% ( P < 0.01), whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a cyclooxygenase-2 inhibitor) had no significant effect. The NOX-dependent increase in O2−was due to the NOX2 isoform; a short interfering (si)RNA against NOX2 blunted ANG II-induced increases in O2−, whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O2−production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O2−production in the MD primarily via AT1-dependent activation of NOX2. Rac1 is required for the full activation of NOX2. This pathway may be an important component of ANG II enhancement of tubuloglomerular feedback.

Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase

2009 ◽  
Vol 296 (4) ◽  
pp. C693-C700 ◽  
Author(s):  
Caroline N. White ◽  
Gemma A. Figtree ◽  
Chia-Chi Liu ◽  
Alvaro Garcia ◽  
Elisha J. Hamilton ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
Pump Current ◽  
Oxidase Inhibitor ◽  
Ang Ii ◽  
Inhibitory Peptide ◽  
Myocyte Function ◽  
Nadph Oxidase Activation

The sarcolemmal Na+-K+ pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to ε-protein kinase C (εPKC), previously implicated in ANG II-induced Na+-K+ pump inhibition. A role for εPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of εPKC with the receptor for the activated kinase and with the cytosolic p47 phox subunit of NADPH oxidase. ANG II decreased electrogenic Na+-K+ pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by εPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na+-K+ pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The α1-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22 phox and cytosolic p47 phox NADPH oxidase subunits at baseline. ANG II had no effect on α1/caveolin 3 or α1/p22 phox interaction, but it increased α1/p47 phox coimmunoprecipitation. We conclude that ANG II inhibits the Na+-K+ pump via PKC-dependent NADPH oxidase activation.

scholarly journals Angiotensin II (AT1) Receptors and NADPH Oxidase Regulate Cl− Current Elicited by β1 Integrin Stretch in Rabbit Ventricular Myocytes

2004 ◽  
Vol 124 (3) ◽  
pp. 273-287 ◽  
Author(s):  
David M. Browe ◽  
Clive M. Baumgarten
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Ventricular Myocytes ◽  
Large Fraction ◽  
Oxidase Inhibitor ◽  
Β1 Integrin ◽  
At1 Receptors ◽  
Downstream Signaling ◽  
Nadph Oxidase Inhibitor ◽  
Paramagnetic Beads

Direct stretch of β1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl− current (Cl− SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl− SAC resemble those of the volume-sensitive Cl− current, ICl,swell. Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for β1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 μM), an AT1R competitive antagonist, blocked Cl− SAC but did not significantly alter the background Cl− current in the absence of integrin stretch. AT1R signaling is mediated largely by H2O2 produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 μM), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl− SAC elicited by stretch and the background Cl− current. A structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl− SAC as well as a large fraction of the background Cl− current. With continuing integrin stretch, Cl− SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl− current that was rapidly and completely blocked by DPI (60 μM). Moreover, exogenous H2O2 (10, 100, and 500 μM), the eventual product of NADPH oxidase activity, also activated Cl− SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H2O2 scavenger, attenuated the response to stretch. Application of H2O2 during NADPH oxidase inhibition by either DPI (60 μM) or AEBSF (0.5 mM) did not fully reactivate Cl− SAC, however. These results suggest that stretch of β1-integrin in cardiac myocytes elicits Cl− SAC by activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl− SAC, providing a second regulatory pathway.

scholarly journals Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells

2001 ◽  
Vol 280 (3) ◽  
pp. L442-L449 ◽  
Author(s):  
Karol R. Smith ◽  
Linda R. Klei ◽  
Aaron Barchowsky
Keyword(s):  
Endothelial Cells ◽  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Vascular Endothelial Cells ◽  
Oxidase Inhibitor ◽  
Vascular Endothelial ◽  
Effect Analysis ◽  
Aortic Endothelial Cells ◽  
Necrosis Factor

Low-level arsenite treatment of porcine aortic endothelial cells (PAEC) stimulated superoxide accumulation that was attenuated by inhibitors of NAD(P)H oxidase. To demonstrate whether arsenite stimulated NADPH oxidase, intact PAEC were treated with arsenite for up to 2 h and membrane fractions were prepared to measure NADPH oxidase activity. Arsenite (5 μM) stimulated a twofold increase in activity by 1 h, which was inhibited by the oxidase inhibitor diphenyleneiodonium chloride. Direct treatment of isolated membranes with arsenite had no effect. Analysis of NADPH oxidase components revealed that p67phoxlocalized exclusively to membranes of both control and treated cells. In contrast, cytosolic Rac1 translocated to the membrane fractions of cells treated with arsenite or angiotensin II but not with tumor necrosis factor. Immunodepletion of p67phoxblocked oxidase activity stimulated by all three compounds. However, depleting Rac1 inhibited responses only to arsenite and angiotensin II. These data demonstrate that stimulus-specific activation of NADPH oxidase in endothelial cells was the source of reactive oxygen in endothelial cells after noncytotoxic arsenite exposure.

scholarly journals Angiotensin II-induced upregulation of SGLT1 and 2 contributes to human microparticle‐stimulated endothelial senescence and dysfunction: protective effect of gliflozins

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Sin-Hee Park ◽  
Eugenia Belcastro ◽  
Hira Hasan ◽  
Kensuke Matsushita ◽  
Benjamin Marchandot ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Angiotensin Ii ◽  
Endothelial Dysfunction ◽  
Nadph Oxidase ◽  
Sglt2 Inhibitor ◽  
Ang Ii ◽  
Galactosidase Activity ◽  
Protein Levels ◽  
No Formation ◽  
Beta Galactosidase

Abstract Background Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduced cardiovascular risk in type 2 diabetes patients independently of glycemic control. Although angiotensin II (Ang II) and blood-derived microparticles are major mediators of cardiovascular disease, their impact on SGLT1 and 2 expression and function in endothelial cells (ECs) and isolated arteries remains unclear. Methods ECs were isolated from porcine coronary arteries, and arterial segments from rats. The protein expression level was assessed by Western blot analysis and immunofluorescence staining, mRNA levels by RT-PCR, oxidative stress using dihydroethidium, nitric oxide using DAF-FM diacetate, senescence by senescence-associated beta-galactosidase activity, and platelet aggregation by aggregometer. Microparticles were collected from blood of patients with coronary artery disease (CAD-MPs). Results Ang II up-regulated SGLT1 and 2 protein levels in ECs, and caused a sustained extracellular glucose- and Na+-dependent pro-oxidant response that was inhibited by the NADPH oxidase inhibitor VAS-2780, the AT1R antagonist losartan, sotagliflozin (Sota, SGLT1 and SGLT2 inhibitor), and empagliflozin (Empa, SGLT2 inhibitor). Ang II increased senescence-associated beta-galactosidase activity and markers, VCAM-1, MCP-1, tissue factor, ACE, and AT1R, and down-regulated eNOS and NO formation, which were inhibited by Sota and Empa. Increased SGLT1 and SGLT2 protein levels were observed in the rat aortic arch, and Ang II- and eNOS inhibitor-treated thoracic aorta segments, and were associated with enhanced levels of oxidative stress and prevented by VAS-2780, losartan, Sota and Empa. CAD-MPs promoted increased levels of SGLT1, SGLT2 and VCAM-1, and decreased eNOS and NO formation in ECs, which were inhibited by VAS-2780, losartan, Sota and Empa. Conclusions Ang II up-regulates SGLT1 and 2 protein expression in ECs and arterial segments to promote sustained oxidative stress, senescence and dysfunction. Such a sequence contributes to CAD-MPs-induced endothelial dysfunction. Since AT1R/NADPH oxidase/SGLT1 and 2 pathways promote endothelial dysfunction, inhibition of SGLT1 and/or 2 appears as an attractive strategy to enhance the protective endothelial function.

Abstract 410: Mitochondrial Uncoupling with CCCP Reverses Acute Hyperglycemia Induced Endothelial Dysfunction in Human Arterioles

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Jingli Wang ◽  
David D Gutterman ◽  
Michael E Widlansky
Keyword(s):  
Endothelial Dysfunction ◽  
Nadph Oxidase ◽  
Endothelial Function ◽  
Mitochondrial Membrane ◽  
Oxidase Inhibitor ◽  
List Type ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Uncoupling ◽  
Acute Hyperglycemia ◽  
Mitochondrial Superoxide

Background : Acute hyperglycemia (HG) impairs endothelial function, which may be related to hyperpolarization of the inner mitochondrial membrane and excessive production of mitochondrial superoxide (ROS). Mitochondrial uncoupling agents could potentially reverse endothelial dysfunction due to HG in intact human arterioles. Methods : Human adipose arterioles were incubated in either euglycemic (NG, 5mM) or hyperglycemic (HG, 33mM) buffer for 4 hours. Vessels were then exposed to increasing doses of acetylcholine (ACh) in the presence and absence of NADPH oxidase inhibitor gp91ds-tat or CCCP, (100 nM), a mitochondrial membrane uncoupling agent. Vascular superoxide levels were also measured after incubation with NG and HG using a chemiluminescent probe (L-012) in the absence and presence of 1 and 2 above. Results : ACh-induced vasodilation was impaired by HG vs. NG control (P<0.001). This impairment was modestly reversed by gp91 ds-tat (P< 0.001), but endothelial function was completely restored to the level observed under euglycemic conditions by CCCP (P=0.008 vs. HG, P>0.90 vs. NG). Endothelium-dependent vasodilation in HG vessels was greater after CCCP compared to gp91ds-tat (P<0.001). L-NAME inhibited all dilation to ACh indicating endothelium-dependence (data not shown). HG induced an increase in superoxide relative to NG (P<0.001), which was similarly suppressed by gp91ds-tat (P = 0.001) and CCCP (P=0.002). Conclusion : HG-induced endothelial dysfunction is reversed with partial uncoupling of mitochondrial oxidative phosphorylation. Mitochondrial uncoupling more than NADPH oxidase blockade improves endothelial dysfunction due to HG.

Eff ects of hyperbaric oxygen on NLRP3 infl ammasome activation in the brain aft er carbon monoxide poisoning

2020 ◽  
pp. 607-619
Author(s):  
Ya’nan Qi ◽  
◽  
Zhibao Guo ◽  
Huijun Hu ◽  
Xiang’en Meng ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Nadph Oxidase ◽  
Hyperbaric Oxygen ◽  
Nlrp3 Inflammasome ◽  
Carbon Monoxide Poisoning ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inflammasome Activation ◽  
Protein Levels ◽  
Nlrp3 Inflammasome Activation ◽  
Myelin Injury

Neuroinflammation plays an important role in brain damage after acute carbon monoxide poisoning (ACOP). The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing (NLRP) 3 inflammasome triggers the activation of inflammatory caspases and maturation of interleukin (IL)-1β and -18, and has been linked to various human autoinflammatory and autoimmune diseases. In this study we investigated the effects of hyperbaric oxygen (HBO2) on NLRP3 inflammasome activation after ACOP. Mice were randomly divided into four groups: sham group (exposure to normobaric air – i.e., 21% O2 at 1 atmosphere absolute); HBO2-only group; CO + normobaric air group; and CO + HBO2 group. Cognitive function was evaluated with the Morris water maze; myelin injury was assessed by Fluoro-Myelin GreenTM fluorescent myelin staining and myelin basic protein (MBP) immunostaining; and mRNA and protein levels of NLRP3 inflammasome complex proteins were measured by quantitative real-time PCR and Western blot, respectively. Additionally, serum and brain levels of IL-1β and -18 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were determined by enzyme-linked immunosorbent assay. It was found that HBO2 improved learning and memory, and alleviated myelin injury in mice subjected to acute CO exposure. Furthermore, HBO2 decreased NLRP3, absent in melanoma 2 (AIM2), caspase-1, and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain mRNA and protein levels, and reduced brain and serum concentrations of IL-1β and -18 and NADPH oxidase. These results indicate that HBO2 suppresses the inflammatory response after ACOP by blocking NLRP3 inflammasome activation, thereby alleviating cognitive deficits.

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